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https://openalex.org/W2135934178	https://bmcpsychiatry.biomedcentral.com/counter/pdf/10.1186/1471-244X-10-68	English	null	Acetylcholinesterase Inhibitors (AChEI's) for the treatment of visual hallucinations in schizophrenia: a case report	BMC psychiatry	2,010	cc-by	2,407	CASE REPORT Open Access * Correspondence: sukhi.shergill@kcl.ac.uk 2Kings College London, Institute of Psychiatry, De Crespigny Park, London, SE5 8AF, UK Full list of author information is available at the end of the article © 2010 Patel et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Visual hallucinations are commonly seen in various neurological and psychiatric disorders including schizophrenia. Current models of visual processing and studies in diseases including Parkinsons Disease and Lewy Body Dementia propose that Acetylcholine (Ach) plays a pivotal role in our ability to accurately interpret visual stimuli. Depletion of Ach is thought to be associated with visual hallucination generation. AchEI’s have been used in the targeted treatment of visual hallucinations in dementia and Parkinson’s Disease patients. In Schizophrenia, it is thought that a similar Ach depletion leads to visual hallucinations and may provide a target for drug treatment Case Presentation: We present a case of a patient with Schizophrenia presenting with treatment resistant and significantly distressing visual hallucinations. After optimising treatment for schizophrenia we used Rivastigmine, an AchEI, as an adjunct to treat her symptoms successfully. Conclusions: This case is the first to illustrate this novel use of an AchEI in the targeted treatment of visual hallucinations in a patient with Schizophrenia. Targeted therapy of this kind can be considered in challenging cases although more evidence is required in this field. Acetylcholinesterase Inhibitors (AChEI’s) for the treatment of visual hallucinations in schizophrenia: a case report Sachin S Patel1, Azizah Attard1, Pamela Jacobsen1, Sukhi Shergill2* Sachin S Patel1, Azizah Attard1, Pamela Jacobsen1, Sukhi Shergill2* Patel et al. BMC Psychiatry 2010, 10:68 http://www.biomedcentral.com/1471-244X/10/68 Patel et al. BMC Psychiatry 2010, 10:68 http://www.biomedcentral.com/1471-244X/10/68 Case Presentation Mrs A is a 43 year old female with a diagnosis of schi- zoaffective disorder. She was transferred to the National Psychosis Unit, a tertiary referral in-patient service which specialises in the management of treatment resis- tant psychotic illness. On admission she presented as dishevelled, agitated, thought disordered and labile in mood. She expressed grandiose and paranoid delusions, 3rd person auditory hallucinations and visual hallucina- tions of large wild cats. Negative features included apathy and withdrawal. Mrs A had little insight into her illness. These symptoms had persisted largely unchanged despite in-patient management and compliance with antipsychotic and mood stabilising medications for the previous 6 months. These visual experiences were evi- dent during the day in clear daylight and consciousness, but worse at night when she was alone in her bedroom; on admission, she would choose to sleep in the corridor so as to avoid these creatures- and had been doing so for over 6 months. A decision was made by the multidisciplinary team to begin an AChEI, Rivastigmine to target visual hallucina- tion symptoms. Rivastigmine patches at 4.6 mg/24 hrs was initiated. No changes were made to all other psy- chotropic medications. PANSS rating scales and MMSE scores were done on two occasions following the addi- tion of rivastigmine patches to therapy. In addition a tai- lored visual hallucination rating scale was developed, adapted from the Psychotic Symptom Rating Scales for auditory hallucinations (PSYRATS) [9]. This consisted of 3 items measuring frequency, vividness and distress associated with the hallucinations. Items were scored 0-4 (frequency and distress) or 0-3 (vividness), and were clinician-rated. Ratings were taken daily by the primary or allocated nurse in the two weeks prior to treatment with rivastigmine and during treatment. Mrs A first became unwell with features of a schizoaf- fective disorder at the age of 19. Following treatment and discharge there was a period of relative stability over the next 20 years during which she was under the care of her local community mental health team (CMHT). At the age of 40, Mrs A was again admitted following a breakdown in her ability to function in the community due to deterioration in her mental state. Various treatment strategies were utilised during this period, including clozapine, following failure of combi- nations of other atypical antipsychotics and mood stabi- lisers. Case Presentation She had responded well to a combination of clozapine, aripiprazole and escitalopram in terms of a reduction in persecutory delusions and auditory halluci- nations, however her visual hallucinations remained vivid. These had then taken greater prominence in Mrs A’s mental state and this subsequently led to more sub- jective distress. Socially she was quite isolative and did not maintain any relations with family or friends Her presentation was not thought to be related to non com- pliance, drug and alcohol misuse or psychosocial stres- sors. Physical Investigations were unremarkable. MRI and EEG were reported as normal and blood indices including thyroid function tests, copper, caeruloplasmin and autoantibody screens were negative. Mrs A continued to show an improvement in her functioning, demonstrated by the fact she now slept consistently in her own bedroom at night, was indepen- dent in her self-care, and started to participate in com- munity outings and OT activities. Mrs A’s level of occupational and psychological therapy input remained stable throughout the introduction of rivastigmine patches, and focused on reducing the distress and inter- ference with daily activities associated with the visual hallucinations. No further medication changes were made to her pharmacological therapy. Mrs A suffered no untoward side effects from the rivastigmine patches. Two weeks following the addition of rivastigmine patches her PANSS total score was 45 (P 13, N 10, GP 22) and this improvement was maintained as her PANSS total score at 7 weeks was 43 (P 11, N 10, GP 22). Over the baseline assessment period, Mrs A contin- ued to report distressing visual hallucinations through- out the day. After the rivastigmine treatment was initiated, after 3 weeks of treatment, reporting of visual hallucinations was decreased to once a day on average, and the level of distress was significantly reduced. This one appearance a day was usually reported as seeing a lion or tiger in her bedroom when she woke up in the middle of the night. Slowly, even this report became much more ambiguous; the animals were more unclear at night and she had more difficulty making them out. Subjectively, Mrs. A reported that she thought the patches were helpful and she was seeing the animals less frequently than before. She was discharged from Mrs A’s PANSS score on admission was 79 (p30, n15, g34) and MMSE was 30/30. Background surroundings [1,2]. Acetylcholine is thought to play a pivotal role in modulating this pathway with low levels correlating to a greater degree of context driven visual representations and thus contextual inaccuracy [3]. This contextual inaccuracy could explain visual hallucinations as images would be perceived despite their absence in external space. Visual hallucinations occur in a variety of neurological and psychiatric disorders and are prominent in the dementias and psychotic illness. The treatment of this distressing symptom often targets the underlying illness rather than the symptom. The pathophysiology of visual hallucinatory generation however remains unclear and more recent research has focused on acetylcholine depletion and its association with visual hallucinations. Diseases with significant Ach depletion include the dementias (in particular Lewy Body Dementia) and Par- kinson’s Disease. Drug therapies to increase levels of Ach are readily available (AchEI’s) and there is evidence to suggest their efficacy in the treatment of visual hallu- cinations in these conditions [4-8] Utilising current models of visual hallucination generation and evidence for the use of AchEI’s in related disorders it would appear that Ach depletion also plays a similar role in Schizophrenia. To be able to better understand visual hallucinatory experience, we must first consider how normal cognitive processing enables the brain to process elementary visual stimuli and convert them into meaningful per- cepts. Bayesian statistical principles offer an elegant model on which to conceptualise the visual pathway. It is proposed that ascending stimulus driven and descend- ing context driven pathways combine in an iterative manner to produce an accurate visual experience of our We present below a case of a patient with treatment resistant schizophrenia presenting with distressing visual hallucinations who we successfully treated with an AchEI, Rivastigmine. Patel et al. BMC Psychiatry 2010, 10:68 http://www.biomedcentral.com/1471-244X/10/68 Patel et al. BMC Psychiatry 2010, 10:68 http://www.biomedcentral.com/1471-244X/10/68 Page 2 of 3 Patel et al. BMC Psychiatry 2010, 10:68 http://www.biomedcentral.com/1471-244X/10/68 therapy, Mrs A’s mental state stabilised with marked improvement in her delusions and auditory hallucina- tions, stable mood and better function. Her PANSS rat- ing improved to a total score of 52 (p14, n13, g 25). Despite these improvements on clozapine, Mrs A con- tinued to experience vivid visual hallucinations of tigers and lions. Author details 1N l P h 1National Psychosis Unit, South London and Maudsley NHS Foundation Trust, Bethlem Royal Hospital, Monks Orchard Rd, Beckenham, BR33BX, UK. 2Kings College London, Institute of Psychiatry, De Crespigny Park, London, SE5 8AF, UK. Authors’ contributions SS contributed to planning, supervision and writing the report. SP reviewed the literature. SP, PJ and AA each contributed to writing the case presentation. Pre-publication history This case illustrates a novel use for AchEI’s in the tar- geted treatment of visual hallucinations in Schizophre- nia. Often the most challenging cases faced in clinical psychiatry are those with treatment resistant symptoms which can prove distressing to patients. Our approach in this case was to combine current thinking in neuro- physiology and therapeutic evidence in related disorders and then to apply these to clinical practice in a targeted way. We appreciate that this is a single case and a novel therapeutic use however we feel that further research in this field is indicated. The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-244X/10/68/prepub doi:10.1186/1471-244X-10-68 Cite this article as: Patel et al.: Acetylcholinesterase Inhibitors (AChEI’s) for the treatment of visual hallucinations in schizophrenia: a case report. BMC Psychiatry 2010 10:68. doi:10.1186/1471-244X-10-68 Cite this article as: Patel et al.: Acetylcholinesterase Inhibitors (AChEI’s) for the treatment of visual hallucinations in schizophrenia: a case report. BMC Psychiatry 2010 10:68. doi:10.1186/1471-244X-10-68 Cite this article as: Patel et al.: Acetylcholinesterase Inhibitors (AChEI’s) for the treatment of visual hallucinations in schizophrenia: a case report. BMC Psychiatry 2010 10:68. Case Presentation The pharmacological man- agement plan was to commence and maintain semi- sodium valproate within therapeutic plasma levels, reduce and discontinue her clonazepam and to restabi- lise on clozapine therapy. Following 4 months of this therapy with clozapine at a dose of 450 mg per day and in combination with psychological and occupational Patel et al. BMC Psychiatry 2010, 10:68 http://www.biomedcentral.com/1471-244X/10/68 Patel et al. BMC Psychiatry 2010, 10:68 http://www.biomedcentral.com/1471-244X/10/68 Page 3 of 3 Patel et al. BMC Psychiatry 2010, 10:68 http://www.biomedcentral.com/1471-244X/10/68 Patel et al. BMC Psychiatry 2010, 10:68 http://www.biomedcentral.com/1471-244X/10/68 8. Bullock R, Cameron A: Rivastigmine for the treatment of dementia and visual hallucinations associated with Parkinson’s disease: a case series. Current Medical Research and Opinion 2002, 18(5):258-64. 9. Haddock G, McGarron J, Tarrier N, Faragher EB: Scales to measure dimensions of hallucinations and delusions: the psychotic symptom rating scales (PSYRATS). Psychol Med 1999, 29:879-889. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-244X/10/68/prepub doi:10.1186/1471-244X-10-68 Cite this article as: Patel et al.: Acetylcholinesterase Inhibitors (AChEI’s) for the treatment of visual hallucinations in schizophrenia: a case report. BMC Psychiatry 2010 10:68. 8. Bullock R, Cameron A: Rivastigmine for the treatment of dementia and visual hallucinations associated with Parkinson’s disease: a case series. Current Medical Research and Opinion 2002, 18(5):258-64. 9. Haddock G, McGarron J, Tarrier N, Faragher EB: Scales to measure dimensions of hallucinations and delusions: the psychotic symptom rating scales (PSYRATS). Psychol Med 1999, 29:879-889. hospital and at 6 month follow up was living indepen- dently quite successfully, with support from her locality mental health team, and remaining free from visual hal- lucinations and continuing her rivastigmine. Consent Written informed consent was obtained from the patient for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests Authors have no competing interests to declare that are relevant to the content of this submission. Received: 30 July 2010 Accepted: 7 September 2010 Published: 7 September 2010 References References 1. Kersten D, Mamassian P, Yuille A: Object perception as bayesian inference. Annu Rev Psychology 2004, 55:271-304. 2. Friston K: A theory of cortical responses. Phil Trans R Soc B 2005, 360:815-836. 3. Yu D, Dayan P: Acetylcholine in cortical inference. Neural Networks 2002, 15:719-730. 4. Edwards K, Royall D, Hershey L, Lichter D, Hake A, Farlow M, Pasquier F, Johnson S: Efficacy and safety of galantamine in patients with dementia with Lewy bodies: a 24 week open-label study. Dementia and Geriatric Cognitive Disorders 2007, 23(6):401-5. 5. Fabbrini G, Barbanti P, Aurilia C, Pauletti C, Lenzi GL, Meco G: Donepezil in the treatment of hallucinations and delusions in Parkinson’s disease. Neurological Sciences 2004, 23(1):41-43. 6. Cummings JL: Cholinesterase Inhibitors: A new Class of Psychotropic Compounds. Am J Psychiatry 2000, 157:1, 4-15. 7. Cummings JL, Askin-Edgar S: Evidence for Psychotropic Effects of Acetylcholinesterase Inhibitors. CNS Drugs 2000, 13(6):385-395. 1. Kersten D, Mamassian P, Yuille A: Object perception as bayesian inference. Annu Rev Psychology 2004, 55:271-304. 2. Friston K: A theory of cortical responses. Phil Trans R Soc B 2005, 360:815-836. 3. Yu D, Dayan P: Acetylcholine in cortical inference. Neural Networks 2002, 15:719-730. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 4. Edwards K, Royall D, Hershey L, Lichter D, Hake A, Farlow M, Pasquier F, Johnson S: Efficacy and safety of galantamine in patients with dementia with Lewy bodies: a 24 week open-label study. Dementia and Geriatric Cognitive Disorders 2007, 23(6):401-5. • Convenient online submission 5. Fabbrini G, Barbanti P, Aurilia C, Pauletti C, Lenzi GL, Meco G: Donepezil in the treatment of hallucinations and delusions in Parkinson’s disease. Neurological Sciences 2004, 23(1):41-43. g 6. Cummings JL: Cholinesterase Inhibitors: A new Class of Psychotropic Compounds. Am J Psychiatry 2000, 157:1, 4-15. 7. Cummings JL, Askin-Edgar S: Evidence for Psychotropic Effects of Acetylcholinesterase Inhibitors. CNS Drugs 2000, 13(6):385-395.
https://openalex.org/W3187390191	https://www.research-collection.ethz.ch/bitstream/20.500.11850/541570/2/msac009.pdf	English	null	Purifying selection determines the short-term time dependency of evolutionary rates in SARS-CoV-2 and pH1N1 influenza	medRxiv (Cold Spring Harbor Laboratory)	2,021	cc-by	7,541	ETH Library Originally published in: Originally published in: Molecular Biology and Evolution 39(2), https://doi.org/10.1093/molbev/msac009 This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. Abstract High-throughput sequencing enables rapid genome sequencing during infectious disease outbreaks and provides an opportunity to quantify the evolutionary dynamics of pathogens in near real-time. One difﬁculty of undertaking evo- lutionary analyses over short timescales is the dependency of the inferred evolutionary parameters on the timespan of observation. Crucially, there are an increasing number of molecular clock analyses using external evolutionary rate priors to infer evolutionary parameters. However, it is not clear which rate prior is appropriate for a given time window of observation due to the time-dependent nature of evolutionary rate estimates. Here, we characterize the molecular evolutionary dynamics of SARS-CoV-2 and 2009 pandemic H1N1 (pH1N1) inﬂuenza during the ﬁrst 12months of their respective pandemics. We use Bayesian phylogenetic methods to estimate the dates of emergence, evolutionary rates, and growth rates of SARS-CoV-2 and pH1N1 over time and investigate how varying sampling window and data set sizes affect the accuracy of parameter estimation. We further use a generalized McDonald–Kreitman test to estimate the number of segregating nonneutral sites over time. We ﬁnd that the inferred evolutionary parameters for both pandemics are time dependent, and that the inferred rates of SARS-CoV-2 and pH1N1 decline by 50% and 100%, respectively, over the course of 1 year. After at least 4months since the start of sequence sampling, inferred growth rates and emergence dates remain relatively stable and can be inferred reliably using a logistic growth coalescent model. We show that the time dependency of the mean substitution rate is due to elevated substitution rates at terminal branches which are 2–4 times higher than those of internal branches for both viruses. The elevated rate at terminal branches is strongly correlated with an increasing number of segregating nonneutral sites, demonstrating the role of purifying selection in generating the time dependency of evolutionary parameters during pandemics. Article 09523 by ETH Zürich Key words: substitution rate, molecular clock, clock rate, purifying selection. Mahan Ghafari,1 Louis du Plessis,1 Jayna Raghwani ,1 Samir Bhatt,2 Bo Xu,3 Oliver G. Pybus ,1 and Aris Katzourakis,1 1Department of Zoology, University of Oxford, Oxford, United Kingdom 2MRC Centre for Global Infectious Disease Analysis, Jameel Institute for Disease and Emergency Analytics, Imperial College London, London, United Kingdom 1Department of Zoology, University of Oxford, Oxford, United Kingdom 2MRC Centre for Global Infectious Disease Analysis, Jameel Institute for Disease and Emergency Analytics, Imperial College London, London, United Kingdom 1Department of Zoology, University of Oxford, Oxford, United Kingdom 2MRC Centre for Global Infectious Disease Analysis, Jameel Institute for Disease and Emergency Analytics, Imperial College London, London, United Kingdom 3Department of Earth System Science, Tsinghua University, Beijing, China infectious disease outbreaks and provides an r real-time. One difﬁculty of undertaking evo- d evolutionary parameters on the timespan of analyses using external evolutionary rate priors ior is appropriate for a given time window of timates. Here, we characterize the molecular ) inﬂuenza during the ﬁrst 12months of their te the dates of emergence, evolutionary rates, ow varying sampling window and data set sizes zed McDonald–Kreitman test to estimate the d evolutionary parameters for both pandemics N1 decline by 50% and 100%, respectively, equence sampling, inferred growth rates and using a logistic growth coalescent model. We evated substitution rates at terminal branches uses. The elevated rate at terminal branches is ral sites, demonstrating the role of purifying during pandemics. ction. nt population models, changes in natural selection, plication rates in various host reservoirs, or se- rrors), misspecification of clock models and satu- nucleotide changes can result in rate ation (Clark and Whittam 1992; Sullivan and Duch^ene et al. 2014; Ghafari et al. 2021). This arity can render findings that are confusing or such that an estimated substitution rate over ndow is not transferable to the analysis of another 2011). For example, during the 2014–2016 West i id i th t d Article Downloaded from https://academic.oup.com/mbe/article/39/2/msac009/6509523 by ETH Zürich user on 27 April 2022 Corresponding author: E-mail: aris.katzourakis@zoo.ox.ac.uk. Corresponding author: E-mail: aris.katzourakis@zoo.ox.ac.uk. Corresponding author: E-mail: aris.katzourakis@zoo.ox.ac.uk. Associate editor: Harmit Malik The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons. org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Open Access Mol. Biol. Evol. 39(2): msac009 doi:10.1093/molbev/msac009 Advance Access publication January 17, 2022 1 Purifying Selection Determines the Short-Term Time Dependency of Evolutionary Rates in SARS-CoV-2 and pH1N1 Influenza Mahan Ghafari,1 Louis du Plessis,1 Jayna Raghwani ,1 Samir Bhatt,2 Bo Xu,3 Oliver G. Pybus ,1 and Aris Katzourakis,1 Mahan Ghafari,1 Louis du Plessis,1 Jayna Raghwani ,1 Samir Bhatt,2 Bo Xu,3 Oliver G. Pybus ,1 and Aris Katzourakis,1 Introduction of coalescent population models, changes in natural selection, differing replication rates in various host reservoirs, or se- quencing errors), misspecification of clock models and satu- ration of nucleotide changes can result in rate underestimation (Clark and Whittam 1992; Sullivan and Joyce 2005; Duch^ene et al. 2014; Ghafari et al. 2021). This nonstationarity can render findings that are confusing or conflicting such that an estimated substitution rate over one time window is not transferable to the analysis of another (Ho et al. 2011). For example, during the 2014–2016 West Africa Ebolavirus epidemic, there was controversy and con- cern regarding the virus’ “mutation rate,” because early esti- mates of the substitution rate from the epidemic appeared to be approximately twice the average rate across multiple user on 27 April 2022 Rapid whole-genome sequencing has become part of patho- gen surveillance systems and is important to both infection control and enables a detailed investigation of the epidemi- ological and evolutionary characteristics of pathogens. Quantifying infectious disease evolution enables the inference of parameters such as times of origin, epidemic growth rates, and evolutionary rates (Fraser et al. 2009; Smith et al. 2009; Lu et al. 2020). One of the perils of making such inferences over short time periods (i.e., a few months or years) is that the inferred param- eters of interest may vary over the timespan of observation (Meyer et al. 2015). Although several factors can lead to the misestimation of substitution rates (such as misidentification The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons. org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Open Access Mol. Biol. Evol. 39(2): msac009 doi:10.1093/molbev/msac009 Advance Access publication January 17, 2022 1 Open Access 1 MBE Ghafari et al. . doi:10.1093/molbev/msac009 molecular clock rate, time of origin, growth rate, and number of nonneutral sites for different data sets that represent dif- ferent timescales of genomic observation. We show that time dependency in estimates of the mean substitution rate is dominated by elevated rates at terminal branches, whereas the estimated rate of substitution at internal branches does not exhibit a time-dependent decay. Results We use Bayesian phylogenetic methods implemented in BEAST v.1.10 to estimate the molecular clock rate, times of origins, and growth rates of the SARS-CoV-2 and pH1N1 pandemics. The data sets varied in size and temporal sam- pling range and were created to reflect the way in which real data sets accumulate in size and diversity during the course of an epidemic. Specifically, we aggregate all available samples up to each respective month and infer the parameters of interest (fig. 1 and supplementary fig. S1, Supplementary Material online). We first compare the results for SARS-CoV-2 using two coalescent growth priors: exponential growth and logistic growth. Although this is not a comprehensive comparison between all phylogenetic models that can be used to explain the evolutionary dynamics, it allows us to find the better- fitting model that introduces less error in parameter estima- tion—the demographic model is effectively a nuisance vari- able. Our analysis suggests, except for the month of January, the logistic growth coalescent tree prior is a better fit to the data (table 1). As reported in previous studies, we find that there is not enough temporal signal in the data to reliably infer the evolutionary parameters during the first 2 months of the SARS-CoV-2 pandemic (Duch^ene et al. 2020). This results in the underestimation of substitution rates as well as high statistical uncertainty for the parameter estimates. The lack of temporal signal in the SARS-CoV-2 samples is also evident from the number of new singletons (i.e., single nucleotide variations in the data set compared with the ancestral se- quence) added the data set per month during the first 2– 3months (supplementary fig. S2, Supplementary Material on- line). On the other hand, for pandemic H1N1 (pH1N1), the molecular clock rate is up to two times higher for the first 3 months than for the following months (fig. 1A and B). Although the inferred substitution rate of SARS-CoV-2 tends to decrease as we increase the timespan of measurement, the rate for pH1N1 does not change considerably after the first 3– 4months of measurement, in agreement with previous find- ings (Meyer et al. 2015). More specifically, there is extensive evidence that purifying selection in viruses can result in varied estimates for rates of substitution and evolutionary rate ratio, dN/dS, across differ- ent timescales (Sharp et al. 2001; Holmes 2003; Hughes and Hughes 2007). Introduction We then use a general- ized McDonald–Kreitman test based on nucleotide site fre- quencies that allows purifying selection to be quantified over short timescales and demonstrate that there is a strong cor- relation between the elevated rates at terminal branches and the high number of low frequency nonneutral sites in both SARS-CoV-2 and pH1N1 genomes. outbreaks (Gire et al. 2014; Holmes et al. 2016). Confusion such as this is also common in the SARS-CoV-2 literature and arises in part due to an incomplete understanding of esti- mated substitution rates by users of phylogenetic analysis software. Given the importance of phylogenetic dating and clock estimation for all SARS-CoV-2 genomic epidemiology worldwide, understanding the pattern of inferred substitution rate over time and investigating the potential underlying mechanisms involved in creating time-dependent rate effects in viruses can shed light on the molecular evolutionary dy- namics of viruses. Another major obstacle is that during an ongoing out- break, the level of sequence diversity may be so low that the amount of accrued evolutionary change is not sufficient to make informative phylogenetic inference possible (Duch^ene et al. 2020). Several statistical tests have been de- veloped to ensure enough temporal signal is present in a set of temporally sampled genome sequences to reliably infer evolutionary parameters (Duch^ene et al. 2015; Murray et al. 2016; Duch^ene S and Duch^ene DA 2020). Results It is likely that a greater proportion of poly- morphisms observed among genomic sequences sampled early in an epidemic are segregating deleterious mutations, which will only persist for a limited time before being elimi- nated by purifying selection (Lam et al. 2016). The duration of this time-dependent effect may be prolonged due to incom- plete purifying selection in populations with very large effec- tive population sizes (Woodhams 2006). p p ( ) Although evidence of strong purifying selection has mostly been reported at the species level and over very long time- scales (i.e., thousands to millions of years), using inference methods based on the dN/dS ratio (Ho et al. 2005; Pybus et al. 2007; Wertheim and Pond 2011), there have been few studies at the intra-population level and over short time- scales, mainly because there is often no opportunity to collect sufficiently large numbers of samples through time to track low-frequency variants (Hedge et al. 2013; Meyer et al. 2015). Crucially, although purifying selection has been put forward as a possible explanation for the time dependency of substi- tution rates over such timescales, there have not been any systematic studies to investigate the role of purifying selection and quantify its contribution to altering the inferred substi- tution rate of viruses over time. Furthermore, using standard phylogenetic methods to compute the dN/dS ratio for con- specific sequences sampled from a single population over short timescales may be inappropriate as the differences be- tween sequences over such timescales represent segregating polymorphisms as opposed to fixed substitutions along inde- pendent lineages. The former has been shown to produce very different estimates of the dN/dS ratio over time (Rocha et al. 2006; Kryazhimskiy and Plotkin 2008). Both the inferred times of origin and growth rates of SARS- CoV-2 and pH1N1 in early months have wide credible inter- vals due to the high uncertainty associated with small sample sizes and narrow sampling windows. The precision of these two inferred parameters increases with the addition of data In this study, we aim to identify and quantify the source of time dependency of virus substitution rate estimates over short time periods and characterize estimates of the 2 A B C D E F FIG. 1. Results (A, B) Inferred rates, (C, D) times of origin, and (E, F) growth rates of SARS-CoV-2 (left column) and pH1N1 inﬂuenza (right column) using an exponential (red) and a logistic (black) growth model. Open circles represent nonconvergence for at least one parameter in the Bayesian analysis. Note that y-axes are not on the same scale for SARS-CoV-2 and pH1N1. Evolutionary Rates in SARS-CoV-2 and pH1N1 Influenza . doi:10.1093/molbev/msac009 MBE MBE Evolutionary Rates in SARS-CoV-2 and pH1N1 Influenza . doi:10.1093/molbev/msac009 A B B A Downloaded from https://academic.oup.com/mbe/article/39/2/msac009/6509523 by ETH Zürich user on 27 April 2022 Downloaded from https://academic.oup.com/mbe/article/39/2/msac009/6509523 by ETH Zürich user on 27 April 2022 C D D C F E E F E F FIG. 1. (A, B) Inferred rates, (C, D) times of origin, and (E, F) growth rates of SARS-CoV-2 (left column) and pH1N1 inﬂuenza (right column) using an exponential (red) and a logistic (black) growth model. Open circles represent nonconvergence for at least one parameter in the Bayesian analysis. Note that y-axes are not on the same scale for SARS-CoV-2 and pH1N1. expectation after 6months of sampling and yield unreliable estimates (fig. 1C and D). This is likely because after the first few months, the growth rate declines and the population dynamics deviate from the exponential growth model (fig. 1E and F). This, in turn, results in the underestimation of growth rate of SARS-CoV-2 and inferring an older time of origin. We also note that, from a molecular epidemiology perspective, using the exponential growth coalescent model would not be appropriate nor realistic particularly over longer timescales (i.e., >6months) as the pandemic dynamics did from later months and remains roughly consistent after the first 4–5months of measurement (fig. 1C–F). Using the logis- tic growth model, we find the estimated times of origin for SARS-CoV-2 and pH1N1 to be October 28, 2019 (95% HPD: September 30, 2019, November 24, 2019) and January 18, 2009 (95% HPD: December 14, 2008, February 22, 2009), re- spectively, which is also in agreement with previous studies (Smith et al. 2009; Hedgeet al. 2013; Lu et al. 2020). However, under the exponential growth model, the inferred time of origin of SARS-CoV-2 samples significantly diverge from the from later months and remains roughly consistent after the first 4–5months of measurement (fig. 1C–F). Results Using the logis- tic growth model, we find the estimated times of origin for SARS-CoV-2 and pH1N1 to be October 28, 2019 (95% HPD: September 30, 2019, November 24, 2019) and January 18, 2009 (95% HPD: December 14, 2008, February 22, 2009), re- spectively, which is also in agreement with previous studies (Smith et al. 2009; Hedgeet al. 2013; Lu et al. 2020). However, under the exponential growth model, the inferred time of origin of SARS-CoV-2 samples significantly diverge from the 3 MBE Ghafari et al. . doi:10.1093/molbev/msac009 Table 1. Log-Marginal Likelihoods of Exponential and Logistic Growth Models with Increasing Temporal Ranges of Sampling Dates. Month of Sampling Number of Samples Log-Marginal Likelihood Exponential Growth Model Log-Marginal Likelihood Logistic Growth Model Bayes Factor Jan 41 241,022.77 241,025.56 22.79 Feb 121 243,062.00 243,047.11 114.88 Mar 181 244,627.77 244,607.95 119.78 Apr 241 246,524.75 246,506.25 118.53 May 301 248,587.39 248,548.15 139.24 Jun 361 252,310.61 252,248.23 162.38 Jul 421 255,138.43 255,036.60 1101.83 Aug 481 258,580.94 258,430.09 1150.85 Sep 541 262,101.22 261,805.93 1295.29 Oct 601 265,433.20 265,005.09 1428.11 Nov 661 269,121.03 268,921.98 1199.05 Dec 721 273,094.26 272,389.69 1704.57 NOTE.—Taking exponential growth as the null model, we select the logistic growth model for any data set with a positive Bayes factor. odels with Increasing Temporal Ranges of Sampling Dates Downloaded from https://academic.oup.com/mbe/article/39/2/msac009/6509523 by ETH Zürich user on 27 April 2022 NOTE.—Taking exponential growth as the null model, we select the logistic growth model for any data set with a positive Bayes factor. replacement sites is always greater than silent sites in ORF1ab whereas, for HA, silent sites are in majority in most months (fig. 2C and D). The total number of nonneutral sites in ORF1ab is higher because it is a much longer gene (21,000nt) compared with HA (1,800nt). not continue to grow exponentially (i.e., the growth rate started to drop after the first few months due to various behavioral changes in the population and the implementa- tion of nonpharmaceutical interventions). Despite the differ- ence in the estimated time of origin, both the exponential and logistic growth models infer very similar clock rates for SARS-CoV-2 over time. One of the impacts of purifying selection over short time- scales is that the number of replacements on terminal branches should be higher than that on internal branches. Results To measure this effect during the SARS-CoV-2 and pH1N1 epidemics, we used a branch-specific two-parameter molec- ular clock model in BEAST and infer separate rates of substi- tution for terminal and internal branches (see fig. 2E and F). Our results show that for both viruses, the rates of substitu- tion on terminal branches are 2–4 times higher than on in- ternal branches. The difference is more dramatic in SARS- CoV-2 where the average ratio of substitution rates at termi- nal branches are four times higher than that of internal branches whereas it is only two times higher in HA during the first 12month of observation. Figure 2E and F also shows that the mean and credible intervals of the inferred substitu- tion rate at terminal and internal branches are nonoverlap- ping during several months of rate measurement which demonstrates that there is a significant difference between the rate of evolution at these two branch categories. This also agrees with the observation of a higher proportion of non- neutral sites in SARS-CoV-2 samples compared with pH1N1 because there is going to be more deleterious mutations at terminal branches of SARS-CoV-2. Further, there is a clear decline in the estimated rate at terminal branches as the sampling window increases. In contrast, the estimated rate at internal branches gradually increases through time (with the exception of the first 2–3months for pH1N1). Purifying selection has been often cited as one of the main evolutionary processes contributing to the elevation of esti- mated molecular evolutionary rates over short timescales (Hedge et al. 2013). The argument is that low-frequency del- eterious mutations can segregate in a population for some time before being purged because of purifying selection. Therefore, the proportion of all changes that are deleterious is high when the phylogenetic tree is short, and lower when the tree is longer as it takes more time for shared neutral or advantageous changes to accrue with respect to the ancestral state. To evaluate this hypothesis, we investigate the correla- tion between the number of nonneutral polymorphisms and estimated substitution rates over time. Our results show that there is a higher proportion of segregating mutations at low frequencies (<15%) during the first few months of observa- tion, in both the ORF1ab of SARS-CoV-2 and the HA of pH1N1 influenza (fig. 2A and B). Results In particular, the proportion of low-frequency nonneutral sites in ORF1ab was higher dur- ing the first 4–5months of observation and had a roughly 4- fold drop during that period compared with a 2-fold drop in HA gene. After the first 5months, the proportion of low frequency nonneutral sites remains roughly constant at around 15% in ORF1ab, whereas it shows an uptick from 15% to 17% in HA toward the beginning of 2010. The slight increase in the proportion of nonneutral sites in HA during this period is also in agreement with a rise in the relative genetic diversity of pH1N1 around the world in late 2009/ early 2010 which may also have resulted in an increase in the number of segregating deleterious mutations in the popula- tion (Su et al. 2015). We note that although it is unlikely for mutations in the low frequency class to have strongly positive fitness effects, they may also contain neutral or adaptive mutations that have not reached sufficiently high frequencies yet. We also find that the number of low frequency We also investigate the role of sequencing error as a po- tential confounder for the elevated rates at terminal branches. By masking more than 110 sites of SARS-CoV-2, in addition to our standard filtering step (see Materials and Methods sec- tion), which are suggested to be prone to recurrent sequenc- ing error and appear to be highly homoplastic, we recalculate the rate of evolution at terminal and internal branches (sup- plementary fig. S3, Supplementary Material online). We find that although the overall inferred rates drop for the 4 MBE Evolutionary Rates in SARS-CoV-2 and pH1N1 Influenza . doi:10.1093/molbev/msac009 A B C D E F G H n o n f o n oitc a r F -neutral sites Substuon rate at terminal branches Substuon rate at terminal branches Fracon of non-neutral sites n o n f o n oitc a r F -neutral sites Fracon of non-neutral sites Inferred substuon rate (SSY) )Y S S( e t a r n oit u tits b u s d e rr ef nI FIG. 2. (A, B) Number of nonneutral sites over time for SARS-CoV-2 (left column) and pH1N1 (right column). (C, D) Number of replacement (dashed blue line), silent (solid blue line) sites, and their ratio (red line) over time. Results (E, F) Mean clock rate (black), and the rates at the terminal (red) and internal (blue) branches. The MCMC chains for the ﬁrst month of sampling SARS-CoV-2 and the ﬁrst three months of pH1N1 do not converge using the logistic growth coalescent model. Instead, the exponential growth coalescent model was used. (G, H) Correlation coefﬁcient between the substitution rate at terminal branches and number of nonneutral sites—excluding the estimates from the ﬁrst month of sampling due to inadequate temporal signal and signiﬁcant uncertainty in the inferred parameters. Downloaded from https://academic.oup.com/mbe/article/39/2/msac009/6509523 by ETH Zürich user on 27 April 2022 A n o n f o n oitc a r F -neutral sites B Fracon of non-neutral sites B A n o n f o n oitc a r F -neutral sites Downloaded from https://academic.oup.com/mbe/article/39/2/msac009/6509523 by ETH Zürich user on 27 April 2022 D C C D E )Y S S( e t a r n oit u tits b u s d e rr ef nI F Inferred substuon rate (SSY) E F G Substuon rate at terminal branches n o n f o n oitc a r F -neutral sites H Substuon rate at terminal branches Fracon of non-neutral sites H G FIG. 2. (A, B) Number of nonneutral sites over time for SARS-CoV-2 (left column) and pH1N1 (right column). (C, D) Number of replacement (dashed blue line), silent (solid blue line) sites, and their ratio (red line) over time. (E, F) Mean clock rate (black), and the rates at the terminal (red) and internal (blue) branches. The MCMC chains for the ﬁrst month of sampling SARS-CoV-2 and the ﬁrst three months of pH1N1 do not converge using the logistic growth coalescent model. Instead, the exponential growth coalescent model was used. (G, H) Correlation coefﬁcient between the substitution rate at terminal branches and number of nonneutral sites—excluding the estimates from the ﬁrst month of sampling due to inadequate temporal signal and signiﬁcant uncertainty in the inferred parameters. 5 Ghafari et al. . doi:10.1093/molbev/msac009 Our results provided further evidence that short-term rate estimates are subject to time-dependent rate effects largely due to transient polymorphisms (Hedgeet al. 2013; Meyer et al. 2015). In addition, our estimated mean substitution rates, times of origin, and growth rates agree with previous studies of SARS-CoV-2 (Lu et al. 2020; Volz et al. 2020; Duch^ene et al. 2020; du Plessis et al. q y g Material online). Furthermore, in the pH1N1 data set, we can see a dip in the inferred rate at terminal branches in December 2009 (fig. 2F). There is also a sudden loss of low frequency genetic diversity in the data set during the same period which is partially recovered in February and March (see the trajectory of newly added singletons and low frequency variants in sup- plementary fig. S2D and F, Supplementary Material online). On the other hand, the pattern of substitution rate at internal branches is nonmonotonic over time. In particular, we see an increase in the rate at internal branches of SARS-CoV-2 from August onward. Similarly, there is a steady rise in the rate at internal branches of pH1N1 from February onward. By dividing the data into successively longer temporal intervals, we showed that the overabundance of deleterious mutations at terminal branches is the main reason behind the gradual decay in mean substitution rate over time in SARS- CoV-2 and pH1N1 influenza. Further work can be done to quantify the longer-term effect of purifying selection on the time dependency of substitution rates. Discussion We found that the overabundance of deleterious mutations during the early stages of both the SARS-CoV-2 and pH1N1 influenza pandemics strongly correlates with higher substitu- tion rates at terminal branches relative to internal branches of inferred phylogenetic trees. Once there is enough temporal signal in the data to reliably estimate relevant epidemiological and evolutionary parameters, the mean substitution rates decline over the course of 1 year of rate measurement, whereas the estimated time of origin and growth rate are more stable and remain roughly the same after the first 4– 5months of measurement. We found that this declining pat- tern in mean substitution rate is caused by the rate decay at terminal branches whereas the rate at internal branches does not exhibit such a pattern over time. In particular, the gradual increase in the substitution rate at internal branches over the span of several months for both SARS-CoV-2 and pH1N1 influenza could be related to signatures of adaptive evolution. We also found that sites that are prone to recurrent sequenc- ing error and appear to be highly homoplastic do not make a significant contribution to the time-dependent pattern of substitution rates. Although both sequencing error and del- eterious mutations appear as singletons or low-frequency variations in the data, deleterious mutations are strongly bi- ased toward changing amino acids, whereas sequencing errors are equally likely to be synonymous or nonsynony- mous. Therefore, they do not make similar contributions to altering the inferred substitution rates over time. Results 2021) and pH1N1 influ- enza (Rambaut and Holmes 2009; Smith et al. 2009; Hedge et al. 2013; Meyer et al. 2015). We found that although the time-dependent rate effect for pH1N1 is less pronounced after the first 3months of sampling, the same effect in SARS-CoV-2 is more pronounced and continues even after 12months of sampling. This may be the result of a much higher mean substitution rate in pH1N1 compared with SARS-CoV-2. We also found a higher proportion of low fre- quency nonneutral sites for the SARS-CoV-2 data set during the first 8months of observations and a higher ratio of re- placement to silent sites during the entire 1 year of observa- tion compared with pH1N1. The same method used in this study to infer the number of low frequency nonneutral sites has also been used to characterize the molecular evolutionary dynamics of other RNA viruses including seasonal coronavi- ruses and emerging avian influenza viruses (Bhatt et al. 2013; Lu et al. 2018; Kistler and Bedford 2021). alignments with masked sites (due to their lower genetic variation compared with unmasked alignments), the time- dependent rate drop at terminal branches is retained (sup- plementary fig. S3B, Supplementary Material online). Therefore, sequencing error cannot be the underlying source for time dependency of evolutionary rates. In figure 2G and H, we further show that there is an overall significant correlation between the rate of substitution at terminal branches for the unmasked alignments and number of nonneutral sites in both SARS-Cov-2 (R2 ¼ 0.74 and P< 0.001) and pH1N1 (R2 ¼ 0.53 and P< 0.01) (fig. 2G and H). By examining the site frequency spectrum for both data sets, we can also see that most of the variation comes from the low-frequency regime (<15%) with very limited variation in the mid- to high- frequency regimes (supplementary fig. S2, Supplementary Material online). alignments with masked sites (due to their lower genetic variation compared with unmasked alignments), the time- dependent rate drop at terminal branches is retained (sup- plementary fig. S3B, Supplementary Material online). Therefore, sequencing error cannot be the underlying source for time dependency of evolutionary rates. Results In figure 2G and H, we further show that there is an overall significant correlation between the rate of substitution at terminal branches for the unmasked alignments and number of nonneutral sites in both SARS-Cov-2 (R2 ¼ 0.74 and P< 0.001) and pH1N1 (R2 ¼ 0.53 and P< 0.01) (fig. 2G and H). By examining the site frequency spectrum for both data sets, we can also see that most of the variation comes from the low-frequency regime (<15%) with very limited variation in the mid- to high- frequency regimes (supplementary fig. S2, Supplementary Material online). alignments with masked sites (due to their lower genetic variation compared with unmasked alignments), the time- dependent rate drop at terminal branches is retained (sup- plementary fig. S3B, Supplementary Material online). Therefore, sequencing error cannot be the underlying source for time dependency of evolutionary rates. In figure 2G and H, we further show that there is an overall significant correlation between the rate of substitution at terminal branches for the unmasked alignments and number of nonneutral sites in both SARS-Cov-2 (R2 ¼ 0.74 and P< 0.001) and pH1N1 (R2 053 d P 001) (fi 2G d H) B i i h i ¼ 0.53 and P< 0.01) (fig. 2G and H). By examining the site frequency spectrum for both data sets, we can also see that most of the variation comes from the low-frequency regime (<15%) with very limited variation in the mid- to high- frequency regimes (supplementary fig. S2, Supplementary Material online). Materials and Methods We downloaded all SARS-CoV-2 sequences from GISAID and pH1N1 influenza sequences from GenBank and aligned them using MUSCLE v3.8.425 (Edgar 2004)—a complete metadata table acknowledging the authors, originating and submitting laboratories of the SARS-CoV-2 sequence data is available in supplementary table S1, Supplementary Material online. For the SARS-CoV-2 data set, we mask the first 54 and last 240 sites of the alignments and only include complete sequences that are more than 29,000nt long with high coverage as de- termined by GISAID’s default search option (i.e., entries with <1% Ns and <0.05% unique amino acid substitutions). Furthermore, for part of the analysis where we investigate the role of sequencing error in time-dependent rate effects at terminal branches for SARS-CoV-2, we mask an additional 114 sites that appear to be highly homoplastic or prone to recurrent sequencing error (De Maio et al. 2020, https://viro- logical.org/t/issues-with-sars-cov-2-sequencing-data/473). We specifically select pH1N1 sequences from GenBank for the full coding region of the hemagglutinin (HA) and ensure that none of the SARS-CoV-2 and pH1N1 samples have under- gone serial passaging. To investigate the effects of the temporal range of sam- pling dates on the accuracy of parameter estimation, we in- crementally increase the size of each data set by adding 60 6 MBE Evolutionary Rates in SARS-CoV-2 and pH1N1 Influenza . doi:10.1093/molbev/msac009 genomes, chosen randomly, for every additional month of sampling. Thus, after 12 months of sampling since the first sequence was uploaded on GISAID and/or GenBank, we have 720 samples. We note that due to a lack of temporal signal in the early SARS-CoV-2 samples (i.e., most of the early samples were almost completely identical) and failure of the Markov chain Monte Carlo (MCMC) chains for the phylogenetic anal- yses to converge, we only used 41 samples collected between December 24, 2019 and January 31, 2020 (labeled as “January sequences” in our analysis) and took 20 additional samples during the next month to match with the 120 samples used for pH1N1 by the end of the second month of sampling (see supplementary fig. S1, Supplementary Material online). We use the adapt-a-rate package (Bhatt et al. 2010, 2011; Raghwani et al. 2016), a generalized McDonald–Kreitman test, to estimate the number of nonneutral sites by assuming that deleterious mutations are mostly confined to the low frequency range (0–15%), neutral mutations to the mid fre- quency range (15–75%), and adaptive mutations to the high frequency range (75–100%). Acknowledgments This work was supported by the Biotechnology and Biological Science Research Council (BB/M011224/1 to M.G) and the European Research Council (101001623- PALVIREVOL to A. K.). For each data set, we perform MCMC runs for 100 million steps, sample trees every 10,000 steps, and remove the first 10% of the steps as burn-in. We ensure that the effective sample size for every parameter of interest is >200 using Tracer v1.7 (Rambaut et al. 2018). For the second part of the analysis, we use a two-parameter molecular clock model with one strict clock rate for terminal branches and one strict clock for internal branches, using the same priors as before. This molecular clock model is a version of a fixed local clock whereby rather than having a single global rate on all branches, the terminal and internal branches are allowed to evolve according to different evolutionary rates whereas rate constancy is assumed along the respective branches (Yoder and Yang 2000; Drummond and Suchard 2010). We use an MCMC chain of length 50 million steps, sampling every 1,000 steps and evaluate sampling of the parameters of interest using Tracer v1.7. Phylogenetic Analyses Phylogenetic Analyses We use BEAST v1.10 (Suchard et al. 2018) for the Bayesian phylogenetic analysis of the entire data set using an HKYþ C substitution model with a Laplace prior (mean¼ 0 and scale¼ 100) on the coalescent growth rate, a Lognormal prior (mean¼ 1 and SD¼ 2) on the coalescent population size, and a continuous time Markov chain prior on the evolution- ary clock rate. For the first part of the analysis, we use a strict clock model and exponential and logistic growth coalescent demographic models of SARS-CoV-2 evolution and only the logistic growth model for the HA segment of pH1N1. To quantify the relative fit of the two coalescent models for SARS-CoV-2, we compute their log marginal likelihoods using the generalized stepping-stone sampling method and com- pare their Bayes factors (Fan et al. 2011; Baele and Lemey 2014; Baele et al. 2016). We note that although the growth coefficient of the two coalescent models is not expected to converge to the same value (as they correspond to intrinsi- cally different population dynamics), the two models are for- mally nested, and the likelihood function of the exponential model exists as a limit of the likelihood function of the logistic model. Therefore, even though the growth rate is effectively a nuisance variable for our purposes, the likelihoods of the two models can be compared via a likelihood ratio test to select the better-fitting one (Pybus and Rambaut 2002). We use the ORF1ab of SARS-CoV-2 and HA of pH1N1 influenza for this analysis. We also carried out a similar analysis for the other genes of SARS-CoV-2, including the S gene. However, because of the limited genetic diversity present in the sequences and their relatively short size, our generalized McDonald–Kreitman test using adapt-a-rate is unable to es- timate the number of nonneutral sites. The reason for choos- ing to analyze the HA is primarily because of the availability of a large number of sequences from GenBank for the full coding region of HA which allows us to randomly sample 60 align- ments per month from April 2009 to March 2010. m/mbe/article/39/2/msac009/6509523 by ETH Zürich user on 27 April 2022 Supplementary Material Supplementary Material Supplementary data are available at Molecular Biology and Evolution online. Data Availability Data sets required to reproduce the analyses are available at https://github.com/mg878/twoclock_rate. The adapt-a-rate package is also available at https://github.com/jnarag/ teaspoon. Materials and Methods We then estimate the site fre- quency spectra by comparing the main alignments to an ancestral sequence. For SARS-CoV-2, the ancestral sequence is the earliest sample collected from Wuhan, Wuhan/ IPBCAMS-WH-01/2019, and for pH1N1 it is the earliest sam- ple from Mexico, ACQ99614jA/Mexico/4108/2009. 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https://openalex.org/W1990765490	https://dash.harvard.edu/bitstream/1/4728500/1/2150640.pdf	English	null	Improving Sedative-Hypnotic Prescribing in Older Hospitalized Patients: Provider-Perceived Benefits and Barriers of a Computer-Based Reminder	Journal of general internal medicine	2,007	cc-by	4,115	Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:4728500 Citation Agostini, Joseph V., John Concato, and Sharon Inouye. 2007. Improving sedative-hypnotic prescribing in older hospitalized patients: Provider-perceived benefits and barriers of a computer-based reminder. Journal of General Internal Medicine 23(Suppl 1): 32-36. Published Version doi:10.1007/s11606-007-0238-9 doi:10.1007/s11606-007-0238-9 Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility JGIM JGIM BRIEF REPORTS PARTICIPANTS AND SETTING: Thirty-six house staff physicians at a university hospital. PARTICIPANTS AND SETTING: Thirty-six house staff physicians at a university hospital. MEASUREMENTS: Information was collected regarding the experiences of prescribing an SH using a computer order entry system with a reminder intervention. Clini- cians were asked about their perceptions of the re- minder and what they found most and least useful about it. Responses were analyzed using grounded theory methodology. Computerized systems could improve the quality of health care by providing data to physicians at the point of clinical care,8,9 and increasing the use of such systems has been shown to improve the safety of drug prescribing behavior.10–12 Computer-based reminders, in particular, may play an impor- tant role to augment other ongoing quality improvement efforts to improve prescribing, such as clinical pharmacist consulta- tions. We conducted a qualitative study to explore how clinicians made their prescribing decisions and to determine attitudes towards a computer-based reminder in routine practice that has been previously demonstrated13 to be effective for reducing inappropriate SH drug use. Computerized systems could improve the quality of health care by providing data to physicians at the point of clinical care,8,9 and increasing the use of such systems has been shown to improve the safety of drug prescribing behavior.10–12 RESULTS: The 36 participants (including 29 interns) had prescribed an SH medication for a hospitalized patient over age 65 years. Three themes associated with benefits of a computer reminder were identified: increasing awareness of safety, including risk of delirium, falls, and general patient safety risks; use- fulness of information technology; and the value of the educational content, including geriatric pharmacology review and nonpharmacologic treatment options. Bar- riers included the demands of the reminder with regard to time needed to read the reminder, the role of clinician experience with regard to preserving clinical autonomy, and the information content of the reminder, including its being too basic or not relevant for a particular patient. The mean satisfaction rating for the reminder was 8.5 (±0.9 SD), with 10 indicating high satisfaction. Computer-based reminders, in particular, may play an impor- tant role to augment other ongoing quality improvement efforts to improve prescribing, such as clinical pharmacist consulta- tions. We conducted a qualitative study to explore how clinicians made their prescribing decisions and to determine attitudes towards a computer-based reminder in routine practice that has been previously demonstrated13 to be effective for reducing inappropriate SH drug use. Improving Sedative-Hypnotic Prescribing in Older Hospitalized Patients: Provider-Perceived Benefits and Barriers of a Computer-Based Reminder 1Clinical Epidemiology Research Center 151B, VA Connecticut Healthcare System, 950 Campbell Avenue, West Haven, CT 06516, USA; 2Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA; 3Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Aging Brain Center, Hebrew SeniorLife, Boston, MA, USA. KEY WORDS: point-of-care systems; medical order entry systems; sedatives and hypnotics; aged; computers. J Gen Intern Med 23(Suppl 1):32–6 DOI: 10.1007/s11606-007-0238-9 © Society of General Internal Medicine 2007 BACKGROUND: Older adults are commonly prescribed sedative-hypnotic (SH) medications when hospitalized, yet these drugs are associated with important adverse effects such as falls and delirium. OBJECTIVE: To identify provider-perceived benefits or barriers of a computer-based reminder regarding ap- propriate use of SH medications. BACKGROUND DESIGN: Qualitative study using semi-structured interviews. DESIGN: Qualitative study using semi-structured interviews. Sedative-hypnotic (SH) medication use in older adults is associated with potentially deleterious adverse effects, includ- ing increased risk of delirium, falls, over-sedation, and carry- over effects.1–3 Despite these risks, one third or more of older patients are prescribed an SH drug during hospitalization.4,5 Use of these drugs is predominantly elective, based on the discre- tion of the prescribing physician, although SH drugs are not rec- ommended as first-line therapies for insomnia in older adults.6,7 METHODS Description of original study A pre/post-intervention study13 was conducted to develop and evaluate a feasible point-of-care computer-based reminder that (1) provided a brief educational review of potential adverse effects of SH medications and (2) offered recommendations for a nonpharmacologic approach for insomnia in older adults. The reminder was incorporated CONCLUSIONS: Improving decision support systems involves an understanding of how clinicians respond to real-time strategies encouraging better prescribing. 32 Agostini et al.: Benefits and Barriers of a Computerized Reminder 33 JGIM within an existing computer system (Eclipsys, Boca Raton, FL) at a large academic medical center (Yale-New Haven Hospital) that requires electronic entry of all laboratory tests, medications, and other patient care orders. The reminder was designed over 1 year, following input from several constituencies, including the pharmacy staff, hospital computer programmers, and Internal Medicine faculty. within an existing computer system (Eclipsys, Boca Raton, FL) at a large academic medical center (Yale-New Haven Hospital) that requires electronic entry of all laboratory tests, medications, and other patient care orders. The reminder was designed over 1 year, following input from several constituencies, including the pharmacy staff, hospital computer programmers, and Internal Medicine faculty. value of the reminder, and opportunities to improve the computer ordering process. The interview questions were semi-structured regarding participants’ perceptions of the reminder and were followed with open-ended questions and probes (e.g., “Any other [benefits, barriers] of the reminder?”). In addition, satisfaction with the educational reminder was rated using a score from 0 to 10, with higher scores indicating a greater degree of satisfaction. Participants were no longer recruited when no new themes were elicited during interviews (i.e., the point of thematic saturation). When diphenhydramine or diazepam was ordered, a screen appeared that sought confirmation whether the indication for the order was for sleep. If so, a text reminder appeared on a new screen with several statements: “In older patients, confu- sion, delirium, falls, and ineffective sleep are associated with SHs. Their use is not recommended.” The reminder then notified the physician of the patient’s age with the statement: “Your patient is [age inserted] years old.” The same screen also highlighted a nonpharmacologic treatment (i.e., a warm bev- erage) for insomnia. If diazepam or diphenhydramine were being ordered, and the nonpharmacologic treatment was not selected, then lorazepam or trazodone was suggested as more appropriate alternatives (with relatively fewer adverse effects). RESULTS Thirty-six clinicians who had prescribed an SH were approached, and all agreed to be interviewed. Twenty-nine (81%) were interns in their first postgraduate year, and 23 (64%) were providing cross-coverage for a patient (Table 1). The most common indication for prescribing an SH was a patient requesting a sleeping medication; most physicians who or- dered an SH did not know whether the patient had been taking the same drug at home. All participants acknowledged having read the reminder screen. Clinicians rated their overall satisfaction with the reminder with a mean score of 8.5 (±0.9 SD), with 10 indicating high satisfaction. Scores did not differ based on postgraduate year of training or cross-coverage status of the physician (data not shown). In 95% of prescribing episodes during the year after activation of the computer reminder, “safer” ordering was observed, defined as a nonpharmacologic intervention or an order for lorazepam or trazodone (rather than for diphenhy- dramine or diazepam). In 5% of cases, users continued to order either diphenhydramine or diazepam for sleep in their older patients. (A 1-year follow-up period was chosen to assess a meaningful response because it was hypothesized that clini- cians may be more likely to respond to a reminder in the first weeks or months of its use and “wane” thereafter). Overall, during the pre-intervention period, prescriptions for one or more SHs were ordered for 2,208 of 12,356 (18%) patients aged 65 years and older. In contrast, the combined prescription rate for the four drugs of interest during the post-intervention year was 1,832 of 12,153 (15%) patients, an 18% risk reduction (p< 0.001) in SH orders. METHODS Alternatively, a prescriber could “back out” of the process and order no medication or continue to order the original medica- tion selected, e.g., diphenhydramine. The institutional review board of the Yale School of Medicine approved the protocol. Themes were identified by open coding of the text relating to benefits and barriers of the reminder, which were then compared within and across interviews according to the constant comparative method of analysis.14 Codes were combined and synthesized into broader, recurrent themes based on consensus of two investigators (JVA, SKI) with further input from a third investigator (JC) producing the final version. Awareness of Patient Safety Risks Demands of reading the reminder Clinicians’ concerns about the reminder generally focused on the time it took to read and the presence of an additional screen to review while prescribing. For example, the reminder was “just another screen to scroll through,” “takes too long to go through at first,” “clicking through screens was burdensome,” and “[it] slows me down.” Risk of delirium Clinicians commonly mentioned the value of recognizing risks of acute mental status changes and cognitive decline in older patients given SHs. Quotes included “delirious patients are difficult to manage,” “delirium is so harmful that it’s good to know,” and “no one likes to have to deal with...or get called for delirious patients.” Risk of falls House staff recognized that falls are an important problem in hospitalized patients and reported that the computer reminder was helpful because “falls increase [length of] hospital stays,” and “I don’t want my patient to fall because he’s [already] unsteady now.” Role of clinical experience Some participants commented on the possible intrusiveness of the reminder in a general context and the erosion of clinicians’ prescribing autonomy. Comments included “if the patient needs sleep I can decide whether to order a drug or not,” and the reminder screen “makes [him/her] feel watched.” One participant felt that “clinicians should know side effects—don’t give a reminder for everything.” General patient safety risks The reminder was reportedly useful in prompting clinicians to rethink indications for ordering a medication for sleep difficulty because of the potential patient safety risk. One participant noted that when called with an SH request, “Usually I just order something, now I thought about it.” Other quotes include “first do no harm, that is what we are taught,” and “if giving drugs makes them [older patients] stay longer due to complications, that’s bad for the patient and everyone.” Information content of the reminder In a more specific context, some clinicians disagreed with the actual content of the reminder itself. Benefits Important Themes Relating to Benefits of and Barriers to use of a Computer-Based Reminder Themes Benefits Heightened awareness of patient safety risks Delirium Falls General patient safety risks Usefulness of computers, information technology Value of educational content of the reminder Geriatric pharmacology review Nonpharmacologic treatment options Barriers Demands of reading the reminder The role of clinician experience Information content of the reminder 1 patients was an important component of the computerized reminder. Quotes include “it reminded me of sensitivity of old patients to drugs,” “it made me lower the dosage of what I usually prescribe,” and “it made me think about the patient’s age and drug use.” One participant noted, “I called the nurse back to ask whether the patient took a sleeper [sleeping medication] at home,” after learning about the appropriateness of SHs in older patients, and another reported, “I forget about how geriatrics patients react to medications; it’s good to remind me.” Nonpharmacologic treatment options Because the reminder included a prominent opportunity to order a nonpharmacologic treatment (warm beverage), clinicians had a feasible and easy method to bypass the use of traditional SH drugs. For example, “the tea idea is cool (although I didn’t do it),” or “I never used tea or warm milk before for sleep, although people do at home.” (subthemes of delirium, falls, and general patient safety); usefulness of computer technology; and clinical value of receiving reminder information (subthemes of benefits of geriatric pharmacology review and benefits of a nonpharma- cologic sleep treatment). The range of responses was similar based on level of training and whether the patient was being cross-covered (data not shown). Awareness of Patient Safety Risks These clinicians felt the reminder was “too simplistic,” or “the patient is already on [diphenhydramine] at home and has no side effects.” Because lorazepam was a recommended alternative medication to diphenhydramine and diazepam, some participants felt the reminder was incorrect: “I thought lorazepam caused delirium also.” One SH prescriber noted that “withdrawing sleep medications might harm the patient—screen didn’t say that.” Usefulness of Computer Technology House staff discussed the benefits of having an available aide in the form of the computer. Quotations include “computers should be used to help doctors,” “I like using computers to help doctors be safer,” “I’m surprised that there aren’t more of these things [computerized reminders],” and “it makes me rethink in the middle of the night when I’m called [...].” Benefits Three major themes for the benefits of the reminder system were identified (Table 2): awareness of patient safety risks Three major themes for the benefits of the reminder system were identified (Table 2): awareness of patient safety risks Current study After the computer-based reminder had been in place for 12 months, we used semi-structured interviews to collect data from house staff physicians on the medical or surgical service who, after having viewed a computer-based reminder, prescribed an SH medication to a patient aged 65 years and older. The interviews were conducted in person or over the telephone, on the morning after an SH was ordered (and after the reminder screen was recently viewed). Interviews were conducted by the principal investigator (JVA) and lasted 10 minutes or less. The first several interviews were audiotaped, with the interviewer also recording data on pencil and paper; all subsequent interviews were recorded on paper after the comparability of the data collection methods was verified. Comments were transcribed during the interview and verified for accuracy after the interview was completed. Table 1. Clinician and Prescribing Characteristics for the 36 Participating Physicians Characteristic N % House staff level Postgraduate year 1 29 80.6 Postgraduate year 2 or higher 7 19.4 Providing cross-coverage for patient 23 63.9 Reason for sedative-hypnotic prescriptiona Patient requested 22 61.1 Nurse requested 11 30.6 No reason 2 5.6 Other reason 12 33.3 Patient prescribed the same sedative-hypnotic used at home Don’t know 29 80.6 Yes 3 8.3 No 4 11.1 Usefulness of the intervention (0, low; 10, high) Mean±SD 8.5±0.9 aSome participants cited more than one reason. Table 1. Clinician and Prescribing Characteristics for the 36 Participating Physicians Data collection included demographic and prescribing data (e.g., level of training, whether the clinician was providing cross-coverage), as well as clinicians’ perspectives regarding barriers and facilitators to improving SH prescribing. The interview guide was focused on the reasons for prescription of an SH, clinician knowledge of SH use before admission, the Agostini et al.: Benefits and Barriers of a Computerized Reminder 34 JGIM Table 2. Barriers Three main barriers to appropriate prescribing (Table 2) were demands of reading the reminder; the role of clinical experi- ence in deciding whether to prescribe an SH; and the information content of the reminder. DISCUSSION Although a computerized reminder system elicited both posi- tive and negative feedback from participating house staff, they rated it highly overall, with a mean satisfaction rating of 8.5 on a 10-point scale. The use of computer-based decision support systems is likely to expand in the future and represents an Value of Educational Content of the Reminder One study using decision support tools in geriatrics found that clinical prescribing improved when recommended dosages and drug selections for psychoactive medications were seamlessly integrated into the ordering process.16 Few other studies, however, have reported on the use of computerized reminders in geriatrics. A major strength of this study is the open-ended structure of the interview guide, which permitted participants to report both likes and dislikes of the reminder. Because participants had recently viewed the screen overnight while ordering an SH drug, we minimized recall bias. In addition, we interviewed house staff who write the majority of orders in the hospital and thus were intimately familiar with the existing strengths and weaknesses of the computerized order entry system. Responses of providers who prescribed an SH could not be compared to providers who were dissuaded by the reminder from ordering an SH because the latter group did not “leave information” in the computer records in the absence of a prescription. In summary, understanding provider-perceived benefits and barriers to the use of information technology in the form of a computer-based reminder may facilitate better methods to ensure that vulnerable older patients are exposed to the safest and most rational use of medications. Acknowledgments: This research was supported by a Merck/ American Federation for Aging Research Award in Geriatric Clinical Pharmacology and a VA Health Services Research Career Develop- ment Award (JVA). This work is supported in part by Grant K24AG00949 (SKI) and the Claude D. Pepper Older Americans Independence Center at Yale University School of Medicine (P30AG21342) from the National Institute on Aging. Dr. Inouye is supported in part by grant R21AG025193 from the National Institute on Aging and by the Milton and Shirley F. Levy Family Chair. The views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the Department of Veterans Affairs. A limitation of the study was that it was a convenience sample drawn from the staff of one teaching hospital with a large geriatrics program and with other ongoing geriatrics interventions, such as the Yale Hospital Elder Life Program.17 Most participants were house staff, which may limit general- izability of the results to all physicians, but we assessed the physicians who actually order the SH drugs in our hospital, in real time. Value of Educational Content of the Reminder Value of Educational Content of the Reminder Geriatrics pharmacology review Most participants reported that reviewing appropriate prescribing criteria for geriatric Geriatrics pharmacology review Most participants reported that reviewing appropriate prescribing criteria for geriatric Agostini et al.: Benefits and Barriers of a Computerized Reminder 35 JGIM important tool in improving patient safety during the drug ordering process. The high degree of satisfaction with this system by the users holds substantial promise for designing future systems. The findings of this study also suggest several opportunities for improving the SH reminder in this study and perhaps other similar reminders. Linkages to medical literature and data supporting the recommendations described in the reminder may facilitate greater trust in the information content of the reminder, at least for those who seek more empirical evidence. Greater customization of the recommendation based on patients’ real-time comorbidities and concomitantly prescribed drugs, or incorporation of a patient’s cognitive status (e.g., presence of delirium or dementia symptoms), might be helpful to enhance clinicians’ decisions to decide whether to prescribe an SH, yet still preserve their clinical autonomy. Finally, making the reminder screen(s) as short and focused as possible may address concerns about the time required to read the reminder itself. The computer ordering system in use at Yale-New Haven Hospital already includes required elec- tronic ordering for all inpatient orders, and consequently, reminders should fit reasonably into the workflow of patient care. Other health care organizations, such as the Veterans Affairs Healthcare System, use reminders even more widely in inpatient and outpatient settings.18 Accordingly, building on the strengths and weaknesses of computer reminders in a variety of health care environments may enhance their overall usefulness as part of long-term quality improvement strategies. Our findings also confirm the results of several studies describing the effects of computer-based behavior changes. Although the educational content of the reminder tended to simplify the message, previous investigators have noted that directed messages received in real time (or at the point of order entry) work best.15 Some clinicians complained that the reminder was an additional screen to read at the time of ordering, suggesting that methods to incorporate the reminder into the workflow of the order entry process would be important to explore. Conflict of Interest: None disclosed. Conflict of Interest: None disclosed. Corresponding Author: Joseph V. Agostini, MD; Clinical Epidemi- ology Research Center 151B, VA Connecticut Healthcare System, 950 Campbell Avenue, West Haven, CT 06516, USA (e-mail: joseph. agostini@yale.edu). Value of Educational Content of the Reminder Different perceptions of the utility of computer-based reminders may exist among various providers (e.g., community- based physicians) and in various settings (e.g., in nonteaching hospitals), such as lower levels of satisfaction or greater perceived barriers if attending physicians are the main prescribers. We also did not explore benefits and barriers from the perspectives of nurses, who often play a role in the decision to prescribe an SH. Whether the perceived benefits and barriers to use of this computer-based reminder are generalizable to other medication classes such as antibiotics or anticoagulants is unknown. In addition, the qualitative focus of this work does not yield estimates of the prevalence of specific responses regarding the benefits and barriers of the reminder, but rather reports on the range of answers that were elicited (until thematic saturation was reached). REFERENCES 1. Leipzig RM, Cumming RG, Tinetti ME. Drugs and falls in older people: a systematic review and meta-analysis: I. Psychotropic drugs. J Am Geriatr Soc. 1999;47:30–9. Future iterations of this computer-based reminder should build on the findings of the current study and incorporate the point-of-care advantages of information technology used at the time of medication prescription. These efforts may complement overall quality improvement efforts, by aiming to improve outcomes of care, decrease medication errors, and minimize unnecessary prescribing; and doing so at minimal cost when computer systems are already in place. 2. Gray SL, Lai KV, Larson EB. Drug-induced cognition disorders in the elderly: incidence, prevention and management. Drug Saf. 1999;21: 101–22. 3. Gurwitz JH, Field TS, Avorn J, et al. Incidence and preventability of adverse drug events in nursing homes. Am J Med. 2000;109:87–94. 4. O’Reilly R, Rusnak C. The use of sedative-hypnotic drugs in a university teaching hospital. CMAJ. 1990;142:585–9. Agostini et al.: Benefits and Barriers of a Computerized Reminder 36 JGIM 5. Meissner HH, Riemer A, Santiago SM, et al. Failure of physician documentation of sleep complaints in hospitalized patients. West J Med. 1998;169:146–9. 12. Chertow GM, Lee J, Kuperman GJ, et al. Guided medication dosing for inpatients with renal insufficiency. JAMA. 2001;286:2839–44. 13. Agostini JV, Zhang Y, Inouye SK. Use of a computer-based reminder to improve sedative-hypnotic prescribing in older hospitalized patients. J Am Geriatr Soc. 2007;55:43–8. 6. Lenhart SE, Buysse DJ. Treatment of insomnia in hospitalized patients. 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Arch Intern Med. 2005;165:802–7. 9. Institute of Medicine. REFERENCES Crossing the quality chasm: a new health system for the 21st century. Washington, DC: National Academy Press; 2001. 17. Inouye SK, Bogardus ST Jr, Baker DI, Leo-Summers L, Cooney LM. The Hospital Elder Life Program: a model of care to prevent cognitive and functional decline in older hospitalized patients. Hospital Elder Life Program. J Am Geriatr Soc. 2000;48:1697–706. 10. Evans RS, Pestotnik SL, Classen DC, et al. A computer-assisted management program for antibiotics and other antiinfective agents. N Engl J Med. 1998;338:232–8. 11. Bates DW, Teich JM, Merchia PR, Schmiz JL, Kuperman GJ, Spurr CD. Effects of computerized physician order entry on prescribing practices. Arch Intern Med. 2000;160:2741–7. 18. Demakis JG, Beauchamp C, Cull WLR, et al. Improving residents’ com- pliance with standards of ambulatory care: results from the VA Cooper- ative Study on Computerized Reminders. JAMA 2000;284:1411–6.
https://openalex.org/W4376056058	https://cerdika.publikasiindonesia.id/index.php/cerdika/article/download/114/204	Indonesian	null	Gambaran Fungsi Kognitif Yang di Ukur dengan MMSE Pada Pasien Riwayat Stroke di Poli Saraf RSUD Dr. H. Abdul Moeloek Bandar Lampung Tahun 2021	Cerdika	2,021	cc-by-sa	4,111	Cerdika: Jurnal Ilmiah Indonesia, Juni 2021, 1 (6), 612-621 p-ISSN: 2774-6291 e-ISSN: 2774-6534 Available online at http://cerdika.publikasiindonesia.id/index.php/cerdika/index GAMBARAN FUNGSI KOGNITIF YANG DI UKUR DENGAN MMSE PADA PASIEN RIWAYAT STROKE DI POLI SARAF RSUD DR. H. ABDUL MOELOEK BANDAR LAMPUNG TAHUN 2021 Akhmad Kheru11, Fitriyani2, Dharmawita3, Putri Fadillah4 1Departemen Fakultas Kedokteran Universitas Malahayati1 Departemen Neurologi Fakultas Kedokteran Universitas Malahayati2, 3 Program Studi Kedokteran Fakultas Kedokteran Universitas Malahayati4 akhmadkheru@gmail.com1 ; dr.fitriyani@yahoo.co.id2; dharmawita@yahoo.com3 ; putrifadillaah@gmail.com4 Abstract description of cognitive function measured with MMSE in patient history of stroke at the nerve poli dr. h. abdul moeloek bandar lampung year 2021. Stroke is a disease that can cause disability and mortality (death) in the third place in the world (WHO, 2019). Morbidity of post-stroke patients can be in the form of physical, psychological and cognitive problems. This increases the risk of decreased cognitive function. One of the cognitive function tests that is often used is the mini mental state examination (MMSE). This study aims to determine cognitive dysfunction as measured by MMSE in patients with a history of stroke at RSUD DR. H. Abdul Moeloek Bandar Lampung in 2021. This type of research is a quantitative study with a descriptive research design and a cross sectional research design. The number of samples was 30 subjects who met the research criteria with consecutive sampling technique. The research data were obtained from medical records by filling in the MMSE questionnaire and processed with the help of SPSS. The results showed that 9 respondents (30%) had severe cognitive impairment in patients aged 56- 65 years (Late Elderly), as many as 8 respondents (26.7%). There were 10 respondents (33.3%) who had severe cognitive weight in patients with primary school education, 8 respondents (26.7%) had damage to the bilateral hemispheres (26.7%), and 11 respondents (36.7%) had infarct lesions.There is severe cognitive impairment in patients with a history of stroke with a component of cognitive function that has a high mean score, namely the orientation component 7 (0-10). Received: Revised : Accepted: 10-06-2021 23-06-2021 26-06-2021 ( ) Keywords: Stroke Histor; Cognitive Function; MMSE. Keywords: Stroke Histor; Cognitive Function; MMSE. Abstrak gambaran fungsi kognitif yang di ukur dengan MMSE pada pasien riwayat stroke di poli saraf RSUD dr.h.abdul moeloek bandar lampung tahun 2021. stroke merupakan salah satu penyakit yang dapat mengakibatkan disabilitas dan mortalitas (kematian) terbanyak dengan urutan ketiga di dunia (who, 612 10.36418/cerdika.v1i6.114 Akhmad Kheru1, Fitriyani, Dharmawita, Putri Fadillah/Cerdika: Jurnal Ilmiah Indonesia, 1(6), 612-621 Akhmad Kheru1, Fitriyani, Dharmawita, Putri Fadillah/Cerdika: Jurnal Ilmiah Indonesia, 1(6), 612-621 Akhmad Kheru1, Fitriyani, Dharmawita, Putri Fadillah/Cerdika: Jurnal Ilmiah Indonesia, 1(6), 612-621 2019). morbiditas pasien pasca stroke dapat berupa masalah fisik, psikis dan kognitif. hal ini meningkatkan resiko berupa penurunan fungsi kognitif. salah satu pemeriksaan fungsi kognitif yang sering digunakan adalah mini mental state examination (MMSE). penelitian ini ditujukan untuk mengetahui gangguan fungsi kognitif yang di ukur dengan MMSE pada pasien riwayat stroke di rsud dr.h.abdul moeloek bandar lampung tahun 2021. jenis penelitian yang digunakan adalah penelitian kuantitatif dengan desain penelitian deskriptif dan rancangan penelitian cross sectional. jumlah sampel 30 subyek yang memenuhi kriteria penelitian dengan teknik consecutive sampling. data penelitian diperoleh dari catatan rekam medis dengan pengisian kuesioner MMSE dan diolah dengan bantuan SPSS. dari hasil penelitian menunjukkan bahwa pada pasien yang berjenis kelamin perempuan yaitu sebanyak 9 responden (30%) pasien memiliki gangguan kognitif berat pada rentang usia 56-65 tahun (lansia akhir) yaitu sebanyak 8 responden (26,7%), gangguan kognitif berat pada pasien dengan jenjang pendidikan sd yaitu sebanyak 10 responden (33,3%) terjadi kerusakan pada hemisfer bilateral sebanyak 8 responden (26,7%), lesi infark sebanyak 11 responden (36,7%). terdapat gangguan kognitif berat pada pasien riwayat stroke dengan komponen fungsi kognitif yang memiliki rerata nilai yang tinggi, yaitu komponen orientasi 7 (0-10). Kata kunci: Riwayat Stroke; Fungsi Kognitif; MMSE. Kata kunci: Riwayat Stroke; Fungsi Kognitif; MMSE. Kata kunci: Riwayat Stroke; Fungsi Kognitif; MMSE. Coresponden Author : Putri Fadillah Email : putrifadillaah@gmail.com g Gambaran Fungsi Kognitif Yang Di Ukur Dengan Mmse Pada Pasien Riwayat Stroke Di Poli Saraf Rsud Dr. H. Abdul Moeloek Bandar Lampung Tahun 2021 613 PENDAHULUAN Stroke yakni penyakit yang menyerang langsung pada otak, stroke ditandai dengan adanya tanda klinis fokal maupun global yang terjadi secara mendadak, mengganggu fungsi serebral dan berlangsung selama >24 jam atau menimbulkan kematian, tanpa penyebab vascular (Sacco et al., 2013). Stroke terdiri dari dua jenis yaitu iskemik dan hemoragik (Bhatti et al., 2013). Menurut WHO stroke ini merupakan salah satu penyakit yang dapat mengakibatkan disabilitas dan mortalitas (kematian) terbanyak dengan urutan ketiga di dunia setelah penyakit jantung dan kanker yang menduduki urutan kedua di dunia. Mayoritas seseorang dapat mengalami stroke setiap 40 detik dan mengalami kematian setiap 4 menit (Asfar & Rusniyanti, 2018). Data WHO menunjukkan bahwa setiap tahunnya ada 13,7 juta kasus baru stroke dan sekitar 5,5 juta kematian terjadi akibat penyakit stroke. Sekitar 70% penyakit stroke dan 87% kematian dan disabilitas akibat stroke terjadi pada negara berpendapatan rendah dan menengah. Lebih dari empat dekade terakhir, kejadian stroke pada negara tersebut meningkat lebih dari dua kali lipat (Johnson et al., 2016) Berdasarkan data nasional Indonesia menunjukkan peningkatan insiden stroke yaitu pada tahun 2007 dari 8,3/1000 penduduk menjadi 12,1/1000 penduduk pada tahun 2013 (Perdossi, 2013). Menurut riset kesehatan dasar Kabupaten/Kota di Provinsi Lampung, prevalensi kejadian stroke berkiasar antara 2,2%-10,5%. Kotamadya Bandar Lampung mempunyai prevalensi lebih tinggi dibandingkan dengan Kotamadya/Kabupaten yang ada di Provinsi Lampung, baik berdasarkan diagnosis 613 Akhmad Kheru1, Fitriyani, Dharmawita, Putri Fadillah/Cerdika: Jurnal Ilmiah Indonesia, 1(6), 612-621 Akhmad Kheru1, Fitriyani, Dharmawita, Putri Fadillah/Cerdika: Jurnal Ilmiah Indonesia, 1(6), 612-621 maupun berdasarkan gejalanya (Riskesdas, 2019). Morbiditas pasien pasca stroke dapat berupa masalah fisik, psikis dan kognitif (Gorelick & Aiyagari, 2011). Salah satu pemeriksaan fungsi kognitif bagi pasien pasca stroke yang sering digunakan yakni dengan kuesioner MMSE (mini mental state examinanation). Pengukuran dengan MMSE ini merupakan suatu alat yang digunakan secara sistematis untuk penilaian status mental pada seorang pasien. maupun berdasarkan gejalanya (Riskesdas, 2019). Morbiditas pasien pasca stroke dapat berupa masalah fisik, psikis dan kognitif (Gorelick & Aiyagari, 2011). Salah satu pemeriksaan fungsi kognitif bagi pasien pasca stroke yang sering digunakan yakni dengan kuesioner MMSE (mini mental state examinanation). Pengukuran dengan MMSE ini merupakan suatu alat yang digunakan secara sistematis untuk penilaian status mental pada seorang pasien. Gambaran Fungsi Kognitif Yang Di Ukur Dengan Mmse Pada Pasien Riwayat Stroke Di Poli Saraf Rsud Dr. H. Abdul Moeloek Bandar Lampung Tahun 2021 614 METODE PENELITIAN Jenis penelitian yang digunakan adalah penelitian deskriptif kuantitatif melalui desain cross sectional. Penelitian ini dilakukan di Poli Saraf RSUD DR.H.Abdul Moeloek Bandar Lampung, sedangkan waktu penelitiannya adalah Maret 2021.Sampel dalam penelitian ini adalah pasien dengan riwayat stroke di Poli Saraf RSUD DR.H.Abdul Moeloek Bandar Lampung yang dihitung dengan rumus slovin yaitu sebanyak 30 sampel. Kriteria inklusi pada penelitian ini adalah pasien stroke iskemik maupun hemoragik yang rawat jalan di poli saraf dalam keadaan sadar, pasien dewasa dengan usia >18 tahun dan kondisi pasien kesadaran penuh composmentis dan bersedia mengikuti penelitian. Sedangkan kriteria eksklusinya yaitu pasien dengan data pendukung tidak lengkap dan asien dengan kelemahan gerak akibat infeksi, tumor, dan trauma kepala. A. Hasil Penelitian Tabel 1. Distribusi Frekuensi Gambaran Fungsi Kognitif Berdasarkan Jenis Kelamin Pasien Riwayat Stroke di RSUD DR.H.Abdul Moeloek Bandar Lampung tahun 2021 Fungsi Kognitif Normal Ringan Berat Total Jenis Kelamin Laki-laki n 2 7 7 16 % 6,7 % 23,3 % 23,3 % 53,3 % Perempuan n 3 2 9 14 % 10 % 6,7 % 30 % 46,7 % Total n 5 9 16 30 % 16,7 % 30 % 53,3 % 100 % Berdasarkan tabel di atas diketahui bahwa dari 30 responden riwayat stroke yang berjenis kelamin laki-laki didapatkan sebanyak 2 responden (6,7%) dengan fungsi kognitif normal, sebanyak 7 responden (23,3) dengan gangguan kognitif ringan dan sebanyak 7 responden (23,3) dengan gangguan kognitif berat. Sedangkan responden yang berjenis kelamin perempuan didapatkan sebanyak 3 responden (10%) dengan fungsi kognitif normal, sebanyak 2 responden (6,7%) dengan gangguan kognitif ringan dan sebanyak 9 responden (30%) dengan gangguan kognitif berat. Tabel 1. Distribusi Frekuensi Gambaran Fungsi Kognitif Berdasarkan Jenis Kelamin Pasien Riwayat Stroke di RSUD DR.H.Abdul Moeloek Bandar Lampung tahun 2021 Berdasarkan tabel di atas diketahui bahwa dari 30 responden riwayat stroke yang berjenis kelamin laki-laki didapatkan sebanyak 2 responden (6,7%) dengan fungsi kognitif normal, sebanyak 7 responden (23,3) dengan gangguan kognitif ringan dan sebanyak 7 responden (23,3) dengan gangguan kognitif berat. Sedangkan responden yang berjenis kelamin perempuan didapatkan sebanyak 3 responden (10%) dengan fungsi kognitif normal, sebanyak 2 responden (6,7%) dengan gangguan kognitif ringan dan sebanyak 9 responden (30%) dengan gangguan kognitif berat. 614 Akhmad Kheru1, Fitriyani, Dharmawita, Putri Fadillah/Cerdika: Jurnal Ilmiah Indonesia, 1(6), 612-621 Tabel 2. Distribusi Frekuensi Gambaran Fungsi Kognitif Berdasarkan Usia Pasien Riwayat Stroke di RSUD DR.H.Abdul Moeloek Bandar Lampung tahun 2021 Fungsi Kognitif Normal Ringan Berat Total Usia 26-35 Tahun N 1 0 0 1 % 3,3 % 0 % 0 % 3,3 % 36-45 Tahun N 3 1 2 6 % 10 % 3,3 % 6,7 % 20 % 46-55 Tahun N 1 1 2 4 % 3,3 % 3,3 % 6,7 % 13,3 % 56-65 Tahun N 0 4 8 12 % 0 % 13,3 % 26,7 % 40 % 65 Tahun- sampai atas N 0 3 4 7 % 0 % 10 % 13,3 % 23,3 % Total N 5 9 16 % 16,7 % 30 % 53,3 % 100 % Tabel 2. g g f g g y Saraf Rsud Dr. H. Abdul Moeloek Bandar Lampung Tahun 2021 615 Gambaran Fungsi Kognitif Yang Di Ukur Dengan Mmse Pada Pasien Riwayat Stroke Di Poli S f s d d d p g 615 g g f g g Saraf Rsud Dr. H. Abdul Moeloek Bandar Lampung Tahun 2021 Akhmad Kheru1, Fitriyani, Dharmawita, Putri Fadillah/Cerdika: Jurnal Ilmiah Indonesia, 1(6), 612-621 Akhmad Kheru1, Fitriyani, Dharmawita, Putri Fadillah/Cerdika: Jurnal Ilmiah Indonesia, 1(6), 612-621 Berdasarkan tabel di atas diketahui bahwa dari 30 responden riwayat stroke dengan pendidikan responden yang tidak sekolah didapatkan sebanyak 5 responden (16,7%) dengan gangguan kognitif berat, SD didapatkan sebanyak 1 responden (3,3%) dengan fungsi kognitif normal, sebanyak 3 responden (10%) dengan gangguan kognitif ringan dan sebanyak 6 responden dengan gangguan kognitif berat. Responden dengan pendidikan SMP didapatkan sebanyak 3 responden (10%) dengan gangguan kognitif ringan dan sebanyak 3 responden (10%) dengan gangguan kognitif berat. Responden dengan pendidikan SMA didapatkan sebanyak 4 responden (13,3%) dengan fungsi kognitif normal, sebanyak 2 responden (6,7%) dengan gangguan kognitif ringan, sebanyak 2 responden (6,7%) dengan gangguan kognitif berat. Responden dengan pendidikan perguruan tinggi didapatkan sebanyak 1 responden (3,3%) dengan gangguan kognitif ringan. Sedangkan. Sebagian besar responden memiliki gangguan kognitif berat pada responden dengan jenjang pendidikan SD. Tabel 4. Distribusi Frekuensi Karakteristik Responden Berdasarkan Hemisfer dengan Fungsi Kognitif pada Hasil CT-Scan Pasien Riwayat Stroke di RSUD DR.H.Abdul Moeloek Bandar Lampung tahun 2021 Fungsi Kognitif Normal Ringan Berat Total Hemisfer Kanan n 2 4 6 12 % 6,7 % 13,3 % 20 % 40 % Kiri n 3 4 2 9 % 10 % 13,3 % 6,7 % 30 % Bilateral n 0 1 8 9 % 0 % 3,3 % 26,7 % 30 % Total n 5 9 16 30 % 16,7 % 30 % 53,3 % 100 % Tabel 4. Distribusi Frekuensi Karakteristik Responden Berdasarkan Hemisfer dengan Fungsi Kognitif pada Hasil CT-Scan Pasien Riwayat Stroke di RSUD DR.H.Abdul Moeloek Bandar Lampung tahun 2021 Berdasarkan tabel di atas diketahui bahwa dari 30 responden riwayat stroke dengan hemisfer kanan didapatkan sebanyak 2 responden (6,7%) dengan fungsi kognitif normal, sebanyak 4 responden (13,3%) dengan penurunan kognitif ringan dan sebanyak 6 responden (20%) dengan gangguan kognitif berat. Responden riwayat stroke dengan hemisfer kiri didapatkan sebanyak 3 responden (10%) dengan fungsi kognitif normal, sebanyak 4 responden (4,4%) dengan gangguan kognitif ringan dan sebanyak 2 responden (6,7%) dengan gangguan kognitif berat. Sedangkan, responden riwayat stroke dengan hemisfer bilateral didapatkan sebanyak 1 responden (3,3%) dengan gangguan kognitif ringan dan sebanyak 8 responden (26,7%) dengan gangguan berat. Sebagian besar responden terjadi kerusakan pada hemisfer bilateral. Gambaran Fungsi Kognitif Yang Di Ukur Dengan Mmse Pada Pasien Riwayat Stroke Di Poli Saraf Rsud Dr. H. Abdul Moeloek Bandar Lampung Tahun 2021 616 A. Hasil Penelitian Distribusi Frekuensi Gambaran Fungsi Kognitif Berdasarkan Usia Pasien Riwayat Stroke di RSUD DR.H.Abdul Moeloek Bandar Lampung tahun 2021 Fungsi Kognitif Berdasarkan tabel di atas diketahui bahwa dari 30 responden riwayat stroke dengan rentang 26-35 tahun didapatkan sebanyak 1 responden (3,3%) dengan fungsi kognitif normal. Pada rentang usia 36-45 tahun didapatkan sebanyak 3 responden (10%) dengan kognitif normal, sebanyak 1 orang (3,3%) dengan gangguan kognitif ringan dan sebanyak 2 responden (3,3%) dengan gangguan kognitif berat. Pada rentang usia 46-55 tahun didapatkan sebanyak 1 responden (3,3%) dengan fungsi kognitif normal, sebanyak 1 responden (3,3%) dengan gangguan kognitif ringan dan sebanyak 2 responden (6,7%) dengan gangguan kognitif berat. Pada rentang usia 56-65 tahun didapatkan sebanyak 4responden (13,3%) dengan gangguan kognitif ringan dan sebanyak 8 responden (26,7%) dengan gangguan kognitif berat serta pada usia 65 tahun-sampai atas didapatkan sebanyak 3 responden (10%) dengan gangguan kognitif ringan dan sebanyak 4 responden (13,3%) dengan gangguan kognitif berat Sebagian besar responden memiliki gangguan kognitif berat pada rentang usia 56-65 tahun (Lansia Akhir). Gambaran Fungsi Kognitif Yang Di Ukur Dengan Mmse Pada Pasien Riwayat Stroke Di Poli Saraf Rsud Dr. H. Abdul Moeloek Bandar Lampung Tahun 2021 6 5 Tabel 3. Distribusi Frekuensi Gambaran Fungsi Kognitif Berdasarkan Pendidikan Pasien Riwayat Stroke di RSUD DR.H.Abdul Moeloek Bandar Lampung tahun 2021 Fungsi Kognitif Normal Ringan Berat Total Pendidikan Tidak Sekolah n 0 0 5 5 % 0 % 0 % 16,7 % 16,7 % SD n 1 3 6 10 % 3,3 % 10 % 20 % 33,3 % SMP n 0 3 3 6 % 0 % 10 % 10 % 20 % SMA n 4 2 2 8 % 13,3 % 6,7 % 6,7 % 26,7 % Perguruan Tinggi n 0 1 0 1 % 0 % 3,3 % 0 % 3,3 % Total n 5 9 16 30 % 16,7 % 30 % 53,3 % 100 % Tabel 3. Distribusi Frekuensi Gambaran Fungsi Kognitif Berdasarkan Pendidikan Pasien Riwayat Stroke di RSUD DR.H.Abdul Moeloek Bandar Lampung tahun 2021 615 Akhmad Kheru1, Fitriyani, Dharmawita, Putri Fadillah/Cerdika: Jurnal Ilmiah Indonesia, 1(6), 612-621 Fungsi Kognitif Normal Ringan Berat Total Lesi Infark n 5 7 5 17 % 16,7 % 23,3 % 16,7 % 56,7 % Hemoragik n 0 2 11 13 % 0 % 6,7 % 36,7 % 43,3 % Total n 5 9 16 30 % 16,7 % 30 % 53,3 % 100 % Berdasarkan tabel diatas bahwa dari 30 responden riwayat stroke dengan lesi infark didapatkan sebanyak 5 responden (16,7%) dengan fungsi kognitif normal, sebanyak 7 responden (23,3%) dengan gangguan kognitif ringan, dan sebanyak 5 616 Akhmad Kheru1, Fitriyani, Dharmawita, Putri Fadillah/Cerdika: Jurnal Ilmiah Indonesia, 1(6), 612-621 Akhmad Kheru1, Fitriyani, Dharmawita, Putri Fadillah/Cerdika: Jurnal Ilmiah Indonesia, 1(6), 612-621 Akhmad Kheru1, Fitriyani, Dharmawita, Putri Fadillah/Cerdika: Jurnal Ilmiah Indonesia, 1(6), 612-621 responden (16,7%) dengan gangguan kognitif berat. Pasien lesi hemoragik didapatkan sebanyak 2 responden (6,7%) dengan gangguan kognitif ringan dan sebanyak 11 responden (36,7%) dengan gangguan kognitif berat. Sebagian besar responden mengalami lesi hemoragik. Tabel 6. Distribusi Frekuensi Karakteristik Responden Berdasarkan Fungsi Tabel 6. Distribusi Frekuensi Karakteristik Responden Berdasarkan Fungsi Kognitif pasien riwayat stroke di RSUD DR.H.Abdul Moeloek Bandar Lampung tahun 2021 Fungsi Kognitif Jumlah Presentase Normal 5 16,7 % Gangguan Kognitif Ringan 9 30 % Gangguan Kognitif Berat 16 53,3 % Total 30 100 % Berdasarkan tabel di atas diketahui bahwa dari 30 responden riwayat stroke berdasarkan fungsi kognitif didapatkan sebanyak 5 responden (16,7%) dengan fungsi kognitif normal, sebanyak 9 responden (30%) dengan gangguan kognitif ringan dan sebanyak 16 responden (53,3%) dengan gangguan kognitif berat. Sebagian besar responden memiliki gangguan kognitif berat. Tabel 7. Rerata Komponen Fungsi Kognitif Komponen Fungsi Kognitif Hasil Pengukuran Mean (Min- Max) Orientasi 7 (0-10) Registrasi 2 (0-3) Atensi dan Kalkulasi 3 (0-7) Mengingat Kembali 0 (0-3) Bahasa 3 (0-7) Tabel 7. Rerata Komponen Fungsi Kognitif Tabel 7. Rerata Komponen Fungsi Kognitif Berdasarkan tabel di atas menunjukkan bahwa dari lima komponen fungi kognitif yang diperiksa terdapat sebaran data yang tidak normal. Terdapat komponen fungsi kognitif yang memiliki rerata nilai yang tinggi, yaitu komponen orientasi 7 (0-10). Gambaran Fungsi Kognitif Yang Di Ukur Dengan Mmse Pada Pasien Riwayat Stroke Di Poli Saraf Rsud Dr. H. Abdul Moeloek Bandar Lampung Tahun 2021 617 Akhmad Kheru1, Fitriyani, Dharmawita, Putri Fadillah/Cerdika: Jurnal Ilmiah Indonesia, 1(6), 612-621 Secara teoritis angka harapan hidup wanita lebih tinggi daripada laki-laki, sehingga keberadaan perempuan lebih banyak daripada laki-laki. Hasil penelitian ini mendukung hasil penelitian sebelumnya dari (Prasetyo, 2012), yang menemukan bahwa terdapat hubungan jenis kelamin dengan gangguan kognitif. Pembagian dua jenis kelamin yang ditentukan secara biologis dan anatomis yang melekat pada jenis kelamin tertentu. Perbedaan jenis kelamin juga menyebabkan proses stroke dan gangguan kognitif yang terjadi menjadi berbeda antara pria dan wanita. Wanita lebih banyak menderita stroke kardioemboli sedangkan pria lebih banyak menderita stroke lakunar, dimana hal tersebut menjelaskan bahwa wanita memiliki resiko gangguan kognitif lebih besar dari pria (Sun et al., 2014) Hasil penelitian ini sesuai dengan penelitian yang dilakukan oleh Tumiwa, dkk di Rumah Sakit Bethesda Yogyakarta diperoleh hasil nilai p = 0,007 (p < 0,05) yang menandakan bahwa terdapat hubungan usia dengan gangguan fungsi kognitif pada pasien pasca stroke iskemik. Hal ini sama dengan hasil penelitian (Aiyagari & Gorelick, 2016). bahwa terjadinya gangguan fungsi kognitif pasca stroke akan meningkat seiring dengan tingginya usia. Dalam penelitian tersebut disebutkan bahwa orang dengan usia lebih dari 50 tahun berisiko mengalami demensia vascular. Sesuai dengan pernyataan Potter dan Perry (2010) pada usia 45-59 tahun atau dewasa pertengahan yang mengalami perubahan kognitif dan fisiologis (mengalami perubahan pada pembuluh darah sehingga terhambatnya pasokan oksigen dalam darah ke jaringan). Usia dewasa menengah sudah mengalami perubahan struktur pada pembuluh darah dan penurunan keelastisan pada pembuluh darahnya sehingga mengakibatkan aliran darah ke organ lain mengalami keterhambatan. Apabila keterhambatan terjadi pada otak dapat menyebabkan stroke. Perubahan struktur pembuluh darah responden ini yang kemudian menjadi faktor penyebab banyaknya responden yang mengalami stroke pada usia 51-61 tahun. Hasil penelitian ini sejalan dengan penelitian yang dilakukan oleh (Gustami et al., n.d.) di empat Rumah Sakit Pendidikan Universitas Mazandaran, Iran mendapatkan level pendidikan subjek yang terbanyak adalah SD. Hasil ini menunjukkan bahwa usia dan tingkat pendidikan berpengaruh pada nilai tes MMSE. Penelitian yang dilakukan oleh Rasquin, dkk yang menyatakan tingkat pendidikan rendah merupakan salah satu predictor gangguan kognitif setelah mengalami stroke. Penelitian oleh Lindsay, dkk dikutip oleh Agustina, menyatakan bahwa tingkat pendidikan mempengaruhi fungsi kognitif, dimana faktor risiko terjadinya gangguan fungsi kognitif bersamaan dengan serangan stroke pada tingkat pendidikan < 6 tahun meningkat dibandingkan dengan tingkat pendidikan >10 tahun. Hal ini mungkin karena pengetahuan yang didapat cenderung kurang sehingga sulit menangkap informasi serta kurangnya kesadaran untuk melakukan kontrol teratur. Gambaran Fungsi Kognitif Yang Di Ukur Dengan Mmse Pada Pasien Riwayat Stroke Di Poli Saraf Rsud Dr. H. Abdul Moeloek Bandar Lampung Tahun 2021 618 B. Pembahasan Hasil penelitian ini sejalan dengan penelitian yang dilakukan (Wu et al., 2016) di Binhai New Area di Tianjin, China yang meneliti gangguan fungsi kognitif dengan studi case countrol yang menggunakan sampel sebanyak 112 subjek kelompok kasus yang menderita gangguan fungsi kognitif dibandingkan dengan 115 subjek sebagai kelompok kontrol didapatkan sebanyak 79 subjek perempuan di kelompok kasus dan 55 subjek perempuan di kelompok kontrol. Hal ini secara signifikan menggambarkan bahwa subjek perempuan pada kelompok kasus lebih tinggi (70,54%) dibandingkan subjek laki-laki. p p p p gg ( ) g j Sama halnya dengan penelitian yang dilakukan oleh Dinata, dkk di Bagian Rawat Inap Penyakit Dalam RSUD Kabupaten Solok Selatan tahun 2013 mendapatkan dari total pasien stroke sebanyak 96 orang didapatkan penderita perempuan (54,1%) lebih banyak daripada laki-laki (45,9%). Menurut penelitian (Asfar & Rusniyanti, 2018), menjelaskan bahwa wanita lebih banyak beresiko terkena stroke pada lanjut usia dan kemungkinan besar meninggal dunia karena penyakit stroke, sedangkan serangan stroke pada laki-laki 1,25% lebih tinggi dari pada wanita yang usianya muda. 617 KESIMPULAN Berdasarkan hasil penelitian yang dilakukan, maka dapat disimpulkan bahwa karakteristik pasien riwayat stroke di RSUD DR. H. Abdul Moeloek Bandar Lampung Tahun 2021, sebagian besar berjenis kelamin laki-laki yaitu sebanyak 16 responden (53,3%), berusia 56-65 tahun (Lansia Akhir) yaitu sebanyak 12 responden (40%), pendidikan SD yaitu sebanyak 10 responden (33,3%), dengan letak lesi di hemisfer kanan yaitu sebanyak 12 responden (40%) dan didapatkan hasil CT-scan dengan adanya infark 17 responden (56,7%). Sebagian besar responden memiliki gangguan kognitif berat pada pasien yang berjenis kelamin perempuan yaitu sebanyak 9 responden (30%), pasien memiliki gangguan kognitif berat pada rentang usia 56-65 tahun (Lansia Akhir) yaitu sebanyak 8 responden (26,7%), gangguan kognitif berat pada pasien dengan jenjang pendidikan SD yaitu sebanyak 10 responden (33,3%) terjadi kerusakan pada hemisfer bilateral sebanyak 8 responden (26,7%), lesi infark sebanyak 11 responden (36,7%) dan komponen fungsi kognitif yang memiliki rerata nilai yang tinggi, yaitu komponen orientasi 7 (0-10). Gambaran Fungsi Kognitif Yang Di Ukur Dengan Mmse Pada Pasien Riwayat Stroke Di Poli Saraf Rsud Dr. H. Abdul Moeloek Bandar Lampung Tahun 2021 619 Akhmad Kheru1, Fitriyani, Dharmawita, Putri Fadillah/Cerdika: Jurnal Ilmiah Indonesia, 1(6), 612-621 g p g y Status kognitif yang buruk pasca stroke bergantung pada derajat stroke yang diderita serta lokasi dan luasnya lesi. Hasil dari penelitian Duering et.al menyatakan bahwa stroke iskemik sering mengenai daerah ganglia basalis dan lobus frontotemporal hingga ke subkorteks. Infark lakunar di bagian talamus dan ganglia basal menyebabkan penurunan fungsi kognitif yang lebih besar dibandingkan dengan infark di area substansia alba (Putri et al., 2017). Penelitian yang dilakukan oleh (Wende et al., 2020) menunjukkan bahwa lesi pada hemisfer kiri memiliki nilai yang lebih rendah dibandingkan dengan lesi dihemisfer kanan. Hasil ini didukung oleh penelitian yang dilakukan oleh Sitaresmi di tahun 2014, Hal ini dikarenakan hemisfer kiri memiliki dominasi pengaturan untuk fungsi proporsi verbal linguistic sedangkan hemisfer kanan untuk fungsi non verbal-visuopasial dan emosional. Jika terdapat gangguan bahasa, pemeriksaan kognitif seperti seperti memori verbal dan fungsi eksekutif akan mengalami kesulitan. Sehingga pada gangguan berbahasa akan menurunkan skor fungsi kognitif karena domain fungsi kognitif yang lain juga terganggu. 618 Gambaran Fungsi Kognitif Yang Di Ukur Dengan Mmse Pada Pasien Riwayat Stroke Di Poli Saraf Rsud Dr. H. Abdul Moeloek Bandar Lampung Tahun 2021 620 BIBLIOGRAFI Aiyagari, v., & gorelick, p. B. (2016). Hypertension and stroke: pathophysiology and management. Springer. Asfar, a., & rusniyanti, d. (2018). Faktor yang berhubungan dengan fungsi kognitif penderita stroke non hemoragik di rumah sakit umum daerah kota makassar tahun 2018. 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Hubungan derajat stroke terhadap status kognitif pada pasien stroke iskemik di poliklinik saraf rumah sakit umum daerah dr . Zainoel abidin banda aceh relationship degree stroke on the cognitive status patients ischemic stroke. 2(1), 61–67. 619 Akhmad Kheru1, Fitriyani, Dharmawita, Putri Fadillah/Cerdika: Jurnal Ilmiah Indonesia, 1(6), 612-621 Riskesdas. (2019). Prevalensi psikosis di indonesia berdasarkan riset kesehatan dasar 2018. Jurnal penelitian dan pengembangan pelayanan kesehatan, 9–16.DOI: https://doi.org/10.22435/jpppk.v3i1.1882 Sacco, r. L., kasner, s. E., broderick, j. P., caplan, l. R., connors, j. J., culebras, a., elkind, m. S. V, george, m. G., hamdan, a. D., & higashida, r. T. (2013). An updated definition of stroke for the 21st century: a statement for healthcare professionals from the american heart association/american stroke association. Stroke, 44(7), 2064–2089. Sun, j.-h., tan, l., & yu, j.-t. (2014). Post-stroke cognitive impairment: epidemiology, mechanisms and management. Annals of translational medicine, 2(8). Wende, m. P., wungouw, h. P. L., & damanik, e. M. B. (2020). Perbedaan gangguan fungsi kognitif pada pasien stroke iskemik lesi hemisfer kiri dan kanan di rsud prof. Dr. Wz johannes kupang. Cendana medical journal (cmj), 8(1), 528– 534.https://doi.org/10.35508/cmj.v8i1.2662 Wu, c., zhang, z., fei, y., wu, g., qian, y., & meng, s. (2016). Characteristics of mercury form in soil-rice system and food security assessment in wastewater-irrigated paddy fields of tianjin. Transactions of the chinese society of agricultural engineering, 32(18), 207–212. © 2021 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY ND) license (https://creativecommons.org/licenses/by/3.0/). BIBLIOGRAFI © 2021 by the authors. 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https://openalex.org/W2055407041	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0057561&type=printable	English	null	Effectiveness of Nature Reserve System for Conserving Tropical Forests: A Statistical Evaluation of Hainan Island, China	PloS one	2,013	cc-by	6,277	Introduction would be more accurate to say that only 3% of its previous distribution was protected. Merely assessing the representativeness of the PAs system is not enough to determine whether it provides effective protection for tropical forests. One of the most common conservation strategies in the protection of tropical forests and mitigation of climate change is the establishment of protected areas (PAs) [1–3]. To date, 23% of tropical moist forest and 11% of tropical dry forest around the world are protected [4]. Although the total area set aside for protection continues to increase, it is unclear whether the strategy effectively achieves the stated conservation objectives [5,6]. In the interest of facilitating the conservation of biodiversity, the Convention on Biological Diversity (CBD) decided to evaluate and improve the effectiveness of PAs in 2004 [7]. Considering that much of tropical biodiversity is unlikely to persist in the face of the growing pressure of human activities, assessing the effectiveness of the PA systems in the conservation of tropical forests is one of the most urgent issues in the preservation of remaining tropical biodiversity [4,8]. Recently, more studies have focused on how well biodiversity features are actually protected or conserved [12]. One approach is to predict the deforestation that would have been observed had PAs not been established [13,14]. Empirical studies of this kind are far scarcer than those for representation, and typically rather more limited in scope, largely due to the difficulty of acquiring baseline data [5,6,12]. A commonly adopted method is to compare rates of land-cover clearing inside and outside PAs [1,15–18]. One can conclude that the PAs are partially effective at conserving biodiversity when deforestation rates are lower inside than outside PAs. However, this approach may provide somewhat optimistic evaluations of PAs’ effectiveness. This is because the creation of a PA might displace deforestation activities into neighboring forests through preemptive clearing, relocation of displaced communities, and immigration and development along the PAs’ boundaries (‘‘neighborhood leakage’’) [5,19]. In addition, PAs are often located in relatively inaccessible remote areas, which are mostly at higher elevations, with steep slopes, and far away from main roads and residential sites [20–23]. For these reasons, the Past studies of the effectiveness of the PAs system have focused on improving representativeness by working on system design and identifying features that were inadequately covered relative to specified targets [9]. Abstract Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lijsh@craes.org.cn Effectiveness of Nature Reserve System for Conserving Tropical Forests: A Statistical Evaluation of Hainan Island, China Wei Wang1,2, Peter Pechacek1,2, Mingxia Zhang3,4, Nengwen Xiao1,2, Jianguo Zhu4, Junsheng Li1,2* 1 Chinese Research Academy of Environmental Sciences, Beijing, China, 2 State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, China, 3 The Center for Integrative Conservation, Xishuangbanna Tropical Botanical Garden, The Chinese Academy of Sciences, Menglun, Xishuangbanna, Yunnan, China, 4 Ecology, Conservation and Environment Center, Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming, Yunnan, China Abstract Evaluating the effectiveness of existing nature reserve systems for the conservation of tropical forests is an urgent task to save the remaining biodiversity. Here, we tested the effectiveness of the reserve system on Hainan Island by conducting a three-way comparison of changes in forest area in locations within the reserves, adjacent to the reserves, and far outside of the reserves. We used a general linear model to control for the effects of covariates (historical forest area, elevation, slope, and distance to nearest roads), which may also be correlated with the changes in forest area, to better explain the effectiveness of the reserve system. From 2000 to 2010, the forest area inside Hainan’s nature reserve system showed an increase while adjacent unprotected areas and the wider, unprotected landscape both experienced deforestation. However, the simple inside-outside comparisons may overestimate the protective effect of the reserve system. Most nature reserves (.60%) showed increasing fragmentation. And the risk of rapid deforestation remained high at low elevations, where remaining forests tend to be easily logged and converted to commercial plantations. Future conservation efforts should pay more attention to those sites with less challenging environmental conditions. Citation: Wang W, Pechacek P, Zhang M, Xiao N, Zhu J, et al. (2013) Effectiveness of Nature Reserve System for Conserving Tropical Forests: A Statistical Evaluation of Hainan Island, China. PLoS ONE 8(2): e57561. doi:10.1371/journal.pone.0057561 Editor: Brock Fenton, University of Western Ontario, Canada Received July 10, 2012; Accepted January 26, 2013; Published February 28, 2013 ng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright: 2013 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. nding: The study was supported by a Public Welfare Scientific Research Project, Ministry of Environmental Protection of the People’s R . 201209028). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manus supported by a Public Welfare Scientific Research Project, Ministry of Environmental Protection of the People’s Republic of China (Gran ders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Forests Mapping We used eight Landsat TM/ETM+ images, four from 2000 and four from 2010, covering all of Hainan Island (path/row numbers 123–124/46–47) to obtain information on land cover. We downloaded these images from the International Scientific Data Service Platform, Computer Network Information Center of the Chinese Academy of Sciences website (http://datamirror.csdb.cn). All images had a resolution of 30 m and were geo-referenced to Gauss Kruger/Krasovsky coordinates with a root mean square ,1 pixel. China has been making great efforts toward protecting its natural resources since the first Nature Reserves (NRs) were established in 1956 [25,26]. NRs are the main body of China’s PA system, but little is known about their effectiveness due to a lack of systematic planning and spatial data on their extent and boundaries [26,27]. Here we selected Hainan Island, which harbors the most extensive primary tropical rainforest in China [28], to assess the effectiveness of the NR system in the conservation of natural forests. To do so, we (1) compared changes in forest area and fragmentation patterns among the forest patches inside NRs, in adjacent 10-km unprotected areas, and in the wider unprotected landscape from 2000 to 2010; (2) identified the effects of covariates (historical forest area, elevation, slope, and distance to nearest roads) on observed changes in forest area; and (3) determined the effectiveness of the NR system by comparing deforestation rates in protected and unprotected areas while controlling or not for the effects of those covariates. Our results provide information useful for future conservation efforts to maintain tropical forests in Hainan. In order to collect ground data for both mapping and validation, we employed a stratified method to identify 1225 samples of 1006100 m2 across the whole island. A full description of the sampling process can be found in Zhang et al. [29]. We conducted two field surveys in 2005 and measured the canopy cover and tree height in each sample, using handheld Garmin 72 GPS receivers to record the location for the ground truth data. Using vegetation class definitions issued by the International Geosphere–Biosphere Programme (IGBP), we defined forests as those areas dominated by natural trees with a canopy cover .60% and mean height exceeding 2 m, covering at least 1 ha [34]. Plantations with simple grid-like and homogeneous structures were considered distinct from these ‘‘natural’’ forests and as one type of non-forest land cover. Effectiveness of Nature Reserve System in Tropics effectiveness of the PA systems should be tested taking environ- mental and human impact conditions into account [24]. effectiveness of the PA systems should be tested taking environ- mental and human impact conditions into account [24]. Forests Mapping In the field surveys, we collected only the data that showed minimal change from 2000 to 2005 and ignored other data, using information from local forestry bureaus and nature reserves administrations. We used these ground truth data to develop and assess the 2000 forest map. To develop and assess the 2010 forest map, we compared ground truth data collected in 2005 to high-resolution Google Earth images from 2010 and treated unchanged areas as ground truth data. We randomly selected about half of the ground truth data and kept them as training data for classification. We used the remaining data for assessing the accuracy of the forest maps. Study Area The study was carried out on Hainan Island (Fig. 1), which has an area of about 34,000 km2. Hainan Island is located at the northern edge of the Indo-Malayan rain forest (18u099–20u119 N, 108u369–111u049 E). The island is mountainous in the middle, and flatter in northern and coastal areas. Vegetation is diverse across the island, with a pattern of vertical zonation. In mountainous areas with high rainfall, lowland rainforest occurs below 600 m, montane and ravine rainforest occur between 600 and 1200 m, and evergreen broadleaf forest occurs above 1200 m. Small areas of dwarf mossy forest are distributed on ridges of mountain tops [29]. We used a maximum likelihood classification algorithm to classify 2000 and 2010 images separately using Erdas Imagine 9.0 (Leica Geosystems Geospatial Imaging LLC, 2005) with the aid of training data and Digital elevation model (DEM) from the 1:25,000 topographic maps [35]. We then resampled the classification results into forest maps with a minimum mapping unit (MMU) of 1 ha. Since 1949, human activities have caused serious deforestation and degradation of Hainan’s forests [30]. To protect the intrinsic biodiversity of Hainan, the clear-cutting of all rainforests was banned in 1994 [31]. Although human population increased rapidly during the past 20 years, the reforestation of degraded land and the reduction in logging of natural forest have had a positive effect on maintaining forests [31]. Currently, 8.4% of Hainan is set aside in 54 NRs [32], mainly located in remote and economically less valuable areas [33], which is similar to most NRs of China [26]. Human disturbance within the NRs was limited except for occasional hunting and gathering by the indigenous people in the island [28]. However, it is unclear whether the current NR system effectively protects Hainan’s forest cover or if the protection is merely due to challenging topography consequently associated with less human pressure. This uncertainty makes it difficult to develop flexible management and funding mechanisms for future conservation actions. Introduction However, these studies did not reveal the impact of habitat loss and could have been misleading in terms of historical context [10,11]. For instance, if a particular habitat takes up 10% of an existing PA system, but 70% of that habitat’s original cover had already been lost at the time of observation, it February 2013 \| Volume 8 \| Issue 2 \| e57561 1 PLOS ONE \| www.plosone.org Data Analysis Ch i Changes in forest area. Using buffer analysis in areas around the NRs, we generated layers of adjacent unprotected areas (a 10 km buffer area around the NRs’ boundaries) and the wider unprotected landscape (.10 km from NRs’ boundaries) (Fig. 1). We then measured 2000 and 2010 forest areas inside the NRs, in adjacent unprotected areas, and in the wider unprotected landscape. We chose changes in forest area in the wider unprotected landscape as controls, following [15]. To simplify the dataset and minimize statistical dependence in the dataset, we used random sampling instead of treating the whole island as a study subject. Because the sampling can only provide an estimate of the true outcome of the whole island, a sufficiently large number of sampling plots was required. Considering the minimum NR area in Hainan (100 ha), we used 100 ha quadrats as sampling plots. We randomly sampled 2000 plots of 100 ha across Hainan Island and excluded plots (N = 576) whose boundaries crossed the boundaries of NRs, adjacent unprotected areas, or the wider unprotected landscape. We calculated forest area for 2000 and 2010 for each plot inside NRs (N = 147), in adjacent unprotected areas (N = 626), and in the wider unprotected landscape (N = 651). We conducted a Wilcoxon signed-rank test to evaluate whether In this study, we excluded 20 marine and wetland NRs and six NRs with an area ,100 ha, focusing on the remaining 28 NRs that are primarily dedicated to protecting forest ecosystems (Table S1). These NRs had been established for at least five years, which is long enough for them to reflect recent management activities [6]. In fact, most of them were established between 1974 and 1996, only three were established in 2004 and two were established in early 2006. We delineated boundaries of the NRs as polygons using the NRs’ management plans (from the Department of Land Environment & Resources of Hainan Province), supplemented by measurements taken in the field. February 2013 \| Volume 8 \| Issue 2 \| e57561 PLOS ONE \| www.plosone.org 2 Effectiveness of Nature Reserve System in Tropics Figure 1. Nature reserve system of Hainan Island. Data Analysis Ch i The nature reserve system, adjacent unprotected areas (surrounding lands within 10 km of the nature reserve boundaries) and wider unprotected landscape (more than 10 km away from the nature reserve boundaries) overlaid with natural forest cover in 2010 and digital elevation model (DEM) of Hainan Island, China. doi:10.1371/journal.pone.0057561.g001 Figure 1. Nature reserve system of Hainan Island. The nature reserve system, adjacent unprotected areas (surrounding lands within 10 km of the nature reserve boundaries) and wider unprotected landscape (more than 10 km away from the nature reserve boundaries) overlaid with natural forest cover in 2010 and digital elevation model (DEM) of Hainan Island, China. doi:10.1371/journal.pone.0057561.g001 the total amount of forest area was different between 2000 and 2010 inside NRs, in adjacent unprotected areas, and in the wider unprotected landscape, respectively. We performed three Mann- Whitney U tests before considering the covariates to compare changes in forest areas between (1) NRs and adjacent unprotected areas; (2) NRs and wider unprotected landscape; (3) adjacent unprotected areas and wider unprotected landscape. correlated with each other, so we used partial correlation analysis to measure the degree of association between one covariate and the response variable (change in forest area), controlling for the effects of other variables [36]. We calculated the partial correlation coefficients (rp) with the following four analyses: (1) between forest area in 2000 and changes in forest area (control variables: inside or outside of NRs, elevation, slope, and distance to nearest roads); (2) between elevation and changes in forest area (control variables: inside or outside of NRs, forest area in 2000, slope, and distance to nearest roads); (3) between slope and changes in forest area (control variables: inside or outside of NRs, forest area in 2000, elevation, and distance to nearest roads); (4) between distance to nearest roads and changes in forest area (control variables: inside or outside of NRs, forest area in 2000, elevation, and slope). Forest fragmentation analysis. For each NR and its 10-km adjacent unprotected area, we calculated the values of fragmen- tation indices between 2000 and 2010. The indices we used included the mean patch size (MPS, the average forest patch size, in hectares) and the mean nearest neighbor (MNN, the average edge-edge distance between each forest patch and the nearest neighboring patch, in meters). We conducted a Wilcoxon signed- rank test to detect differences between the two periods of time in forest fragmentation index. Results We produced the final forest maps of Hainan for 2000 and 2010, and they showed overall accuracy of 93.2% and 88.5%, respectively. In 2000, about 18.0% (612,830 ha) of the island was covered by tropical forests. From 2000 to 2010, the overall size of Hainan’s forests was reduced by 6.8% (41,399 ha), whereas the forest area inside the NRs increased (Wilcoxon test: N = 147, W = 55.65, P,0.001). In contrast, adjacent 10-km unprotected areas and the wider unprotected landscapes both experienced deforestation (N = 626, W = 292.68, P,0.001; and N = 651, W = 303.74, P,0.001) (Table 1, 2). The results also indicated that the forest patches inside most NRs were becoming isolated. Of the 28 NRs studied, 12 experienced decreases in MPS and 18 experienced increases in MNN. Although non-parametric testing showed that the MPS of forest patches inside the NRs underwent no changes between 2000 and 2010 (from 128.3 ha to 178.3 ha, Wilcoxon test: N = 28, W = 9.80, P = 0.224), the MNN showed an increase (from 246.3 m to 319.8 m, N = 28, W = 10.75, P = 0.007). The MPS and MNN of forest patches in adjacent 10-km unprotected areas both showed significant changes, from 11.2 ha to 19.7 ha (N = 28, W = 6.25, P,0.001) and from 331.7 m to 360.2 m (N = 28, W = 13.77, P = 0.006), respectively. relative to those in adjacent 10-km unprotected areas (Mann- Whitney U test: U = 31511.00, P,0.001) and with those in the wider unprotected landscape (U = 24474.00, P,0.001). Further- more, adjacent unprotected areas showed lower levels of de- forestation than the wider unprotected landscape (U = 183996.50, P = 0.003). By defining control variables, the first two PCA variables, which had explained most of the variance observed (88.9%) among the four covariates, were selected as new covariates. The analysis of the general linear model still showed that deforestation level was lower inside NRs than those in adjacent unprotected areas and in the wider unprotected landscape (P,0.05 in all cases). However, the mean differences in the pairwise comparisons were all lower than those in the simple non-parametric tests (Table 3). ) p y As for forest area in 2000, elevation, slope, and distance to nearest roads, correlation tests showed that all four covariates had strong or moderately-strong positive relationships with one another (r .0.4). Discussion Evaluating the effectiveness of NRs for the purpose of conserving tropical forests is urgent. Previous reports have analyzed trends at the level of the NR system (rather than at the individual NR level), using analysis to balance the effects of the covariates that might affect changes in forest area and determining whether deforestation activities had been displaced from NRs onto adjacent unprotected areas (rather than a simple inside-outside comparison) [5–7,12–17]. We addressed these points in the case of the tropical Hainan Island by dividing the natural forests into three groups (NRs, adjacent 10-km unprotected areas, and the wider unprotected landscapes) and comparing the differences in changes in forest area among these groups before and after the effects of given covariates (historical area and accessibility of natural forests) were balanced. p Overall, the results suggested that, in terms of preventing deforestation, the NR system offered an effective solution over the past 10 years. Without considering the covariates (the simple non- parametric tests), forest area inside NRs showed an increase First, there were increases in forest area inside Hainan’s NR system, implying that forest recovery could be relatively fast and efficient in the tropics even over a relatively short, 10-year time frame [37–39]. Preconditions of the recovery should include the absence of human disturbance, and proximity of sufficient amount of native trees needed for regeneration [28,31]. The ban on the clear-cutting of all natural forests since 1994 may also have favored the recovery [31]. In contrast, the unprotected areas (including the adjacent unprotected areas and the wider unprotected landscapes) showed an 11.8% loss (1.18% year–1) in forest over, which was not as severe as that observed in other tropical regions [5]. As in the rest of the tropical world, the main reasons were shifting cultivation and illegal logging outside of NRs [40,41]. The tendency was robust regardless of whether the effects of historical forest area and accessibility of natural forests were taken into account or not. Given that the changes in forest area had strong correlation with forest area in 2000 (negative) and elevation Table 1. Changes in tropical forests across Hainan Island. Table 1. Changes in tropical forests across Hainan Island. Table 1. Changes in tropical forests across Hainan Island. Results The strongest correlations were between forest area in 2000 and slope (r = 0.789), followed by forest area in 2000 and elevation (r = 0.785). Elevation and slope were also strongly and positively correlated (r = 0.759) (P,0.001 in all cases). By defining control variables, the partial correlation analyses further and better explained the coefficients (rp) between the response variable (change in forest area from 2000 to 2010) and the covariates (forest area in 2000, elevation, slope, and distance to nearest roads, respectively). The variables that were most strongly correlated with the change in forest area were: forest area in 2000 (rp = –0.552) and elevation (rp = 0.442), followed by slope (rp = 0.235). Distance to nearest roads had a weaker but still significant effect (rp = 0.116) (P,0.001 in all cases). Data Analysis Ch i Testing the effectiveness of nature reserve system. We used the Pearson’s r correlation analysis to evaluate relationships among covariates. We then used principal component analysis (PCA) to convert these potentially correlated variables into a set of values of linearly uncorrelated variables. We selected only the first few principal components that could explain most (.80%) of the observed variance among forest area in 2000, elevation, slope, and distances to main roads, and then reduced the dimensionality of the transformed data. We then used a general linear model, the analysis of covariance, to compare changes in forest area on the condition that the effects of these new covariates be balanced Testing the effect of covariates on forest change. For each sampling plot, we selected forest area in 2000, elevation, slope, and distance from the edge to the nearest main roads as covariates for statistical analyses, following [1,15,23]. We obtained data on elevation and slope from the digital elevation model (DEM) created from 1:25,000 topographic maps. We also created digital layers of main roads (including highways, national roads, provincial roads and county roads) from 1:25,000 topographic maps of 1997. Covariates that affect a response variable may be February 2013 \| Volume 8 \| Issue 2 \| e57561 PLOS ONE \| www.plosone.org 3 Effectiveness of Nature Reserve System in Tropics Table 2. Mean forest area. 2000 2010 Group N Mean Std. Deviation Mean Std. Deviation P–valuea 1 147 73.41 28.12 78.18 27.46 0.000 2 626 30.54 28.24 28.53 31.80 0.000 3 651 22.80 28.39 17.76 28.67 0.000 Comparison of mean forest area (ha) between 2000 and 2010 across different sampling plots of 100 ha on Hainan Island (Group 1: inside nature reserves, Group 2: in adjacent 10-km unprotected areas, Group 3: in the wider unprotected landscape). aWilcoxon Signed Ranks Test (2-tailed). doi:10.1371/journal.pone.0057561.t002 Table 2. Mean forest area. between (1) NRs and adjacent unprotected areas; (2) NRs and the wider unprotected landscape; (3) adjacent unprotected areas and the wider unprotected landscape. Table 2. Mean forest area. Acknowledgments We are grateful to the Department of Land Environmental Resources of Hainan Province, Hainan Provincial Forestry Department, and Hainan’s nature reserves for their support and for access to delineation of nature reserve boundaries, to the International Scientific Data Service Platform, Computer Network Information Center of the Chinese Academy of Sciences for remote sense images. We would like to thank Dr. Rong Di of Rutgers University for copyediting of this manuscript and Prof. Xie Yan of the Institute of Zoology and Prof. Wang Hao of Peking University for sharing data. We also appreciate the valuable comments that we received from Dr. Xiao Wen of Dali University and three anonymous reviewers for their constructive criticism. Supporting Information Table S1 Nature reserves dedicated primarily to the protection of forest ecosystems on Hainan Island. (DOC) The recovered forest within NRs’ boundaries cannot be assumed to have reached the full naturalness of the former mature rainforest [43]. In fact, the results revealed increasing isolation of forest patches among most Hainan’s NRs (.60%). The reasons could be the selective logging at higher elevations and the conversion of smaller forest patches to commercial plantations (e.g. rubber and eucalyptus) in the lowlands [29]. In addition, short term regeneration can only fulfill some of the functions of mature rainforests [44–46]. Carbon sequestration might continue with little changes after regeneration [45], whereas supportive func- tions, such as providing wildlife habitat, may be altered [38,44]. For example, even though some monoculture pine plantations (classified as non-forest in this study) in the Bawangling National Nature Reserve have been restored to mixed native forests, the Hainan gibbon (Nomascus hainanus), one of the world’s rarest apes, still lost some of its prime habitat [29,39]. Discussion The covariates were the first and second components extracted from the principal component analysis of the independent variables (forest area in 2000, elevation, slope, and distance to nearest roads). deforestation. We assume that sites with less challenging environ- mental conditions (e.g. low elevation, flatter slopes) or proximity to roads will have the potential for rapid deforestation because of their attractiveness to logging operations, and that this would be especially true for remaining mature rainforest at lower elevations. Establishing NRs with effective management in the lowlands and preventing illegal selective logging within existing NRs will provide powerful instruments to prevent deforestation. Moreover, large- scale restoration of native forests is required to insure that the regenerated forests within the NR system regain functionality. This could help connect fragmented patches of forest, an issue of special importance to many local threatened and endangered species, such as the Hainan Gibbon [33,39]. (positive), controlling the effects of these covariates is important to better explain the effectiveness of NR system for conserving tropical forests. This result concurred with those of studies in Costa Rica [1] and in Sumatra [15]: the simple inside-outside comparisons may have considerably overestimated the protective effects of NRs, particularly where NRs showed marked topo- graphic differences from their immediate surroundings [42]. The results also suggested the absence of a detrimental ‘‘neighborhood leakage’’ effect on Hainan Island. Even though the adjacent unprotected areas experienced some deforestation, they saw less amounts of deforestation than the wider unprotected landscapes. Population growth, pre-emptive clearing, and the relocation of illegal settlers along the boundaries of the NRs may have a marginal influence on deforestation, as in Sumatra [15]. This could be explained by the fact that most of Hainan’s NRs and their adjacent unprotected areas are located in the central mountainous region, which has high elevations and steep slopes, and these areas are less subject to human activity [33]. Author Contributions In sum, these results suggested that Hainan’s NR system was effective in preventing deforestation over the past 10 years, but attention should be paid to future conservation efforts because other factors (the covariates in our study) were also correlated with Designed the model used in analysis: WW. Conceived and designed the experiments: WW JGZ JSL. Performed the experiments: WW MXZ. Analyzed the data: WW NWX. Contributed reagents/materials/analysis tools: WW MXZ. Wrote the paper: WW PP. 4. Chape S, Spalding M, Jenkins MD (2008) The World’s Protected Areas: UNEP World Conservation Monitoring Centre. Berkeley, CA: University of California Press. Discussion Area of forests (ha) 2000 2010 % change Inside nature reserves 169,169 180,206 +6.5% In adjacent unprotected areas 268,403 250,769 –6.6% In wider unprotected landscape 175,258 140,456 –19.9% Total 612,830 571,431 –6.8% Changes in the area of tropical forests inside nature reserves, in adjacent unprotected areas (within 10 km of nature reserves’ boundaries), and in the wider unprotected landscapes (.10 km from nature reserves’ boundaries) in Hainan, China, from 2000 to 2010. doi:10.1371/journal.pone.0057561.t001 February 2013 \| Volume 8 \| Issue 2 \| e57561 PLOS ONE \| www.plosone.org 4 Effectiveness of Nature Reserve System in Tropics Table 3. Pairwise comparisons. Without covariates After the covariates were balanced Group (I) vs. Group(J) Mean difference (I–J) P–valuea Mean difference (I–J) P–valueb Group 1 vs. Group 2 6.78** 0.000 4.52* 0.006 Group 1 vs. Group 3 9.81 0.000 7.17 0.000 Group 2 vs. Group 3 3.03* 0.003 2.65 0.004 The results of pairwise comparisons before and after the effects of covariates were balanced. The dependent variable was the changes in forest area (ha). Group identity served as the independent variable (Group 1: inside nature reserves, Group 2: in adjacent 10-km unprotected areas, Group 3: in the wider unprotected landscape). The covariates were the first and second components extracted from the principal component analysis of the independent variables (forest area in 2000, elevation, slope, and distance to nearest roads). Th diff i i ifi t t th 0 05 l l The results of pairwise comparisons before and after the effects of covariates were balanced. The dependent variable was the changes in forest area (ha). Group identity served as the independent variable (Group 1: inside nature reserves, Group 2: in adjacent 10-km unprotected areas, Group 3: in the wider unprotected landscape). The covariates were the first and second components extracted from the principal component analysis of the independent variables (forest area in 2000, elevation, slope, and distance to nearest roads). The mean difference is significant at the 0.05 level. **The mean difference is significant at the 0.001 level. The results of pairwise comparisons before and after the effects of covariates were balanced. The dependent variable was the changes in forest area (ha). Group identity served as the independent variable (Group 1: inside nature reserves, Group 2: in adjacent 10-km unprotected areas, Group 3: in the wider unprotected landscape). 1. Andam KS, Ferraro PJ, Pfaff A, Sanchez-Azofeifa GA, Robalino JA (2008) Measuring the effectiveness of protected area networks in reducing deforestation. 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Nagendra H (2008) Do Parks Work? Impact of Protected Areas on Land Cover Clearing. AMBIO: A Journal of the Human Environment 37: 330–337. 27. Sang W, Ma K, Axmacher JC (2011) Securing a Future for China’s Wild Plant Resources. BioScience 61: 720–725. 28. Deng F, Zang R, Chen B (2008) Identification of functional groups in an old- growth tropical montane rain forest on Hainan Island, China. Forest Ecology and Management 255: 1820–1830. 6. Bruner AG (2001) Effectiveness of Parks in Protecting Tropical Biodiversity. Science 291: 125–128. 7. Stoll-Kleemann S (2010) Evaluation of management effectiveness in protected areas: Methodologies and results. Basic and Applied Ecology 11: 377–382. g 29. Zhang M, Fellowes JR, Jiang X, Wang W, Chan BPL, et al. (2010) Degradation of tropical forest in Hainan, China, 1991–2008: Conservation implications for Hainan Gibbon (Nomascus hainanus). Biological Conservation 143: 1397–1404. 8. Leverington F, Hockings M, Costa KL (2008) Management effectiveness evaluation in protected areas: Report for the project ‘Global study into management effectiveness evaluation of protected areas’. The University of Queensland, Gatton, IUCN WCPA, TNC, WWF, AUSTRALIA. ( ) g 30. Meng J, Lu Y, Lei X, Liu G (2011) Structure and floristics of tropical forests and their implications for restoration of degraded forests of China’s Hainan Island. Tropical Ecology 52: 177–191. g p Queensland, Gatton, IUCN WCPA, TNC, WWF, AUSTRALIA 9. Scott JM, Davis F, Csuti B, Noss R, Butterfield B, et al. (1993) Gap Analysis: A Geographic Approach to Protection of Biological Diversity. Journal of Wildlife Management 57(1) supplement: wildlife Monographs No.123: 1–41. 31. Zhang Y, Uusivuori J, Kuuluvainen J (2000) Econometric analysis of the causes of forest land use changes in Hainan, China. Canadian Journal of Forest Research 30: 1913–1921. 10. Jennings MD (2000) Gap analysis: concepts, methods, and recent results. Landscape Ecology 15: 5–20. 32. MEP (2012) The 2011 List of the Nature Reserves, China. Beijing: China Environmental Science Press. 11. Wang W, Ren G, He Y, Zhu J (2008) Habitat Degradation and Conservation Status Assessment of Gallinaceous Birds in the Trans-Himalayas, China. Journal of Wildlife Management 72: 1335–1341. 33. Wu R, Ma G, Long Y, Yu J, Li S, et al. (2011) The performance of nature reserves in capturing the biological diversity on Hainan Island, China. Environmental Science and Pollution Research: 800–810. g 12. Tang Z, Fang J, Sun J, Gaston KJ (2011) Effectiveness of Protected Areas in Maintaining Plant Production. Effectiveness of Nature Reserve System in Tropics PLoS ONE 6: e19116. 34. Hansen MC, Defries RS, Townshend JRG, Sohlberg R (2000) Global land cover classifications at 1 km spatial resolution using a classification tree approach. International Journal of Remote Sensing 21: 1331–1364. 13. Liu J, Linderman M, Ouyang Z, An L, Yang J, et al. (2001) Ecological Degradation in Protected Areas: The Case of Wolong Nature Reserve for Giant Pandas. Science 292: 98–101. 35. Ren G, Zhu AX, Wang W, Xiao W, Huang Y, et al. (2009) A hierarchical approach coupled with coarse DEM information for improving the efficiency and accuracy of forest mapping over very rugged terrains. Forest Ecology and Management 258: 26–34. 14. Chai S-L, Tanner E, McLaren K (2009) High rates of forest clearance and fragmentation pre- and post-National Park establishment: The case of a Jamaican montane rainforest. Biological Conservation 142: 2484–2492. J g 15. Gaveau DLA, Epting J, Lyne O, Linkie M, Kumara I, et al. (2009) Evaluating whether protected areas reduce tropical deforestation in Sumatra. Journal of Biogeography 36: 2165–2175. 36. Kutner MH, Nachtsheim CJ, Neter J, Li W (2005) Applied Linear Statistical Models. New York: McGraw-Hill/Irwin. 37. Dunn RR (2004) Recovery of faunal communities during tropical forest regeneration. Conservation Biology 18: 302–309. g g p y 16. Sanchez-Azofeifa GA, Daily GC, Pfaff A, Busch C (2003) Integrity and isolation of Costa Rica’s national parks and biological reserves: examing the dynamics of land-cover change. Biological Conservation 109: 123–135. 38. Letcher SG, Chazdon RL (2009) Rapid recovery of biomass, species richness, and species composition in a forest chronosequence in Northeastern Costa Rica. Biotropica 41: 608–617. 17. Young JE, Sa´nchez-Azofeifa GA, Hannon SJ, Chapman R (2006) Trends in land cover change and isolation of protected areas at the interface of the southern boreal mixedwood and aspen parkland in Alberta, Canada. Forest Ecology and Management 230: 151–161. p 39. Fellowes JR, Chan BPL, Zhou J, Chen S, Yang S, et al. (2008) Current status of the Hainan gibbon (Nomascus hainanus): progress of population monitoring and other priority actions. Asian Primates Journal 1(1): 2–11. 40. Dalle SP, Pulido MT, De Blois S (2011) Balancing shifting cultivation and forest conservation: lessons from a ‘‘sustainable landscape’’ in southeastern Mexico. Ecological Applications 21: 1557–1572. 18. Southworth J, Nagendra H, Carlson LA, Tucker C (2004) Assessing the impact of Celaque National Park on forest fragmentation in western Honduras. Applied Geography 24: 303–322. g p y 19. References February 2013 \| Volume 8 \| Issue 2 \| e57561 PLOS ONE \| www.plosone.org 5 Effectiveness of Nature Reserve System in Tropics Wittemyer G, Elsen P, Bean WT, Burton ACO, Brashares JS (2008) Accelerated human population growth at protected area edges. Science 321: 123–126. g pp 41. Deng F, Chen Q, Chen X (2007) Compar ison of Ecological Service among natural forest, rubber and eucalyptus plantations. Journal of South China University of Tropical Agriculture 13: 19–23 (in Chinese with English abstract). 20. Joppa LN, Loarie SR, Pimm SL (2008) On the protection of ‘‘protected areas’’. Proceedings of the National Academy of Sciences 105: 6673–6678. y p g g 42. Mas J-F (2005) Assessing protected area effectiveness using surrounding (buffer) areas environmentally similar to the target area. Environmental Monitoring and Assessment 105: 69–80. Proceedings of the National Academy of Sciences 105: 6673–6678 21. Margules CR, Pressey RL (2000) Systematic conservation planning. Nature 405: 243–253. 2011) China’s new forests aren’t as green as they seem. Nature 477: 37 22. Oldfield T (2004) A gap analysis of terrestrial protected areas in England and its implications for conservation policy. Biological Conservation 120: 303–309. 44. Liebsch D, Marques M, Goldenberg R (2008) How long does the Atlantic Rain Forest take to recover after a disturbance? Changes in species composition and ecological features during secondary succession. Biological Conservation 141: 1717–1725. p p y g 23. Joppa LN, Pfaff A (2009) High and far: biases in the location of protected areas. PLoS ONE 4(12): e8273. 24. Joppa LN, Pfaff A (2010) Global protected area impacts. Proceedings of the Royal Society B: Biological Sciences 278: 1633–1638. 45. Silver WL, Kueppers LM, Lugo AE, Ostertag R, Matzek V (2004) Carbon sequestration and plant community dynamics following reforestation of tropical pasture. Ecological Applications 14: 1115–1127. 25. Liu J, Ouyang Z, Pimm SL, Raven PH, Wang X, et al. (2003) Protecting China’s Biodiversity. Science 300: 1240–1241. 46. Turner IM, Corlett RT (1996) The conservation value of small, isolated fragments of lowland tropical rain forest. Trends in Ecology and Evolution 11: 330–333. 26. Wu R, Zhang S, Yu DW, Zhao P, Li X, et al. (2011) Effectiveness of China’s nature reserves in representing ecological diversity. Frontiers in Ecology and the Environment: 383–389. 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https://openalex.org/W4389896950	https://www.scielo.br/j/bak/a/Gh3KCgGYszdxP7XKXDJC8Cc/?lang=pt&format=pdf	Portuguese	null	ERRATA	Bakhtiniana	2,023	cc-by	733	Figura 1 – Corpus de análise da narrativa de Duna. Bakhtiniana, São Paulo, 18 (4): e64318p, out./dez. 2023 Todo conteúdo de Bakhtiniana. Revista de Estudos do Discurso está sob Licença Creative Commons CC - By 4.0 Todo conteúdo de Bakhtiniana. Revista de Estudos do Discurso está sob Licença Creative Commons CC - By 4.0 D.O.I. http://dx.doi.org/10.1590/2176-4573p64318 D.O.I. http://dx.doi.org/10.1590/2176-4573p64318 ERRATA No artigo “A ficção científica: o enunciador hiperperceptivo e a viagem do ponto de vista na referenciação”, com número de D.O.I. http://dx.doi.org/10.1590/2176-4573p61037, publicado no periódico Bakhtiniana. Revista de Estudos do Discurso, 18 (4): e61037p, 2023, na figura 1: Onde se lia: Figura 1 – Corpus de análise da narrativa de Duna. Thufir Hawat entrou de mansinho na sala de treinamento do Castelo Caladan, fechou a porta suavemente. Ficou parado ali um momento, sentindo-se velho, cansado e surrado pelas intempéries. Sua perna esquerda doía no ponto em que o haviam ferido uma vez, a serviço do Velho Duque. Três gerações deles agora, pensou. Olhou para o outro lado do recinto iluminado pela luz do meio-dia que, aos borbotões, atravessava as claraboias, e viu o menino sentado de costas para a porta, absorto em documentos e mapas espalhados sobre uma mesa. Quantas vezes terei de dizer ao garoto para não se sentar de costas para a porta? Hawat limpou a garganta. (...) – Ouvi você chegar pelo corredor – disse Paul. – E ouvi você abrir a porta. Thufir Hawat entrou de mansinho na sala de treinamento do Castelo Caladan, fechou a porta suavemente. Ficou parado ali um momento, sentindo-se velho, cansado e surrado pelas intempéries. Sua perna esquerda doía no ponto em que o haviam ferido uma vez, a serviço do Velho Duque. Olhou para o outro lado do recinto iluminado pela luz do meio-dia que, aos borbotões, atravessava as claraboias, e viu o menino sentado de costas para a porta, absorto em documentos e mapas espalhados sobre uma mesa. Quantas vezes terei de dizer ao garoto para não se sentar de costas para a porta? Hawat limpou a garganta. (...) Quantas vezes terei de dizer ao garoto para não se sentar de costas para a porta? Hawat limpou a garganta. – Ouvi você chegar pelo corredor – disse Paul. – E ouvi você abrir a Leia-se: Figura 1 – Corpus de análise da narrativa de Duna. Thufir Hawat entrou de mansinho na sala de treinamento do Castelo Caladan, fechou a porta suavemente. Ficou parado ali um momento, sentindo-se velho, cansado e surrado pelas intempéries. Sua perna esquerda doía no ponto em que o haviam ferido uma vez, a serviço do Velho Duque. Três gerações deles agora, pensou. Bakhtiniana, São Paulo, 18 (4): e64318p, out./dez. 2023 Todo conteúdo de Bakhtiniana. Revista de Estudos do Discurso está sob Licença Creative Commons CC - By 4.0 D.O.I. http://dx.doi.org/10.1590/2176-4573p64318 Olhou para o outro lado do recinto iluminado pela luz do meio-dia que, aos borbotões, atravessava as claraboias, e viu o menino sentado de costas para a porta, absorto em documentos e mapas espalhados sobre uma mesa. Quantas vezes terei de dizer ao garoto para não se sentar de costas para a porta? Hawat limpou a garganta. (...) – Ouvi você chegar pelo corredor – disse Paul. – E ouvi você abrir a porta. – Os sons que produzo podem ser imitados. – Eu saberia a diferença. É bem possível, Hawat pensou. (...) (Herbert, 2017, p. 50-51). Thufir Hawat entrou de mansinho na sala de treinamento do Castelo Caladan, fechou a porta suavemente. Ficou parado ali um momento, sentindo-se velho, cansado e surrado pelas intempéries. Sua perna esquerda doía no ponto em que o haviam ferido uma vez, a serviço do Velho Duque. Três gerações deles agora, pensou. Olhou para o outro lado do recinto iluminado pela luz do meio-dia que, aos borbotões, atravessava as claraboias, e viu o menino sentado de costas para a porta, absorto em documentos e mapas espalhados sobre uma mesa. Quantas vezes terei de dizer ao garoto para não se sentar de costas para a porta? Hawat limpou a garganta. Quantas vezes terei de dizer ao garoto para não se sentar de costas para a porta? Hawat limpou a garganta. (...) – Ouvi você chegar pelo corredor – disse Paul. – E ouvi você abrir a porta. – Os sons que produzo podem ser imitados. – Eu saberia a diferença. (Herbert, 2017, p. 50-51). Figura 1 – Corpus de análise da narrativa de Duna.
https://openalex.org/W4385074983	https://www.qeios.com/read/78ODHC/pdf	English	null	Review of: "There’s an App for That: Development of a Digital App for Mental Health Issues"	null	2,023	cc-by	270	Qeios, CC-BY 4.0 · Review, July 22, 2023 Qeios ID: 78ODHC · https://doi.org/10.32388/78ODHC Review of: "There’s an App for That: Development of a Digital App for Mental Health Issues" Nor Fariza Mohd Nor1 1 Universiti Kebangsaan Malaysia Nor Fariza Mohd Nor1 1 Universiti Kebangsaan Malaysia Nor Fariza Mohd Nor1 Potential competing interests: No potential competing interests to declare. Potential competing interests: No potential competing interests to declare. This is an interesting study. However, this manuscript lacks academic elements of a journal article. This is more of a business proposal, instead of an academic article. There is no theory that frames this study, hence, the theoretical foundation of this study is missing. The author immediately focused on the context, which is Indonesia in the Introduction section. The authors should begin with mental health as a global problem and refer to past studies in other parts of the world to establish the context of the study. There is no reference made to past studies in other parts of the world about mental health and apps that have been developed to tackle or handle mental health issues. A search in google scholar with the search term "apps for managing mental health', revealed 244,000 hits. As academic, the authors should not only focused on Indonesia but consider similar studies that have been done globally to establish significant and important contribution of the present study to mental health issue. There are many sweeping statements made without supporting evidence, i.e. past studies. There are language error which calls for proofread work. There are language error which calls for proofread work. Qeios ID: 78ODHC · https://doi.org/10.32388/78ODHC 1/1
https://openalex.org/W4312542914	https://www2.ifrn.edu.br/ojs/index.php/HOLOS/article/download/7655/3336	Portuguese	null	A PÓS-GRADUAÇÃO COMO REFERÊNCIA HISTÓRICA À CARREIRA E À SOCIEDADE	Holos	2,020	cc-by	5,891	DELFINO & SOUSA (2022) A PÓS-GRADUAÇÃO COMO REFERÊNCIA HISTÓRICA À CARREIRA DOCENTE E À SOCIEDADE I. A. DE L. DELFINO1, I. S. DE SOUSA2 Universidade Federal de Campina Grande1,2 https://orcid.org/0000-0001-5143-87211 islania.lira@ufcg.edu.br1 Submetido 22/08/2018 - Aceito 20/07/2020 DOI: 10.15628/holos.2022.7655 Submetido 22/08/2018 - Aceito 20/07/2020 DOI: 10.15628/holos.2022.7655 RESUMO utilizou-se bibliografia referente aos temas propostos, além do corpus documental composto por pareceres, leis e ainda os planos nacionais de pós-graduação. Compreende-se que, desde sua criação, a pós-graduação alcançou grandes avanços e representa a parcela mais estruturada e em contínuo desenvolvimento no complexo Sistema de Educação Superior no Brasil, apesar dos desafios ainda enfrentados em termos de financiamento, assimetrias e descompassos, destacando-se em seu horizonte histórico uma forte ameaça de supressão quanto aos incentivos do Estado. Diante das incertezas que rondam o Ensino Superior e as agências de fomento à pesquisa no Brasil, sentimo-nos desafiados a discutir essa temática tão relevante para o desenvolvimento do país. Assim, este estudo tem por objetivo discorrer acerca do sistema de pós-graduação no Brasil, a partir de uma perspectiva histórica, visibilizando seus marcos iniciais, seu desenvolvimento, seu contexto atual e suas perspectivas de continuidade e ampliação. Aborda-se ainda o tema como um dos aspectos supostamente estruturantes da gestão da carreira docente institucionalizada. Adotando-se a abordagem qualitativa, como fontes de informações PALAVRAS-CHAVE: Ensino Superior, Historicidade da Pós-Graduação, Carreira. 1 INTRODUÇÃO A educação pode ser considerada um dos fatores que retratam as diferenças de desenvolvimento e ordenamento de riquezas entre nações. O ensino superior, tendo as universidades como seu elemento central, desponta como um possível sistema condutor desse processo. No Brasil, a criação das primeiras universidades tornou-se realidade principalmente a partir do processo de mobilização da comunidade científica para a renovação do setor educacional, tendo a inserção da atividade de pesquisa como um fundamento básico para sua atuação (Gatti, 2001; Martins, 2003). Martins (2003) destaca ainda que a década de 1920 foi marcada por intensas transformações neste sentido, principalmente ao lembrar que o sistema de ensino superior era nessa época formado por escolas e faculdades isoladas e voltadas às atividades de ensino, constituindo até então os espaços oficiais do conhecimento. A criação da Associação Brasileira de Educação, o Manifesto dos Pioneiros da Educação Nova, o Movimento da Escola Nova no Brasil, a criação da Universidade de São Paulo (USP) e da Sociedade Brasileira para o Progresso da Ciência (SBPC) fizeram parte dessa história, tendo esta última um papel fundamental na defesa da institucionalização da atividade de pesquisa nas universidades, da reforma universitária e da criação dos cursos de pós-graduação no Brasil (Martins, 2003). Sequencial a isso, entre os anos de 1950 e 1960, a criação do Conselho Nacional de desenvolvimento Científico e Tecnológico (CNPq) e da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) fomentou o processo de institucionalização da pesquisa e com ele o surgimento dos primeiros cursos de pós-graduação. Sua origem, em parte foi alavancada por políticas educacionais provenientes do Estado, baseadas em sua concepção autoritária de universidade que servisse ao seu projeto de industrialização e segurança nacionais (Martins, 2003; Silva, 2011; Alves; Oliveira, 2014)e, em parte por um conjunto de iniciativas encabeçadas pela comunidade científica da época. Os avanços para a pós-graduação a partir de então foram se multiplicando e, apesar de afirmar que a idealização de universidade que se tinha quando da sua concepção acabou por nunca se concretizar, Morhy (2003) também concorda com as profundas mudanças perceptíveis quando se compara a universidade atual com a da década de 1950. Hoje, esse espaço temporal de pouco mais de 60 anos traz consigo uma bagagem histórica considerável em termos de transformações, permanências e desenvolvimento da pós- graduação brasileira. ABSTRACT Before the uncertainties surrounding Higher Education and research promotion agencies in Brazil, we are challenged to discuss this theme so relevant to the country's development. Thus, this study aims to discuss the post-graduation system in Brazil, from a historical perspective, inquiring its initial milestones, its development, its current context and its perspectives of continuity and expansion. The theme is also addressed as one of the supposedly structuring aspects of institutionalized teaching career management. Adopting the qualitative approach, we used literature referring to the themes proposed as sources of information, besides the documentary corpus composed of opinions, laws and also the national graduate plans. It is understood that, since its inception, graduate studies have made great strides and represent the most structured and continuous development in the complex System of Higher Education in Brazil, despite the challenges still faced in terms of financing, asymmetries and mismatches, highlighting in its historical horizon a strong threat of suppression of the incentives of the State. Keywords: Higher Education, Postgraduate historicity, Career. Keywords: Higher Education, Postgraduate historicity, Career. 1 HOLOS, Ano 38, v.2, e7655, 2022 Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. DELFINO & SOUSA (2022) Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. DELFINO & SOUSA (2022) da carreira docente. Por uma exigência social implícita – e explícita do ponto de vista do desenvolvimento de carreira dentro das universidades –, constitui hoje um dos seus pilares de ascensão profissional de professores em todos os níveis de ensino. É válido o alerta de Mendes (2003) sobre a maior importância da visão consciente, reflexiva e consistente de mundo a partir do conhecimento, em detrimento de um permanente processo de atualização e reciclagem onde se sujeita à participação sequenciada em modalidades do sistema universitário programados a tornarem-se obsoletos. Assim problematizado o contexto do estudo, parte-se dos seguintes pressupostos: 1) o desenvolvimento histórico da pós-graduação e sua configuração atual representam grande avanço à sociedade brasileira; e 2) a pós-graduação historicamente está inserida na carreira docente institucionalizada nas universidades como propulsora da ascensão profissional. Neste sentido, questiona-se sobre o contexto histórico da pós-graduação no Brasil e qual sua configuração atual como norteadora da carreira docente. Este estudo apresenta, portanto, reflexões teóricas com o objetivo de discorrer acerca do sistema de pós-graduação no Brasil, a partir de uma perspectiva histórica, visibilizando seus marcos iniciais, seu desenvolvimento, seu contexto atual e suas perspectivas de continuidade e ampliação. Aborda-se ainda o tema como um dos aspectos supostamente estruturantes da gestão da carreira docente institucionalizada. Para isso, adotando-se uma abordagem qualitativa, utilizou-se como fonte a bibliografia referente ao tema, além do corpus documental composto por pareceres, leis e ainda os planos nacionais de pós-graduação. A estrutura proposta, além desta introdução e dos aspectos conclusivos, apresenta o contexto histórico e atual envolvendo o ensino superior e a pós-graduação no Brasil, além da sua decorrência na formação da carreira docente. Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. 1 INTRODUÇÃO A posterior criação de outras instituições de fomento, a modernização, a importância e abrangência atual na atuação do CNPq e da CAPES, e a elaboração de documentos norteadores como os Planos Nacionais de Pós-Graduação (PNPG) são demonstrações claras desses avanços. Ressalta-se também, em tempo, que a pós-graduação faz parte do processo de formação Ressalta-se também, em tempo, que a pós-graduação faz parte do processo de HOLOS, Ano 38, v.2, e7655, 2022 HOLOS, Ano 38, v.2, e7655, 2022 2 Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. 2 O ENSINO SUPERIOR NO BRASIL: FORMAÇÃO E DESENVOLVIMENTO A configuração do sistema de ensino superior brasileiro experimentou mudanças expressivas nos últimos cem anos. Este período foi marcado por diversos movimentos sociais e políticas educacionais que contribuíram para sua constante e intensa ampliação. De acordo com Martins (2002), apesar do seu surgimento no início do século XIX, com a chegada da corte real portuguesa e a criação das primeiras escolas formadoras de profissionais liberais, desenvolveu-se com pouca intensidade e ausência de investimentos nesse período. Esse autor acrescenta que somente na década de 1920, em um raro período democrático no país, a comunidade acadêmica brasileira realizou grandes movimentos de mobilização para a criação das universidades. A luta era para que elas fossem instituições independentes dos interesses do Estado, com maior autonomia, visando à conquista do seu caráter científico e a promoção da pesquisa, pois o que se tinha até então eram escolas e faculdades isoladas, voltadas ao ensino para a formação superior da elite da época. 3 HOLOS, Ano 38, v.2, e7655, 2022 Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. DELFINO & SOUSA (2022) Na década de 1930, durante a primeira fase do governo de Getúlio Vargas, vivenciou-se uma ampla reforma educacional intitulada “Francisco Campos”, que promoveu um avanço por meio da criação institucional das universidades (Martins, 2002, p. 5). Esse avanço dos investimentos e políticas em relação ao ensino superior associa-se ao objetivo do Estado em transformar o país em uma nação forte e capaz de se tornar uma grande potência. Alguns fatos relevantes contribuíram neste sentido, pois desempenharam importante papel em prol da institucionalização universitária, como a criação da Associação Brasileira de Educação, em 1924. Seguindo-se a isso, o Manifesto dos Pioneiros da Educação Nova, em 1932, assinado por expoentes educadores como Anísio Teixeira, que recomendava a criação de universidades adequadas à integração entre ensino e pesquisa. Além disso, a criação da Universidade de São Paulo (USP) em 1934, destacou-se como a primeira experiência prática da institucionalização universitária, como lembra Martins (2003). Derivada já dos avanços da atividade científica instituída até então, em 1948, foi criada a Sociedade Brasileira para o Progresso da Ciência (SBPC), atuando na defesa da institucionalização da atividade de pesquisa nas universidades, da reforma universitária e da criação dos cursos de pós-graduação no Brasil (Martins, 2003). Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. 2 O ENSINO SUPERIOR NO BRASIL: FORMAÇÃO E DESENVOLVIMENTO Saviani (2008) critica a descontinuidade histórica no âmbito das políticas nacionais para a estruturação da educação que pode ser percebida ante as inúmeras reformas propostas pelos diferentes governos. Fato este que, no âmbito nacional, deu-se principalmente a partir da Reforma de Capanema, entre 1942 e 1946, a Lei de Diretrizes e Bases da Educação (LDB de 1961), e as reformas do regime militar, em 1968 e 1971. Apesar dessa crítica, Martins (2002, p. 5) lembra que, A reforma de 1968, a despeito de ocorrer em clima de deterioração dos direitos civis, inspirou-se em muitas das ideias do movimento estudantil e da intelectualidade das décadas anteriores: 1 – instituiu o departamento como unidade mínima de ensino, 2 – criou os institutos básicos, 3 – organizou o currículo em ciclos básico e o profissionalizante, 4 – alterou o exame vestibular, 5 – aboliu a cátedra, 6 – tornou as decisões mais democráticas, 7 – institucionalizou a pesquisa, 8 – centralizou decisões em órgãos federais. A partir de 1970, a política governamental para a área foi estimular a pós-graduação e a capacitação docente. Mesmo com todo esse contexto de crescimento, nos anos 1960, havia somente cerca de uma centena de instituições de ensino superior, em sua maioria de pequeno porte, direcionadas às atividades de ensino, atendendo menos de 100 mil estudantes, com predominância do gênero masculino, e detentoras de um corpo docente academicamente frágil (Martins, 2000; 2003). Ressalte-se neste cenário, que a pesquisa somente fazia parte da atividade docente na universidade em raros casos (Gatti, 2001). As três décadas subsequentes, no entanto, representaram mais um período histórico de ampliação e desenvolvimento da educação superior no Brasil, principalmente nas décadas de 1970 e 1990, com o proeminente aumento quantitativo do número de instituições, cursos e alunos – estes últimos, em grande parcela, já integrados ao mercado de trabalho, e interessados em um título acadêmico para ampliar suas possibilidades de carreira (Martins, 2000). Interesse 4 4 HOLOS, Ano 38, v.2, e7655, 2022 Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. DELFINO & SOUSA (2022) despertado pelo próprio sistema de governo à época, com vistas a uma profissionalização do setor industrial, ancorado em base capitalista. despertado pelo próprio sistema de governo à época, com vistas a uma profissionalização do setor industrial, ancorado em base capitalista. 2 O ENSINO SUPERIOR NO BRASIL: FORMAÇÃO E DESENVOLVIMENTO Destaca-se, além disso, a expansão por meio do acesso propiciado por iniciativas governamentais, o que diminuiu em tese a elitização do sistema, mas em contrapartida, representou ainda uma medida insuficiente, por não conseguir romper com o seu caráter excludente, dadas as precárias condições socioeconômicas e consequentes deficiências dos estudantes ao longo da educação básica (Barros, 2015). As manifestações da comunidade científica para a reforma e renovação do ensino superior resultaram também no surgimento da pós-graduação no país, representando outro grande avanço nesse sistema, como se segue. Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. DELFINO & SOUSA (2022) DELFINO & SOUSA (2022) Para estruturar a pós-graduação, foi elaborado o Parecer CFE nº 977/65 em 1965 pelo Conselho Federal de Educação (Almeida Junior, 2005; Alves, Oliveira, 2014). Esta norma distinguiu a pós-graduação stricto sensu e lato sensu. A primeira refere-se à designação dos cursos de mestrado e doutorado, destinados à formação de pesquisadores e docentes para atuação no nível superior, os quais dão seguimento à graduação com objetivos mais aprofundados de natureza acadêmica e científica, mesmo os que atuam em setores profissionais, conferindo no final um grau acadêmico ao aluno (Hostins, 2006). Enquanto a segunda, refere-se a cursos de especialização e aperfeiçoamento, com fins técnico-profissionais específicos sem envolver todo o campo do saber, podendo ser de caráter regular e permanente, e conferindo certificado de aproveitamento. O documento também especificou para os cursos stricto sensu (mestrado e doutorado) sua duração, os trabalhos e atividades referentes a cada um deles, os critérios de seleção e ingresso mais adequados, as características didáticas e metodológicas de condução das disciplinas e demais especificações quanto à orientação, elaboração e defesas de dissertações e teses. Gatti (2001) destaca, quanto à origem desses cursos no Brasil, que eles não são decorrentes do desenvolvimento da pesquisa científica, e sim, de deliberação política estatal. Esta autora também faz menção ao estímulo e financiamento à implantação desses cursos em universidades que apresentavam maior estabilidade institucional. Neste contexto, outra iniciativa a ser destacada como propulsora da regulação da pesquisa científica e tecnológica no Brasil foi a elaboração dos Planos Nacionais de Pós- Graduação (PNPGs) (Martins, 2003; Hostins, 2006). Existiram assim três primeiros planos (o I PNPG, de 1975 a 1979, o II PNPG, de 1982 a 1985, e o III PNPG, de 1986 a 1989) que orientaram o setor para o alcance de alguns resultados como: integração da pós-graduação nas universidades, institucionalização da pesquisa, aumento da capacitação e profissionalização da carreira docente, instituição do sistema de bolsas de pesquisa, política de apoio financeiro e implantação de um sistema nacional de avaliação para os programas (BRASIL, 1975; BRASIL, 1982; BRASIL, 1986). Em 1996, uma comissão foi constituída pela CAPES para definir o IV PNPG, que não foi colocado em prática à época (Hostins, 2006). Este último, apesar de não ter sido efetivamente implantado, existiu e serviu de referência para algumas ações da CAPES. Mesmo assim, Velloso (2004) destaca o notável crescimento da pós-graduação nos anos 1990. Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. 3 A PÓS-GRADUAÇÃO E SEU CONTEXTO HISTÓRICO O contexto histórico da formação e desenvolvimento da pós-graduação no ensino superior brasileiro foi marcado pela constituição do Sistema Nacional de Pós-Graduação. Destaque-se o Estado, os organismos representativos da comunidade científica e o corpo docente das instituições de ensino e pesquisa (existente em escolas isoladas) como os principais atores envolvidos nesse processo, principalmente no intuito de associar o ensino superior no país à atividade de pesquisa (Martins, 2003). Ressalte-se também nesse cenário, em 1951, a criação do Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Esta com o fim de fomentar as atividades do sistema de pós- graduação, por meio da especialização de pessoas que pudessem promover o desenvolvimento econômico e social do país, representando ainda hoje a principal agência nacional neste sentido. O surgimento da CAPES propiciou também a elaboração dos Planos Nacionais de Pós-Graduação (PNPG) para o norteamento desse nível de formação no Brasil (Martins, 2003). A CAPES, o CNPq e outras agências internacionais de fomento, em meados dos anos 1950 e 1960, encabeçaram um conjunto de iniciativas que possibilitou a ida de vários estudantes brasileiros ao exterior para realizarem uma pós-graduação, tendo o retorno desses pesquisadores propiciado a formação de uma massa crítica nacional que influenciou na implantação da pós- graduação no Brasil (Martins, 2003). Este foi considerado um período de expansão e legitimidade, mesmo diante da repressão e do controle militar imposto pelo governo da época (Hostins, 2006). Desta forma, os anos 1960, apesar da estagnação experimentada quanto ao ensino superior, representaram o marco da pós- graduação, quando foram criados os primeiros cursos desse nível no Brasil, tendo seu surgimento atrelado à situação política e social da época. Quanto às questões normativas, a vigente Lei de Diretrizes e Bases da Educação Nacional (Lei nº 9.394, de 20 de dezembro de 1996) aponta como incumbência da União definir as normas gerais sobre cursos de graduação e de pós-graduação. 5 HOLOS, Ano 38, v.2, e7655, 2022 5 Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. DELFINO & SOUSA (2022) por meio do conceito de nucleação, revisão Qualis e criação do Programa de Financiamento às Exportações (PROEX), buscou também ampliar a cooperação internacional e combater as assimetrias entre os programas, além de enfatizar a formação docente para todos os níveis de ensino, e a de técnicos, via mestrados profissionais (Brasil, 2004). por meio do conceito de nucleação, revisão Qualis e criação do Programa de Financiamento às Exportações (PROEX), buscou também ampliar a cooperação internacional e combater as assimetrias entre os programas, além de enfatizar a formação docente para todos os níveis de ensino, e a de técnicos, via mestrados profissionais (Brasil, 2004). O PNPG 2011-2020, por sua vez, foi elaborado idealizando a integração entre universidade, Estado e empresas, introduzindo uma visão sistêmica, e buscando instituir uma “Agenda Nacional de Pesquisa”, onde estão inseridos os temas considerados estratégicos à pesquisa no cenário nacional, sendo eles: água, energia, transporte, controle de fronteiras, agronegócio, Amazônia, mar (Amazônia Azul), saúde, defesa, justiça, segurança pública e criminologia, programa espacial, e desequilíbrio regional (Brasil, 2010, p. 18). Desafios como a internacionalização e cooperação internacional da pós-graduação, seu financiamento, a indução de programas pelas agências de fomento para o desenvolvimento de áreas estratégicas, parcerias institucionais, redução de assimetrias regionais, e melhoria da qualidade geral da pós-graduação são colocados como metas desse PNPG ainda em vigor, consideradas difíceis, mas não impossíveis de se alcançar. Atualmente, o sistema de pós-graduação conta não somente com o CNPq e a CAPES no fomento à pós-graduação, mas também com ações e projetos integrados a elas, como a Plataforma Sucupira (criada em 2013 para subsidiar o processo de avaliação dos programas), o Portal de Periódicos, o Sistema GeoCAPES (que desenvolve informações georeferenciadas para a pesquisa), a criação de MINTER e DINTER (mestrados e doutorados Interinstitucionais para a interiorização da pós-graduação), o Sistema WebQualis (para mensurar a qualidade das publicações provenientes dos programas), dentre outros avanços (Brasil, 2018). Como instrumentais tecnológicos existentes também no Brasil à disposição dos pesquisadores, destaque-se a Plataforma Brasil de Pesquisa (sistema eletrônico do Governo Federal de protocolo para projetos de pesquisa com seres humanos), a Plataforma Lattes (base de dados de currículos que registra a atuação de acadêmicos de todo o país) e o Diretório dos Grupos de Pesquisa do Brasil (base de dados que contêm informações sobre os grupos de pesquisa em atividade). Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. DELFINO & SOUSA (2022) No limiar do Século XXI, a reflexão sobre o seu desenvolvimento no Brasil e suas perspectivas, a importância da qualificação docente para a pesquisa, a interiorização e indução de demandas em áreas consideradas estratégicas, seu financiamento e continuidade, foram destacados por Martins (2003) como atitudes necessárias ao contexto da época visando a maiores avanços. A identificação dessa necessidade pelo Estado levou à formatação do PNPG: 2005-2010, com objetivos voltados à expansão do sistema de pós-graduação, visando maior qualificação do sistema de ensino superior, do sistema de ciência e tecnologia e do setor empresarial (revelando uma lógica de adequação ao sistema capitalista). Este Plano aprimorou o processo de avaliação, 6 HOLOS, Ano 38, v.2, e7655, 2022 6 Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. Assim, percebe-se que a pós-graduação no Brasil, apesar de tardia, representou o limiar de uma universidade onde as atividades de ensino e pesquisa fossem interligadas. De modo que, quando se faz um levantamento quantitativo do número de instituições, cursos e alunos que compõem o Sistema Nacional de Pós-Graduação para efeitos de comparação, percebe-se um aumento considerável desde o seu surgimento. A Tabela 1 apresenta a evolução deste cenário da década de 1960 até o ano de 2017. HOLOS, Ano 38, v.2, e7655, 2022 7 Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. Tabela 1: Evolução da pós-graduação no Brasil Ano Instituições Programas de Mestrado Programas de Doutorado 1960 100 45 17 2002 1.637 1.589 917 2017 2.407 4.101 2.182 Tabela 1: Evolução da pós-graduação no Brasil Ano Instituições Programas de Mestrado Programas de Doutorado 1960 100 45 17 2002 1.637 1.589 917 2017 2.407 4.101 2.182 7 DELFINO & SOUSA (2022) Fonte: Martins, 2003; Brasil, 2018. Fonte: Martins, 2003; Brasil, 2018. Percebe-se o aumento considerável no número de instituições de ensino superior e dos programas de pós-graduação durante todo o período observado, desde a sua criação até os dias atuais. Em estudo aprofundado que analisou dados de cerca de 3,5 mil alunos que concluíram uma pós-graduação, Velloso (2004) identificou que, no Brasil, o tempo decorrido entre a conclusão da graduação e do doutorado, gira em torno de 11 anos, sendo maior que em muitos outros países, e ainda, que essa formação recebida na pós-graduação atende bem às necessidades dos que se voltam às profissões acadêmicas e menos aos que se dedicam a outros campos de atuação. A questão dos mestrados profissionais implantados no Brasil no final do Século XX, apesar de muito combatidos no início do Século XXI, voltam seu foco à prática prifissional, com a conversão, em menor espaço de tempo, dos conhecimentos gerados na universidade para a sociedade (Gatti, 2001; Hostins, 2006). Quando se fala de pós-graduação strictu sensu acadêmica, percebe-se sua demanda basicamente por profissionais engajados ou interessados na carreira docente, com a exigência de mais tempo para conclusão, e direcionamento para a pesquisa acadêmica. Nessa perspectiva, Martins (2003) considera a pós-graduação como o elemento estrutural dentro do Sistema Educacional do país com resultados mais satisfatórios. Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. 4 A PÓS-GRADUAÇÃO E A CARREIRA DOCENTE O surgimento do tema carreira advém da sociedade industrial capitalista liberal, que trazia ideais de igualdade, liberdade para a busca do êxito individual, e progresso econômico e social, com a finalidade de incentivar o indivíduo à construção da sua própria carreira (Chanlat, 1995). Estudos sobre o tema desenvolveram-se consideravelmente ao longo da década de 70, observando-se sua quase ausência na década anterior e abrangendo uma abordagem psicológica e sociológica ao mesmo tempo (Huberman, 2000). No decorrer das últimas décadas, o tema enfrentou mudanças relacionadas às suas principais concepções e atualmente, tem assumido dois direcionamentos. Um deles foca o papel do indivíduo, que se torna responsável pela gestão do seu próprio desenvolvimento profissional. Enquanto o outro evidencia a organização, que assume a responsabilidade de oferecer suporte e condições para o desenvolvimento mútuo de expectativas com relação à carreira dos indivíduos (Dutra, 2007). O planejamento de carreira significa, portanto, a sequência de posições e atividades desenvolvidas por uma pessoa ao longo do tempo em uma organização (Chanlat, 1995). Tradicionalmente, as organizações elaboravam um plano de carreiras para preparar os indivíduos no sentido de galgarem cargos gradativamente superiores. HOLOS Ano 38 v 2 e7655 2022 8 Quanto a carreira docente, em meados da década de 60, quando da criação das primeiras pós-graduações no Brasil, os poucos cursos que concediam a concessão da titulação acadêmica de doutor, representavam mais prestígio que recompensas materiais e financeiras ao professor, visto 8 HOLOS, Ano 38, v.2, e7655, 2022 8 Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. DELFINO & SOUSA (2022) que a carreira docente ainda não era institucionalizada. A exceção, à época, era a USP, na qual o doutorado foi incorporado à carreira (Martins, 2003). que a carreira docente ainda não era institucionalizada. A exceção, à época, era a USP, na qual o doutorado foi incorporado à carreira (Martins, 2003). A institucionalização da carreira docente foi outra luta enfrentada pelos movimentos da comunidade científica quando da criação da pós-graduação no Brasil e foi regulamentada em decorrência da publicação da Lei 5.540/68 (Lei nº 5.540, de 28 de novembro de 1968) como marco da reforma universitária, normatizando a organização e o funcionamento do ensino superior no país. Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. 4 A PÓS-GRADUAÇÃO E A CARREIRA DOCENTE Apesar de a reforma universitária ter sido idealizada pelo governo militar, de modo autoritário e antidemocrático, provocando profundas mudanças sob forte pressão política, ela desencadeou inovações importantes para o ensino superior e para a carreira docente, extinguindo as cátedras, instituindo uma carreira aberta e fundamentada no mérito acadêmico, criando os colegiados de cursos e estabelecendo como unidade mínima de ensino e pesquisa, o departamento. Ressalte-se nessa época, a influência americana na consolidação da pós-graduação e na reforma da educação superior no país (Hostins, 2006). E que a institucionalização da carreira docente aconteceu praticamente na mesma época do surgimento e do desenvolvimento do tema carreira como proposta de investigação acadêmica. Observando-se o contexto atual, a realidade dos docentes de ensino superior reflete exigências constantes à sua formação continuada, bem como a necessidade latente de administrar sua própria carreira profissional. Nascimento (2017), afirma que a pós-graduação representa um âmbito de qualificação profissional, por meio da obtenção gradativa de títulos e graus, ao discorrer sobre suas influências na formação do bacharel docente. A oferta de cursos de mestrado e doutorado profissionais, que ganharam força nos últimos anos, também direcionados à qualificação docente, são evidências quanto às exigências sistemáticas à formação continuada do docente e impactam diretamente na carreira desses profissionais. Mas a carreira docente institucionalizada não deveria abranger mais que somente títulos e graus acadêmicos, leiam-se mestrado e doutorado? Defende-se assim a necessidade da autoformação e da formação continuada, estando o tradicional planejamento de carreira a ceder lugar ao seu autogerenciamento (Rossi; Hunger, 2012; Nóvoa, 2000). Acrescente-se a isso, que a responsabilidade docente com sua autoformação também está totalmente relacionada com seu papel nas universidades. As instituições universitárias são incumbidas de uma atuação na sociedade, determinando objetivos a serem alcançados para isto. Os docentes atuantes em cada instituição têm o dever, também social como consequência, de pensar seus projetos de carreira alinhados ao papel social da instituição que integra. Cientes da inevitabilidade de uma formação continuada, que recai principalmente na pós-graduação, e dos inevitáveis direcionamentos de investigação que cada área de formação e atuação possui, os docentes precisam também atentar-se a necessidade de sua atuação institucional quanto às 9 9 HOLOS, Ano 38, v.2, e7655, 2022 Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. DELFINO & SOUSA (2022) especificidades regionais. Assim, o autodesenvolvimento para gestão de suas próprias carreiras deve ser uma busca constante dos docentes de ensino superior, sendo esse caminho trilhado por meio da pós- graduação, que deve estar alinhada aos objetivos de atuação social da universidade. 5 ASPECTOS CONCLUSIVOS A perspectiva histórica do sistema de pós-graduação no Brasil, o resgate aos seus marcos iniciais, seu desenvolvimento, seu contexto atual e suas perspectivas de continuidade e ampliação, além do seu papel na gestão da carreira docente institucionalizada foram alvos deste estudo. Destaca-se o processo evolutivo da pós-graduação, em parte alavancado por políticas educacionais provenientes do Estado (para cumprir projeto de industrialização do país), e em parte por movimentos encabeçados principalmente pela comunidade científica da época. O país conseguiu criar e alavancar a pós-graduação, mas precisa continuar avançando em termos tanto quantitativos quanto qualitativos, pois necessidades como a flexibilização do modelo, o aperfeiçoamento do sistema de avaliação e a ênfase na internacionalização ainda persistem. Considera-se que a universidade recebe a incumbência de formar cidadãos críticos, mas que lhe chegam despreparados. Assim, existe por parte dela a dificuldade de atingir os próprios objetivos, tendo que suprir as lacunas geradas pelas desigualdades provenientes da precariedade do nível anterior de escolarização. Os docentes universitários, conscientes da necessidade de sua formação continuada, representada principalmente pela pós-graduação, devem direcionar a construção dos seus projetos de carreira às necessidades sociais às quais sua instituição de origem almeja atender, respeitadas as peculiaridades regionais e os temas relevantes na sua área de formação e atuação. Convictos também da importância de direcionar-se cientificamente às demandas identificadas por uma vigente Agenda Nacional de Pesquisa. Percebe-se que, apesar desse contexto de dificuldades historicamente enfrentadas pelas universidades na graduação, a pós-graduação no Brasil constitui um projeto em desenvolvimento, apesar dos desafios que ainda enfrenta em termos de financiamento, assimetrias e descompassos, e representa hoje a parcela mais estruturada e desenvolvida do complexo Sistema de Educação Superior no Brasil. Mas que enxerga em seu horizonte histórico uma forte ameaça por parte de incentivos do Estado, sobretudo no que diz respeito ao seu financiamento. Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. 6 REFERÊNCIAS Almeida Júnior, A., Sucupira, Newton, Salgado, Clóvis, Barreto Filho, José, Silva, Maurício Rocha e, Trigueiro, Durmeval, Lima, Alceu Amoroso, Teixeira, Anísio, & Maciel, Valnir Chagas e Rubens. (2005). Parecer CFE nº 977/65, aprovado em 3 dez. 1965. Revista Brasileira de Educação, (30), 162-173. Recuperado de http://educa.fcc.org.br/scielo.php?script=sci_arttext&pid=S1413- 24782005000300014&lng=pt&tlng=pt. 10 HOLOS, Ano 38, v.2, e7655, 2022 HOLOS, Ano 38, v.2, e7655, 2022 Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. DELFINO & SOUSA (2022) Alves, M. F., & Oliveira, J. F. de. (2015). Pós-Graduação no Brasil: do Regime Militar aos dias atuais. Revista Brasileira De Política E Administração Da Educação, 30(2). https://doi.org/10.21573/vol30n22014.53680 Barros, A. S. X. (2015). Expansão da educação superior no Brasil: limites e possibilidades. Educ. Soc. Campinas, 36(131), 361-390. Recuperado de http://www.scielo.br/pdf/es/v36n131/1678- 4626-es-36-131-00361.pdf Lei n. 5.540, de 28 de novembro de 1968. Fixa normas de organização e funcionamento do ensino superior e sua articulação com a escola média, e dá outras providências. Recuperado de http://www.planalto.gov.br/ Lei n. 9.394, de 20 de dezembro de 1996. Estabelece as diretrizes e bases da educação nacional. Recuperado de http://www.planalto.gov.br/CCIVIL_03/Leis/L9394.htm Brasil. (2018). Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Ações e Programas. Recuperado de: http://www.capes.gov.br/acessoainformacao/acoes-e-programas Brasil. (1079). Ministério da Educação e Cultura, Secretaria de Educação Superior/Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. I Plano Nacional de Pós-Graduação: 1975- 1979. Brasília: MEC/SESU/CAPES. Brasil. (1982). Ministério da Educação e Cultura, Secretaria de Educação Superior/Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. II Plano Nacional de Pós-Graduação: 1982- 1985. Brasília: MEC/SESU/CAPES. Brasil. (1986). Ministério da Educação e Cultura, Secretaria de Educação Superior/Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. III Plano Nacional de Pós-Graduação: 1986- 1989. Brasília: MEC/SESU/CAPES. Brasil. (2004). Ministério da Educação e Cultura, Secretaria de Educação Superior/Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Plano Nacional de Pós-Graduação: 2005-2010. Brasília: MEC/SESU/CAPES. Brasil. (2010). Ministério da Educação e Cultura, Secretaria de Educação Superior/Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Plano Nacional de Pós-Graduação: 2011-2020. v. I, Brasília: MEC/SESU/CAPES. Chanlat, J. F. (1995). Quais Carreiras e para qual sociedade (I). RAE-Revista de Administração de Empresas, 35(6), 67-75. Dutra, J. S. (2007). Administração de Carreiras: uma proposta para repensar a gestão de pessoas. São Paulo: Atlas. Gatti, B. A. (2001). Reflexão sobre os desafios da pós-graduação: novas perspectivas sociais, conhecimento e poder. Revista Brasileira de Educação, 18, 108-116. Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. DELFINO & SOUSA (2022) HOLOS, Ano 38, v.2, e7655, 2022 13 COMO CITAR ESTE ARTIGO: Delfino, I. A. de L., Sousa, I. S. de. (2022). A pós-graduação como referência histórica à carreira e à sociedade. HOLOS, 2, 1-13. Recuperado de https://www2.ifrn.edu.br/ojs/index.php/HOLOS/article/view/7655. SOBRE OS AUTORES I. A. DE L. DELFINO Professora do Centro de Ciências Jurídicas e Sociais da UFCG - Doutoranda em Administração pela USCS. Mestre em Administração pela UFPB. E-mail: islania.lira@ufcg.edu.br ORCID ID: https://orcid.org/0000-0001-5143-8721 I. S. DE SOUSA Professor do Centro de Formação de Professores da UFCG - Doutor em Educação pela UFPB. E-mail: israelhistoria@gmail.com ORCID ID: http://orcid.org/0000-0002-9748-3699 Editor(a) Responsável: Francinaide de Lima Silva Nascimento Pareceristas Ad Hoc: Ana Lúcia Sarmento Henrique e Lenina Lopes Soares Silva Recebido 22 de agosto de 2018 Aceito: 20 de julho de 2020 Publicado: 11 de julho de 2022 DELFINO & SOUSA (2022) COMO CITAR ESTE ARTIGO: Delfino, I. A. de L., Sousa, I. S. de. (2022). A pós-graduação como referência histórica à carreira e à sociedade. HOLOS, 2, 1-13. Recuperado de https://www2.ifrn.edu.br/ojs/index.php/HOLOS/article/view/7655. HOLOS, Ano 38, v.2, e7655, 2022 Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. 6 REFERÊNCIAS 11 HOLOS, Ano 38, v.2, e7655, 2022 Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. DELFINO & SOUSA (2022) Hostins, R. C. L. (2006). Os Planos Nacionais de Pós-graduação (PNPG) e suas repercussões na Pós- graduação brasileira. Perspectiva, 24(1), 133-160. Huberman, M. (2000). O ciclo de vida proﬁssional dos professores. In: NÓVOA, A. (Org.). Vidas de professores. (2 ed), Porto: Porto, pp. 31-61. Martins, A. C. P. (2002). Ensino superior no Brasil: da descoberta aos dias atuais. Acta Cirúrgica Brasileira, 17(3), 04-06. Recuperado de: Martins, A. C. P. (2002). Ensino superior no Brasil: da descoberta aos dias atuais. Acta Cirúrgica Brasileira, 17(3), 04-06. Recuperado de: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S010286502002000900001&lng=en& nrm=iso Martins, C. B. (2000). O ensino superior brasileiro nos anos 90. São Paulo em Perspectiva, 14(1), 41-60. Recuperado de: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0102- 88392000000100006&lng=en&nrm=iso Martins, C. B. (2003). Pós-graduação no contexto do ensino superior brasileiro. In: MORHY, L. (Org.). Universidade em questão. Brasília: Editora Universidade de Brasília, 1, 175-203. Mendes, A. D. (2003). A propósito de amebas, ornitorrincos & humanos, ou a difícil arte de construir currículos. In: MORHY, L. (Org.). Universidade em questão. Brasília: Editora Universidade de Brasília, v. 1, p. 139-142. Nascimento, V. S. O. (2017). O bacharel e a docência: as influências da pós-graduação na carreira profissional. HOLOS, 2, 280-289. Recuperado de: http://www2.ifrn.edu.br/ojs/index.php/HOLOS/article/view/5738 Nóvoa, A. (2000). Os professores e as histórias de sua vida. In: (Org.). Vidas de professores. (2 ed). Porto: Porto, pp. 11-30. Rossi, F. & Hunger, D. (2012). As etapas da carreira docente e o processo de formação continuada de professores de Educação Física. Rev. Bras. Educ. Fís. Esporte, 26(2), 323-338. Recuperado de: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S180755092012000200014&lng=en& nrm=iso Saviani, D. (2008). Política educacional brasileira: limites e perspectivas. Revista de Educação PUC- Campinasm, (24), 7-16. Recuperado de: http://www.faiarapos.com.br/Material/21_10_17_Servico_Social_Politicas_Puplicas/Dermeva lSA VIANI2.pdf Velloso, J. (2004). A pós-graduação no Brasil: formação e trabalho de mestres e doutores no país. Caderno de Pesquisas, São Paulo, 34(122). Recuperado de: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-15742004000200012 HOLOS, Ano 38, v.2, e7655, 2022 12 Este é um artigo publicado em acesso aberto sob uma licença Creative Commons. I. S. DE SOUSA Recebido 22 de agosto de 2018 Aceito: 20 de julho de 2020 Publicado: 11 de julho de 2022 HOLOS, Ano 38, v.2, e7655, 2022 13
https://openalex.org/W3102013076	http://oro.open.ac.uk/74308/1/oaj_issue9_charnley_final-1.pdf	English	null	Art, design and modernity: the Bauhaus and beyond	Open arts journal	2,020	cc-by-sa	9,479	Open Research Online https://oro.open.ac.uk/74308/ License (CC-BY-NC 4.0) Creative Commons: Attribution-Noncommercial 4.0 https://creativecommons.org/licenses/by-nc/4.0/ Policy This document has been downloaded from Open Research Online, The Open University's repository of research publications. This version is being made available in accordance with Open Research Online policies available from Open Research Online (ORO) Policies Versions If this document is identified as the Author Accepted Manuscript it is the version after peer review but before type setting, copy editing or publisher branding This document has been downloaded from Open Research Online, The Open University's repository of research publications. This version is being made available in accordance with Open Research Online policies available from Open Research Online (ORO) Policies Versions Citation Charnley, Kim (2020). Art, design and modernity: the Bauhaus and beyond. Open Arts Journal, 9 pp. 43–56. Versions If this document is identified as the Author Accepted Manuscript it is the version after peer review but before type setting, copy editing or publisher branding 43 43 43 Abstract This essay explores the relationship between art and design in the twentieth century through the Bauhaus, the school which established a revolutionary model for modern art and design education between 1919 and 1933. The Bauhaus vision of design is closely identified with a ‘machine aesthetic’, where the form of an object is governed by its function and adapted to the demands of mass production. The pedagogy of the school, which involved a distinctive and unstable synthesis of art, craft, and design, was inspired by the Gesamtkunstwerk, an idea that was influential among avant-gardes of the early twentieth century, which is usually translated as a synthesis of the arts. This essay explores the utopianism of the Bauhaus, and its relationship to the Gesamtkunstwerk, through a comparison between the ideas of two artist-designers associated with the school: László Moholy-Nagy (1895-1946) and Anni Albers (1899-1994). Although the ‘machine aesthetic’ of industrial design shaped the reception of the Bauhaus, Albers’s work as a weaver, textile artist and textile designer ought to be given equal prominence in evaluation of the school’s design ethos. Once it is, established criticisms of the utopianism of the Bauhaus are called into question, because they take their cue from a narrow and selective account of the activities of the school. This essay concludes by sketching some implications of this shift of perspective for contemporary design. Keywords: Bauhaus, modernist design, Gesamtkunstwerk, utopianism, Anni Albers, László Moholy-Nagy, Jean Baudrillard, Hal Foster, Manfredo Tafuri Full text: https://openartsjournal.org/issue-9/article-4 DOI: http://dx.doi.org/10.5456/issn.2050-3679/2020w04 Keywords: Bauhaus, modernist design, Gesamtkunstwerk, utopianism, Anni Albers, László Moholy-Nagy, Jean Baudrillard, Hal Foster, Manfredo Tafuri Full text: https://openartsjournal.org/issue-9/article-4 DOI: http://dx.doi.org/10.5456/issn.2050-3679/2020w04 Kim Charnley, The Open University Kim Charnley, The Open University This essay uses a direct comparison between László Moholy-Nagy (1895-1946), a ‘master of form’ at the Bauhaus, and Anni Albers (1899-1994), who studied and also taught at the school, to emphasise that a diversity of interpretations of the Gesamtkunstwerk existed at the Bauhaus. This point is significant because neglect of female artists and designers in the critical reception of the school has been rectified only comparatively recently (Müller, 2015; Smith, 2014; Otto & Rössler, 2019). The renewed attention to the achievement of Albers, which was celebrated in an exhibition at Tate Modern in 2019, the centenary of the school’s founding, provides an opportunity to reassess the social utopianism of the Bauhaus. Both Albers and Moholy-Nagy were artist-designers who took a keen interest in new technological developments, though the emphases of their work are entirely different. In particular, Albers’s design philosophy, when compared to Moholy-Nagy’s, illustrates the unstable relationship between art, craft and design at the Bauhaus. This comparison will try to show that an emphasis on the machine aesthetic in critical reception of the Bauhaus tends to overshadow the plural approaches to design that existed in the school. The work of a figure like Albers allows a fresh insight into the achievements and the failings of the Bauhaus as a utopian project. It would be impossible to treat the relationship between art and design in the twentieth century without touching upon the achievement of the Staatliches Bauhaus, better known simply as the Bauhaus (German: ‘Building House’). Opened in Weimar in 1919 and closed in 1933, this school established a powerful legend despite its brief period of activity. Indeed, the Bauhaus has a dual legacy: it was a laboratory for the artistic avant-garde, but it is also seen as the birthplace of modernist design. In this essay, the connection between art and design is considered in relationship to what is generally understood to be the utopianism of the Bauhaus. The modern movement promoted the idea that the arts, design and architecture might catalyse progressive social change. Although this ambition was shaped by diverse intellectual and political influences, one important reference point was the idea of the Gesamtkunstwerk, the ‘total work of art’. This concept, though it plays a key role in the theory and practice of modernist avant-gardes, is notoriously difficult to define. Kim Charnley, The Open University In outline, it suggests both the blurring of boundaries between art and life and the synthesis of different arts into a unified style or collective project. The precise term was first used by Richard Wagner in the middle of the nineteenth century, though it conveys an enthusiasm for cultural renewal that emerged along with Romanticism in the early nineteenth century (Roberts, 2011). As Lutz Koepnick puts it: The Bauhaus: between art and design The Museum of Modern Art played a key role in forming the reputation of the Bauhaus by identifying the formation of the modernist ‘machine aesthetic’ with the school. According to this narrative, which emerged in the 1930s, the Bauhaus developed a purist design language based on the principle that ‘form follows function’. Thus, the design ethos of the Bauhaus is usually identified with products like Marcel Breuer’s chair ‘B3’, also known as ‘The Wassily’ because a prototype was owned by Wassily Kandinsky (Fig.1). In its use of tubular steel, its abstraction from and simplification of the form of an armchair, this object exemplifies a certain ideal of rational design. Clearly, teachers and students at the Bauhaus were also influential in fields including architecture, industrial design, typography, exhibition design, theatrical production, abstract painting and photography. Art and The dream of the Gesamtkunstwerk … figured as a decisive switchboard of various modernist agendas and self-definitions. It illuminates how modernism, by negotiating the dialectics of art and technology, of the aesthetic and the political, of high art and modern mass culture, aspired to couple artistic experimentation to social reform and to reshape the present in the name of a different future. 44 ART, DESIGN AND MODERNITY: THE BAUHAUS AND BEYOND Ki Ch l Th O U 44 indeed, its megalomania and proximity to totalitarian ideology (Tafuri, 1976; Baudrillard, 1981; Foster, 2002; Roberts, 2011; Tonkinwise, 2014). It is very clear that contemporary design is indebted to the Bauhaus, though this is a mixed accolade in so far as design is ‘a cultural phenomenon … linked to consumption’, given that rampant consumerism represents one of the key contributors to climate crisis (Sparke, 2020, p.4). Biographical note Kim Charnley is Staff Tutor at The Open University. His research specialism is contemporary art with a focus on ‘post-object’, socially engaged art such as ‘social practice’, art activism and institutional critique. He is also interested in the intersection between art, design and craft and, especially, the way that avant-gardes have at different times conceived of themselves as collectives. He has published in journals including Art and the Public Sphere, Art Journal, Historical Materialism and The Large Glass and contributed an introduction to Delirium and Resistance: Activist Art and the Crisis of Capitalism, a collection of essays by the artist and theorist Gregory Sholette (Pluto, 2017). A monograph exploring the role of the collective in contemporary art’s politics, titled Sociopolitical Aesthetics: Art, Crisis, Neoliberalism, will be published by Bloomsbury in early 2021. OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 www.openartsjournal.org ISSN 2050-3679 OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 ISSN 2050-3679 www.openartsjournal.org (Koepnick, 2016, p.274) The nature of the ‘future’ that the Bauhaus created has been contentious, however. The aspiration toward total design has been criticised for its elitism, its complicity with consumerism and, OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 ISSN 2050-3679 www.openartsjournal.org 45 design existed at the school in a fluid inter-relationship. Among the teachers, known as ‘masters of form’, were the artists Paul Klee (1879-1940), Wassily Kandinsky (1866-1944), László Moholy-Nagy, Johannes Itten (1888-1967) and Oskar Schlemmer (1888-1943). The most famous students of the Bauhaus often went on to teach at the institution and many of them have dual reputations both as designers and artists, reflecting the border-crossing between art and design that Bauhaus pedagogy encouraged. This latter group includes Anni Albers and Josef Albers (1888-1976), Gunta Stölzl (1897-1983), Marianne Brandt (1893-1983) and Marcel Breuer (1902-1981), among others. a program, a method, an institute, and/or a building’ (Moholy, 2020, p.128). These multiple identities were undoubtedly related to the socially transformative utopianism of the Bauhaus. The founder, the architect Walter Gropius (1883–1969), intended the school to unify and renew the arts, which would serve a new architecture and enable new forms of social life. This Gesamtkunstwerk ideal shaped the structure of the institution and the trajectory of its development. There were three directors of the Bauhaus, all of them architects: Walter Gropius was director until 1928; Hannes Meyer (1889–1954) held the directorship between 1928 and 1930 and Ludwig Mies van der Rohe (1886–1969) led the school for its last three years. The Bauhaus moved twice during its relatively short existence; founded in Weimar, it relocated to Dessau in 1926 and then to Berlin in 1932. These changes of location evidence a constant struggle with sceptical and conservative authorities. A laboratory of avant- garde ideas, the Bauhaus existed precariously during a period of political turbulence, coinciding with the Weimar republic and culminating in the rise of Nazism. Even though the Bauhaus holds such an important position in the canon of modernism, and it has been intensively studied over the best part of a century, it remains enigmatic. In its short period of existence, it seemed to bring together contradictory tendencies and hold them in a dynamic equilibrium. Lucia Moholy, whose photographs of staff and students played a key role in shaping the school’s reception, observed in 1971 that ‘even to the initiated, it could be an idea, Figure 4.1. ‘Wassily’ chair, also known as the Model B3 designed by Marcel Breuer in 1924-25 at the Bauhaus Dessau, Germany. (Image credit: originally posted to Flickr by Lorkan / Creative Commons Attribution 2.0 Generic) Figure 4.1. (Gropius, [1919] 1938, p.18) (Gropius, [1919] 1938, p.18) (Gropius, [1919] 1938, p.18) Gropius refers to the school as a ‘new guild of craftsmen’ in the next line. Yet, the actual organisation of Bauhaus indicates that it was not envisaged simply as a project of craft revivalism. Although each workshop was assigned a technical specialist, called a ‘master of craft’, authority resided in the hands of the ‘master of form’ who oversaw the workshops and were involved in decision-making processes for the school (Wick, 2000, p.36). The ‘masters of form’ were avant-garde artists as already noted. The institutional structure of the school was calculated, therefore, to assimilate avant-garde perspectives into its pedagogic system, while also equipping students with applied skills. Frankly utopian ideals were combined with the pragmatic aims of vocational education. This combination allowed Gropius latitude to pursue a radical agenda, while also representing the avant-garde school as a renewal of tradition, when this kind of argument was necessary to ensure financial support from conservative state authorities (Wick, 2000, p.56). This early conception of the Bauhaus would evolve very quickly. In 1923, after increased contact with Russian constructivism the slogan of the Bauhaus became ‘Art and Industry: a new unity’. The workshops were reorganised to emphasise engagement with mass production especially after the move to Dessau in 1926, where Gropius designed a new building to house the institution. Increasingly, teaching became explicitly oriented toward functionalist design principles (Wick, 2000, p.70). Under Hannes Meyer and Ludwig Mies van der Rohe the primacy of a technical education in design was re-enforced still further. Even so, pedagogical innovations from the early expressionist-influenced phase of the Bauhaus remained important throughout the school’s existence. This excerpt shows something of the intellectual ferment that affected the avant-garde in the aftermath of the First World War. Its imagery is usually said to reflect the utopian ideas of the architect Bruno Taut (1880–1938), who was a key innovator in glass construction, which would become a signature of the International style in architecture. It also makes reference to the gothic ideal as a model for an aesthetic community, inherited from John Ruskin and William Morris: the proclamation was illustrated with a woodcut of a crystal cathedral by Lyonel Feininger (1871-1956). OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 ‘Wassily’ chair, also known as the Model B3 designed by Marcel Breuer in 1924-25 at the Bau (Image credit: originally posted to Flickr by Lorkan / Creative Commons Attribution 2.0 Generic) Figure 4.1. ‘Wassily’ chair, also known as the Model B3 designed by Marcel Breuer in 1924-25 at the Bauhaus Dessau, Germany. (Image credit: originally posted to Flickr by Lorkan / Creative Commons Attribution 2.0 Generic) OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 ISSN 2050-3679 OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 www.openartsjournal.org 46 may cause his work to blossom into art. But proficiency in his craft is essential to every artist. Therein lies a source of creative imagination. It maintained throughout a tenacious commitment to a utilitarian project: that art should contribute to socially useful ends. As a pedagogic institution, the Bauhaus drew upon the tradition of progressive education that stressed teaching through practice. It also inherited the ambitions of the design reform movement in Germany, where schools of art and craft had been founded, drawing on the antecedent example of British art education, with the ambition of renewing the arts through the teaching of handicrafts. The Bauhaus was created from two pre-existing institutions, the Weimar Hochschule fur bildende Kunst (Academy of Art) and the Kunstgewerbeschule (School of Applied Arts). Gropius considered previous attempts to achieve a synthesis of art and craft to have been pedagogic failures because of their relationship to entrenched academic tradition. The Bauhaus was a radical departure in that Gropius was determined to engage with the avant-garde, but it was rooted in ideas that were part of the design reform movement. In the 1919 ‘First Proclamation of the Weimar Bauhaus’ Gropius writes: ‘the new building of the future … will embrace architecture and sculpture and painting in one unity and … rise one day toward heaven from the hands of a million workers like the crystal symbol of a new faith’ (Gropius, [1919] 1938, p.18). (Gropius, [1919] 1938, p.18) It is necessary to address the preliminary course here because it represents the ideals of the Bauhaus very clearly. It was a compulsory period of study, originally of six months, undertaken by all students who entered the Bauhaus before they were permitted to choose a workshop in which to specialise. Yet, Itten’s views on art were a long way from the rationalist and functionalist beliefs that are conventionally thought to have shaped modernist design. As a result, the purpose of the preliminary course went beyond technical instruction: its emphasis, but preserved its essential pedagogic goal, which was the development of the creative individual. One of the emblems of our time is abstraction. It functions, on the one hand, to disconnect components from an existing and persisting whole, either to lead them individually ad absurdum or to elevate them to their highest potential. On the other hand, abstraction can result in generalization and summation, in the construction in bold outline of a new totality. The Gesamtkunstwerk meant not only creating a new unity of the arts, but also breaking up the prevailing beliefs about art, and it was abstraction that made this possible. It will be useful here to say something about drawing at the Bauhaus in order to clarify the implications of this point. As we have seen in Emma Barker’s essay, in seventeenth-century France the change in meaning between dessein and dessin seemed to announce the emergence of a new technical role for drawing. At this point, the theoretical dimension of drawing, established in debates about design, changed its character as drawing became a practice required by nascent forms of industry. At the Bauhaus, the overall pedagogic structure indicated the primacy of utilitarian goals, but the preliminary course allowed drawing and colour studies to be explored as though autonomously, with the idea that this instruction would help students to identify their innate capacities and break free of any pre-existing stylistic assumptions. From the very beginning, my teaching was not directed toward any particular fixed, external goal. The human being itself, as a creature capable of improvement and development, seemed to me to be the task of my pedagogical efforts. Developing the senses, increasing the ability to think and experience spiritually, relaxing and developing the bodily organs and functions – these are the means and paths available to the teacher concerned about education. (Gropius, [1919] 1938, p.18) Furthermore, the idea of the crystal as a principle of multi-faceted unity can be traced back to fin-de-siècle esoteric ideas present in the Darmstadt Artists’ Colony, a utopian community founded in 1899 by Ernest Ludwig, Grand Duke of Hesse (Tafuri and Dal Co, 1976, p.84). The 1919 programme is an unstable synthesis between esotericism and arts and craft utopianism. At this stage the school was, in its ethos, a long way from a machine aesthetic: Rainer Wick’s important study Teaching at the Bauhaus is at pains to emphasise that there was no single pedagogic programme that informed the school in all its phases of activity. Wick takes the view that the complexity of the Bauhaus can only be represented by examining in parallel the different, often competing, commitments of its ‘masters of form’ (Wick, 2000, p.11). Yet, he acknowledges that the most famous and influential pedagogic innovation of the Bauhaus was the preliminary course (Vorkurs) established by Johannes Itten in the early years of the school (Wick, 2000, p.93). After Itten left in 1923, the preliminary course was led by László Moholy-Nagy and Josef Albers, who altered Architects, sculptors, painters, we must all turn to the crafts. Art is not a ‘profession’. There is no essential difference between the artist and the craftsman. The artist is an exalted craftsman. In rare moments of inspiration, moments beyond the control of his will, the grace of heaven OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 ISSN 2050-3679 ISSN 2050-3679 OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 www.openartsjournal.org 47 the different crafts represented in the workshops. As Oskar Schlemmer observed in notebooks written while he was teaching at the Bauhaus: its emphasis, but preserved its essential pedagogic goal, which was the development of the creative individual. It is necessary to address the preliminary course here because it represents the ideals of the Bauhaus very clearly. It was a compulsory period of study, originally of six months, undertaken by all students who entered the Bauhaus before they were permitted to choose a workshop in which to specialise. Yet, Itten’s views on art were a long way from the rationalist and functionalist beliefs that are conventionally thought to have shaped modernist design. As a result, the purpose of the preliminary course went beyond technical instruction: its emphasis, but preserved its essential pedagogic goal, which was the development of the creative individual. (Itten cited in Wick, 2000, p.102) Although the Bauhaus would become famous because of its purist and seemingly rationalist- functionalist approach to design, the teaching that a designer like Marcel Breuer experienced was framed by the expressionist ethos indicated in Itten’s statement. Though Bauhaus pedagogy was vocational, it was not solely technical: intellectual and manual skills were viewed as interdependent and equally important. This became an important legacy of the Bauhaus after Josef and Anni Albers later taught at Black Mountain College, where a version of the preliminary course was incorporated into a liberal arts college (Grawe, 2002). In this context, it has become famous as a conduit of ideas about assemblage to the neo-avant-garde, to Robert Rauschenberg in particular. The meaning of design, at least in the early pedagogy of the Bauhaus, was ambiguous. In The Statutes of the Staatliches Bauhaus of January 1921, ‘instruction in design’ was still associated with painting, composition and modelling, whereas ‘technical drawing’ is listed separately as ‘instruction in projection and construction drawing’ (Wick, 2000, p.67). ‘Design’ at this point was identified with elementary studies in composition in two and three dimensions. Indeed, the teaching was highly theorised in its approach to abstraction, as though in the tradition of disegno as an intellectualised artistic practice. Wassily Kandinsky and Paul Klee each led specialist courses on drawing and colour instruction in the preliminary course, each providing distinctive and idiosyncratic theories of form and colour. An important tension in the structure of the Bauhaus and its reception is evident in the afterlife of the Vorkurs. Whereas the Bauhaus saw individual artistic development as preparation for collective practical study in craft and design, the Vorkurs came to be seen as a preparation for artistic practice alone. By contrast, at the Bauhaus principles of abstraction formed a highly theorised basis for all learning, though students would thereafter be required to commit to study in workshops and to demonstrate their technical proficiency in order to graduate. Although exercises based on abstract art were preliminary, they also provided the elements of a language that could unite Though drawing instruction at the Bauhaus involved many traditional elements – including drawing from the figure, from still life and even analysis of the composition of old master paintings – its implications were always intended to reach beyond the practice of drawing itself. For example, Itten’s instruction in rhythm OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 ISSN 2050-3679 www.openartsjournal.org 48 liberal and mechanical arts. The functionalism that has become the hallmark of modernist design, however, was fashioned in an intellectual atmosphere that emphasised a holistic relationship between mind, body and spirit. Furthermore, the development of the individual was also intended to lay the groundwork for new forms of collective endeavour. The wider context in which this total vision contributed to the emergence of modern design may be addressed through a brief discussion of the pre-history of the Bauhaus. involved physical exercises, because it was deemed important that processes of drawing should be intuited physically as well as visually. Gropius himself considered it important that design should be taught as theory to provide the foundation for a collective ethos: Thus our pupils’ intellectual education proceeded hand in hand with their practical training. Instead of receiving arbitrary and subjective ideas of design they had objective tuition in the basic laws of form and colour, and the primary condition of the elements of each, which enabled them to acquire the necessary mental equipment to give tangible shape to their own creative instincts. Only those who have been taught how to grasp the comprehensive coherence of a larger design, and incorporate original work of their own as an integral part of it, are ripe for active cooperation in building. Gesamtkunstwerk and ‘total design’ It has already been noted that the Gesamtkunstwerk was a reference point for many avant-garde of the turn of the century. The Deutscher Werkbund, an association of German artists and industrialists founded in 1907, represents an important precursor to the Bauhaus in the history of design in Germany not least because of its initiation of a practice of total design. The origin of modernist design is often traced to the work of one of the founding members of this institution, the architect Peter Behrens. Behrens’s work as a consultant to the firm Allgemeine Elektricitäts-Gesellschaft (AEG) in 1907 involved the creation of an integrated identity for the corporation, including the branding, publicity material, products, factory buildings and even the factory clocks (Fig. 4.2). This early example of a fully (Gropius, 1965, p.78) (Gropius, 1965, p.78) This brief and necessarily selective outline of the preliminary course is intended to show that the Bauhaus was in one sense a culmination of the history outlined in the preceding essays. Design was taught through drawing and construction in a way that stimulated intellectual development and sensitivity. Instruction emphasised a reconciliation between OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 www.openartsjournal.org ISSN 2050-3679 Figure 4.2. Peter Behrens. Clock designed for AEG, 1908. (Image credit: Creative Commons Attribution-Share Alike 3.0 Unported / Photo: Christos Vittoratos) Figure 4.2. Peter Behrens. Clock designed for AEG, 1908. (Image credit: Creative Commons Attribution-Share Alike 3.0 Unported / Photo: Christos Vittoratos) Figure 4.2. Peter Behrens. Clock designed for AEG, 1908. (Image credit: Creative Commons Attribution-Share Alike 3.0 Unported / Photo: Christos Vittoratos) 49 as an active principle that might reshape collective experience: integrated corporate identity anticipates practices of industrial design that are now commonplace, where the consistency and integration of communication, across different platforms and media, is deemed centrally important. The ensemble that Behrens created for AEG was conceived by him in the spirit of the Gesamtkunstwerk. AEG was an electrical engineering monopoly at the leading edge of the technological reorganisation of society, producing everything from electrical turbines to lamps and electric kettles and, without strong competitors, was in a position to innovate (Fig. 4.3). The style of a time does not mean particular forms in one or another art; every form is only one of many symbols of inner life, every art only a part of style. Style, however, is the symbol of feeling in common, of the whole conception of the life of a time in its totality, and it only shows itself in the totality formed by all the arts. (Behrens cited in Rancière, 2013, p.149) Behrens employed many celebrated architects in his practice at the beginning of their careers, including Walter Gropius, Ludwig Mies van der Rohe, and Charles-Édouard Jeanneret, ‘Le Corbusier’; thus, two of the three directors of the Bauhaus gained formative experience in his firm. For our purposes, the important issue is that the Gesamtkunstwerk was a flexible ideal: it informed Itten’s pedagogic focus on the shaping of the whole individual, Gropius’s vision of the Bauhaus as a quasi-spiritual community and Behrens’s approach to industrial design, where buildings, products and publications, though they are fashioned in ways appropriate to their function, all participate in an integrated identity. Although there are very different In one sense, Behrens’ work on AEG developed principles laid down by the design reform movement. The AEG turbine factory in Berlin, for example, aimed to overcome the distinction between fine and applied art (Fig. 4.4). Behrens had no time for the Arts and Crafts movement’s hostility to the dehumanising effects of industrial work, however. As Jacques Rancière has succinctly observed: ‘Behrens and his friends of the Werkbund used Ruskin against Ruskin’ (Rancière, 2013, p.147). The reunification of the arts and crafts meant here the celebration of industry, not its rejection. The total work of art tended to invite analogies between aesthetics and social organisation, with style conceived Figure 4.3. Peter Behrens. Three versions of a water kettle designed for AEG, 1.25L, 1L and 0.75L. (Image credit: Creative Commons Attribution-Share Alike 3.0 Unported / Photo: Christos Vittoratos) Figure 4.3. Peter Behrens. Three versions of a water kettle designed for AEG, 1.25L, 1L and 0.75L. (Image credit: Creative Commons Attribution-Share Alike 3.0 Unported / Photo: Christos Vittoratos) OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 ISSN 2050-3679 OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 50 50 Figure 4.4. AEG Turbine Factory, Berlin-Moabit, Germany. Designed by Peter Behrens. Completed in 1909. (Image credit: Creative Commons Attribution-Share Alike 3.0 Unported / Photo: Doris Anthony) Figure 4.4. AEG Turbine Factory, Berlin-Moabit, Germany. Designed by Peter Behrens. Completed in 1909. (Image credit: Creative Commons Attribution-Share Alike 3.0 Unported / Photo: Doris Anthony) to totalitarian’ (Koepnick, 2016, p.274). Although totalitarian governments are usually said to have aestheticized politics, especially by making use of the propaganda power of mass spectacle, Nazi Germany and Stalinist Russia both actively supressed avant- gardes. It seems more reasonable to argue that the Gesamtkunstwerk was susceptible both to progressive and reactionary interpretations and manifestations. This question of utopianism is not confined to matters of historical interpretation, however; it is still common for progressive design theorists to disidentify with the legacy of modernism because of the perceived flaws in its utopianism. Transition design provides an important example of such a movement in design theory, one that faces head on the ‘wicked problems’ that face designers now, such as ‘climate change, loss of biodiversity, depletion of natural resources, and the widening gap between rich and poor’ (Irwin, 2015, p.229). Terry Irwin’s account of this programme advances a highly ambitious and sophisticated conception of ‘design-led transition’ to a more sustainable world. It also involves a critique of design’s engagement with consumerism, which is unsparing. stakes involved in, for example, the creation of a corporate identity and the practice of emancipatory education, they were deemed to be connected in this formative period for the modern movement through the ‘switchboard’ of the Gesamtkunstwerk, to use David Roberts’ metaphor. to totalitarian’ (Koepnick, 2016, p.274). Although totalitarian governments are usually said to have aestheticized politics, especially by making use of the propaganda power of mass spectacle, Nazi Germany and Stalinist Russia both actively supressed avant- gardes. It seems more reasonable to argue that the Gesamtkunstwerk was susceptible both to progressive and reactionary interpretations and manifestations. This question of utopianism is not confined to matters of historical interpretation, however; it is still common for progressive design theorists to disidentify with the legacy of modernism because of the perceived flaws in its utopianism. (Tonkinwise, 2014, n.p.) Is it the case that the Bauhaus initiated an approach to design that expressed this kind of insensitive instrumental rationality? Even a brief overview of Bauhaus pedagogy gives us cause to doubt that this assessment is entirely fair: as we have seen, the Bauhaus employed an enlightened approach to the relationship between intellect, practice and the body, for example. And yet, Tonkinwise is not alone in making this judgement of the Bauhaus: it is a well-established critical position. The art historian Hal Foster, in his essay ‘Design and Crime’, accuses design of being a ‘perverse reconciliation’ of the utopian ideals of modernism, reinterpreted according to ‘the spectacular dictates of the culture industry’ (Foster, 2002, p.19). The focus of his critique is the transition from the total work of art to total design, where the Gesamtkunstwerk is interpreted as a naïve prelude to the manipulative reorganisation of every aspect of human experience. Admittedly, Gropius’s good intentions may be beside the point. Baudrillard is justified in identifying design as a practice through which instrumental rational practices entered a socio-cultural sphere. The architectural theorist Manfredo Tafuri describes the Bauhaus as the ‘decantation chamber of the avant- garde’ to make a comparable point (Tafuri, 1976, p.111). Like Baudrillard, Tafuri views modernism pessimistically; in his account, the utopianism of the avant-garde merely conditions its audiences to accept more readily the anarchic forces of capitalist development. This is a more historically nuanced assessment than Baudrillard’s, benefitting from extensive research into the histories of European architectural modernism (Tafuri and Dal Co, 1976). Tafuri argues that the artists who taught at the Bauhaus unwittingly ‘fulfilled the historic task of selecting from all the contributions of the avant-garde by testing them in terms of the needs of productive reality’ (Tafuri, 1976, p.111). Foster’s argument draws on design and architectural criticism that explores the collapse of modernist utopianism into the logic of capitalist accumulation (Tafuri, 1976; Baudrillard, 1981). Jean Baudrillard’s essay ‘Design and Environment’ provides an important link between this tradition and the reception of the Bauhaus (Baudrillard, 1981; Foster, 2002, p.22). The Bauhaus, Baudrillard argues, was an instigator of a ‘revolution of the object’ (Baudrillard, 1981 p.185). The functionalism of Bauhaus design introduced a new synthesis between material production and communication. OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 Transition design provides an important example of such a movement in design theory, one that faces head on the ‘wicked problems’ that face designers now, such as ‘climate change, loss of biodiversity, depletion of natural resources, and the widening gap between rich and poor’ (Irwin, 2015, p.229). Terry Irwin’s account of this programme advances a highly ambitious and sophisticated conception of ‘design-led transition’ to a more sustainable world. It also involves a critique of design’s engagement with consumerism, which is unsparing. to totalitarian’ (Koepnick, 2016, p.274). Although totalitarian governments are usually said to have aestheticized politics, especially by making use of the propaganda power of mass spectacle, Nazi Germany and Stalinist Russia both actively supressed avant- gardes. It seems more reasonable to argue that the Gesamtkunstwerk was susceptible both to progressive and reactionary interpretations and manifestations. The critique of Bauhaus utopianism Behren’s work for AEG anticipates and perhaps helps to set a trajectory for industrial design in the twentieth century, as the discipline becomes aligned with advertising, branding and public relations. It is perhaps for this reason that the utopianism of the modern movement has since become a lightning rod for critique of modernist design. There exists, first of all, an argument that the ideology of the ‘total artwork’ was complicit with dangerous political developments. David Roberts argues that the ‘total artwork’ has an affinity with totalitarianism, noting that the Gesamtkunstwerk achieved ‘perverted realization’ in Nazism, Fascism and Stalinism (Roberts, 2011, p.2) Koepnick, by contrast, cautions against the ‘rash answers’ that often result when arguments are based on a ‘slippage from total This question of utopianism is not confined to matters of historical interpretation, however; it is still common for progressive design theorists to disidentify with the legacy of modernism because of the perceived flaws in its utopianism. Transition design provides an important example of such a movement in design theory, one that faces head on the ‘wicked problems’ that face designers now, such as ‘climate change, loss of biodiversity, depletion of natural resources, and the widening gap between rich and poor’ (Irwin, 2015, p.229). Terry Irwin’s account of this programme advances a highly ambitious and sophisticated conception of ‘design-led transition’ to a more sustainable world. It also involves a critique of design’s engagement with consumerism, which is unsparing. OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 ISSN 20 OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 www.openartsjournal.org 51 Cameron Tonkinwise, another key theorist of Transition design, includes utopianism under what he terms design’s ‘disorders’, identifying it with ‘megalomania’: elements and the transparency of the process: the famous absolute legibility of signs and messages – the common ideal of all manipulators of codes, whether they be cyberneticians or designers. elements and the transparency of the process: the famous absolute legibility of signs and messages – the common ideal of all manipulators of codes, whether they be cyberneticians or designers. Both the European origin story [of design], centered around the Bauhaus, and the North American version, as expounded by the Streamliners, argued that modern styles of art derived from new machine forms and materials, when applied to everyday products and environments, could de-traditionalize people, accelerating them into more universal, efficient and rational ways of living. For this reason, everything should be (re)designed: total design. (Baudrillard, 1981, p.188) Baudrillard’s argument is perceptive in its identification of the tendency for designed objects to form communicative environments. Clearly, designers associated with the Bauhaus helped to provide the elementary language of this development, alongside other designers and architects of the modern movement. Gropius, after he moved to Harvard Graduate School of Design, also went on to advocated for ‘total architecture’. However, Gropius intended his idea to counter what he saw as a destructive imbalance in modernity, ‘factors of expediency like high-pressure salesmanship, organizational oversimplification and money making as an end in itself’ that impair the individual’s capacity to seek and understand the deeper potentialities of life’ (Gropius, 1962, p.13). Gesamtkunstwerk in practice László Moholy-Nagy was a Hungarian artist whose early work was influenced both by Dada and Russian constructivism. His artistic practice spanned activities including photography, montage, typography, graphic design, lighting and industrial design. He joined the Bauhaus in 1923, replacing Itten as the master of form responsible for the Vorkurs, as well as being responsible for the Metal workshop. Here, I would like to approach Moholy-Nagy’s conception of the Gesamtkunstwerk through three works that he created in 1923, which are often known collectively as the Telephone Pictures. These three images each show an identical abstract motif, each one a different size, made in enamel (Fig. 4.5). Moholy-Nagy claimed to have dictated the instructions for the pictures over the telephone to sign-makers, likening the conversation to playing ‘chess by correspondence’ (Moholy-Nagy, 1947, p.79). Moholy-Nagy’s writings seem to provide some warrant, therefore, for Tonkinwise’s claim that Bauhaus design intended a kind of autocratic intervention into everyday life. Moholy-Nagy thought that it would be possible, and advisable, to ‘to rewire the physiological and neurological hardware of the modern subject, that is, to reconstruct the sensorial apparatus in such a way that society could be changed from the ground up’ (p.282). Everything is not quite as it seems, however. Moholy-Nagy conceived this project as experimental and, most importantly, collective: undertaken in the ‘laboratory’ spirit of the constructivist-influenced avant- garde. At stake in his pedagogy, and his conception of design, was a vision of humanity’s capacity to explore its sensory apparatus and, in so doing, understand shared aesthetic responses. The emancipatory vision of The Telephone Pictures are usually displayed alongside one another, though they are separately titled as EM1, EM2 and EM3. On one level, these works celebrate the authorship at a distance that is part of the routine work of the industrial designer, which Moholy-Nagy saw as a means to extend art’s agency. In his theoretical writings, he advocated for what he called the Figure 4.5. László Moholy-Nagy, Construction in Enamel 1, 2 and 3, 1923–2012. Enamel on steel. 24 x 15cm, 47.5 x 30cm, 94 x 60cm; 9 1/2 x 5 7/8.Edition of 3 + 2 APs. (Image credit: Courtesy of the Estate of the Artist and Almine Rech) Figure 4.5. László Moholy-Nagy, Construction in Enamel 1, 2 and 3, 1923–2012. Enamel on steel. 24 x 15cm, 47.5 x 30cm, 94 x 60cm; 9 1/2 x 5 7/8.Edition of 3 + 2 APs. (Tonkinwise, 2014, n.p.) The clarity of this approach, its ‘rational Esperanto of design’, is framed by Baudrillard as a way-station from the Gesamtkunstwerk to an alienating economic rationale and semiotic code of the designed environment: There are clearly ambiguities in the utopianism of the Bauhaus. It is not entirely wrong to identify in the project of total design ideas that are, at times, autocratic. Yet, the most progressive and ambitious proposals of the Bauhaus are also connected to the implications of the Gesamtkunstwerk ideal. Though it is not possible here to explore this tension in all the detail it demands, it can be briefly treated through a comparison between the ideas of László Moholy-Nagy and Anni Albers, two important exponents of Bauhaus design principles. An ‘aesthetic’ ensemble is a mechanism without lapses, without fault, in which nothing compromises the interconnection of the OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 ISSN 2050-3679 www.openartsjournal.org 52 Gesamtwerk, or ‘total work’. As Koepnick notes, there is a touch of megalomania in Moholy-Nagy’s proclamation from the 1927 publication Painting Photography Film of ‘a synthesis of all the vital impulses spontaneously forming itself into the all-embracing Gesamtwerk (life) which abolishes all isolation, in which all individual accomplishments proceed from a biological necessity and culminate in a universal necessity’ (Moholy-Nagy cited in Koepnick, 2016, p.281). Gesamtwerk, or ‘total work’. As Koepnick notes, there is a touch of megalomania in Moholy-Nagy’s proclamation from the 1927 publication Painting Photography Film of ‘a synthesis of all the vital impulses spontaneously forming itself into the all-embracing Gesamtwerk (life) which abolishes all isolation, in which all individual accomplishments proceed from a biological necessity and culminate in a universal necessity’ (Moholy-Nagy cited in Koepnick, 2016, p.281). Gesamtkunstwerk in practice Born Annelise Fleischmann, Anni Albers studied at the Bauhaus from 1923, and married her fellow student Josef Albers in 1925. Though she became a teacher at the school, instructing students of weaving in design theory and eventually acted as director of the weaving workshop, for many decades her distinctive approach to design, and successful career as an artist, did not receive the critical attention that it deserves. Indeed, it might be argued that Albers’ approach to design is compelling because it managed to overcome obstacles that were set up by the institution of the Bauhaus itself. This project was often hyperbolic. In her memoir of the artist Moholy-Nagy: Marginal Notes, Documentary Absurdities Lucia Moholy – who was married to Moholy- Nagy in the 1920s – claims that he simply handed over diagram for EM1, EM2 and EM3 at the counter of an enamel workshop. In this version of events, Moholy- Nagy was struck after the fact by the possibility that he might have ordered the works by telephone (Kaplan, 1993). This story seems to underline, whether or not it is accurate, the speculative character of Moholy- Nagy’s utopianism. Yet, it is important to note that this attitude was not exactly naïve; rather, it was a response, in Moholy-Nagy’s case, to direct experience of the destructive power of technology in the First World War. Moholy-Nagy knew technology to be capable of wreaking havoc on human beings. This was one of the reasons he was compelled to try to bring it under control. Although permitted to study at the institution, female students were pressured to enter what were considered appropriately feminine workshops on graduation from the preliminary course. Indeed, the numbers of female students were so large that the weaving workshop was set aside as a female-only workshop (Müller, 2015). Walter Gropius encouraged this policy of segregation, seemingly to enforce a distinction between ‘feminine’ and ‘masculine’ design practices (Smith, 2014, p.xxvii). This kind of discrimination was obviously not exceptional at the time; however, it does flatly contradict the pedagogic intention of the Vorkurs, which was to support individual students to identify and follow their innate dispositions as we have seen. The Bauhaus, despite its utopian rhetoric, was organised along rigidly patriarchal lines. As the master of form of the metal workshop between 1923 and 1928, Moholy-Nagy played an important role in moving the Bauhaus in the direction of industrial design. Gesamtkunstwerk in practice (Image credit: Courtesy of the Estate of the Artist and Almine Rech) Figure 4.5. László Moholy-Nagy, Construction in Enamel 1, 2 and 3, 1923–2012. Enamel on steel. 24 x 15cm, 47.5 x 30cm, 94 x 60cm; 9 1/2 x 5 7/8.Edition of 3 + 2 APs. (Image credit: Courtesy of the Estate of the Artist and Almine Rech) OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 www.openartsjournal.org 53 this project was that it might point toward ‘alternative organizations of social space at the level of form, which, in its very changeability, offers the promise of alternative, improved sociality in the future.’ (p.283). room for teaching the basic ideas that keep human content alert and vital. For this we fought and for this we exhausted ourselves. I can no longer keep up with the stronger and stronger tendency toward trade specialisation in the workshops. From the point of view of the present, it is very difficult to read these implications in EM1, EM2 and EM3, however. A more available reading is that the interaction of standardisation and variable size in these works suggest a range of products, comparable to Behrens’ electric kettles (Fig. 4.3). Although Moholy- Nagy regarded these works as experiments in the extension of the agency of the artist, they are now more often interpreted as examples of a deflationary avant-garde strategy, an attack on the mystique invested in easel painting. Indeed, the Telephone Pictures are often compared to Duchamp’s readymade in this spirit (Roberts, 2007). What EM1, EM2 and EM3 seem to indicate, therefore, is the ambivalence of the encounter between art, design and technology in the Bauhaus, where a utopian project engaged with new technologies, with the aim of turning them to progressive ends. (Moholy-Nagy, 1974, p.136) (Moholy-Nagy, 1974, p.136) Although he experimented with authorship at a distance, Moholy-Nagy hated the fragmentation and specialisation of roles which was the reverse of the coin of the complexity of modern manufacturing. Herein is the pathos of Moholy-Nagy’s position; he sought emancipatory possibilities in the reorganisation of production that, impersonal and implacable, undermined the humanist basis of his own project. Anni Albers provides a very different perspective on the Gesamtkunstwerk. Though her work also aims for a holistic conception of art and design, it is not rhetorically committed to the emancipatory potential of new technologies. Gesamtkunstwerk in practice We still deal in weaving, as at the time of its beginning, with a rigid set of parallel threads in tension and a mobile one that traverses it at right angles. The main devices, in turn, have not become obsolete, but still form the nucleus of today’s weaving instruments. This recognition for Stölzl suggests that, though it was clearly patriarchal, the Bauhaus was at least capable of acknowledging outstanding achievement among female students. The reasons for this openness were at least partly economic. The Bauhaus was always short of money and the experience of Weimar made it clear that financial dependence on regional authorities would leave the institution vulnerable. From early on, the products created in the weaving workshop were able to find ready markets among private clients and manufacturers (Rowland, 1988). Under Muche, but especially under Stölzl, the workshop became one of the most financially successful, bridging between textile art and textile design for industry. (Albers, 1965, p.22) (Albers, 1965, p.22) Here technological change is presented not in absolute terms but, rather, it is seen as relative to historical continuities, where some practices cannot be redesigned because they have achieved already their optimal form. For Albers, the weaver revitalises modern industry by reconnecting technological development to pre-historic responses to human needs. While emphasising these connections across time, she also argued that weaving is the closest art to architecture, because it is so intimately involved in problems of construction. In these respects, her ideas may be read almost as an alternative model for the Bauhaus or, at least, one among a number of divergent conceptions of the unification of the arts explored in the school. g y Anni Albers developed her approach to design in this atmosphere of experiment and self-reliance. In her writings on design she argues that direct experimentation on the loom was the best way to overcome the separation of roles between the design and manufacture of textiles, which had become separate processes with the advent of mechanical looms (Smith, 2014). A direct comparison between Albers and Moholy-Nagy presents itself around this point. Whereas Moholy-Nagy’s Telephone Pictures seem to seek emancipatory potential in the separation of conception and execution, Albers prefers to collapse that distance as far as possible through experimentation on the loom, where material qualities can be directly explored in the design process. This comparison shows the diversity of approaches to design at the Bauhaus. Gesamtkunstwerk in practice Under his guidance, the metal workshop created many prototypes that were sold to industry, bringing significant revenues into the school (Wick, 2000). Even so, when Moholy-Nagy left the Bauhaus in 1928, his letter of resignation cites the increasing demands of technical specialisation as the primary reason for his departure: Even so, the school did create opportunities for female students and exceptional individuals were able to seize them. Marianne Brandt, for example, defied convention to become one of the most celebrated and successful designers in the metal workshop. In the As soon as creating an object becomes a speciality, and work becomes trade, the process of education loses all vitality. There must be OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 ISSN 2050-3679 www.openartsjournal.org 54 of new materials, such as cellophane, to understand their aesthetic and functional characteristics in textiles. But Albers did emphasise tactile engagement and material process as the fulcrum of her artistic and design practice in a way that Moholy-Nagy did not. Indeed, Albers’ arguments about the centrality of the loom in her approach to weaving has philosophical implications that allow the comparison to Moholy- Nagy to be extended. Whereas the utopianism of Moholy-Nagy was future-oriented and focused on technological progress, for example, Albers advocated a more nuanced temporality of human technology in her writings. In On Weaving, she reflects on the development of the loom: weaving workshop, the female students took it upon themselves to reinvent the status of their discipline within the institution. At the Weimar Bauhaus Helene Börner, ‘master of craft’ for the weaving workshop, taught traditional techniques and the ‘master of form’, Georg Muche, showed little interest in promoting innovation in what he saw as a women’s artform (Smith, 2014, p.32). Faced with these obstacles, a gifted student Gunta Stölzl took the initiative to develop new approaches and to teach her fellow students, engaging in material experimentation and initiating new areas of practice such as dyeing. When the Bauhaus moved to Dessau, the weaving workshop was provided with new looms and Stölzl was made the first female junior master after Muche left the school in 1927. During the 4,500 years or, in some estimates, 8,000 years that we believe mankind has been weaving, the process itself has been unaffected by the various devices that contributed to speed of execution. Gesamtkunstwerk in practice Although industrial design defines the school’s early reception, it represents only one aspect of a complex utopian engagement between art and technology. Bibliography 1 Albers, A. (1965) On Weaving, London, Studio Vista. 2 Baudrillard, J. (1981) ‘Design and environment’ in For a Critique of the Political Economy of the Sign (trans. Charles Levin), Candor, Telos. g Whereas industrial design dominated the canonical period of the critical reception of the Bauhaus, it is now possible to question these established accounts through a more pluralist understanding of design. At the same time, it is also useful to revisit the critique of Bauhaus utopianism, which tended also to take its cue from industrial design and architecture. Although certain proclamations by Bauhaus artists and designers do suggest a megalomaniacal attempt to redesign the world from scratch, this was not the only interpretation of the Gesamtkunstwerk that existed within the institution; indeed, even Moholy-Nagy’s ideas about the Gesamtwerk are not quite as autocratic as they may appear at first reading. The organisational structures of the Bauhaus were patriarchal undoubtedly. Yet, the expansiveness of Bauhaus pedagogy did empower some students to overcome these limitations. Albers’s concept of design seems still to be relevant to the urgent task that now confronts the design discipline: to fundamentally alter its own relationship to consumerism and to re-envisage the relationship between a fragile environment and the contemporary human world. As designers approach this enormous task, which is perhaps even more ambitious than the horizon that Gropius envisaged for the Bauhaus in 1919, it may be important to hold a nuanced view of the social utopianism of the Bauhaus, which recognised the instability created by a fully technologised world. 3 Findeli, A. and Benton, C. (1991) ‘Design education and industry: The laborious beginnings of the Institute of Design in Chicago in 1944’, Journal of Design History, vol.4, no.2, pp.97–113. 4 Foster, H. (2002 ‘Design and crime’ in Design and Crime (And Other Diatribes), London and New York, Verso. 5 Grawe, G.D. (2000) ‘Continuity and transformation: Bauhaus pedagogy and North America’ in R.K. Wick, Teaching at the Bauhaus (trans. Stephen Mason and Simon Lèbe), Ostfildern-Ruit, Hatje Cantz Verlag. 6 Gropius, W. ([1919] 1938) ‘From the first proclamation’ in Herbert Bayer, Walter Gropius and Ise Gropius (eds) Bauhaus 1919–1928, New York, Museum of Modern Art. 5 Grawe, G.D. (2000) ‘Continuity and transformation: Bauhaus pedagogy and North America’ in R.K. Wick, Teaching at the Bauhaus (trans. Stephen Mason and Simon Lèbe), Ostfildern-Ruit, Hatje Cantz Verlag. 6 Gropius, W. Conclusion The relationship between art, design and utopianism at the Bauhaus is complex. Whereas the critical reception of the school is founded on the celebration of a machine aesthetic, this is only one among several conceptions of design that were explored in the school. Craft played an important part in the Bauhaus throughout its existence, for example. As the weaving workshop demonstrates, handicraft was not left behind as the school developed an increasingly coherent functionalist design ethos; rather, craft continued to play a key role as a site to explore the interactions of art Albers’ design ethos by no means implied the rejection of modern industry. She produced prototypes for mass production throughout her career and, like Stölzl, experimented continually with the properties OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 ISSN 2050-3679 www.openartsjournal.org 55 and industry. This point is important, because it makes room for a plural understanding of the role of design in the Bauhaus and of its designers’ interactions with art. Bibliography Bibliography ([1919] 1938) ‘From the first proclamation’ in Herbert Bayer, Walter Gropius and Ise Gropius (eds) Bauhaus 1919–1928, New York, Museum of Modern Art. 7 Gropius, W. (1962) The Scope of Total Architecture, New York, Collier Books. 7 Gropius, W. (1962) The Scope of Total Architecture, New York, Collier Books. 8 Gropius, W. (1965) The New Architecture and the Bauhaus, Cambridge, Mass., MIT Press. 9 Irwin, T. (2015) ‘Transition design: A proposal for a new area of design practice, study and research’, Design and Culture, vol.7, no.2, pp.229–46. 9 Irwin, T. (2015) ‘Transition design: A proposal for a new area of design practice, study and research’, Design and Culture, vol.7, no.2, pp.229–46. 10 Kaplan, L. (1993) ‘The Telephone Paintings: Hanging up Moholy’, Leonardo, vol.26, no.2, pp.165–68. 10 Kaplan, L. (1993) ‘The Telephone Paintings: Hanging up Moholy’, Leonardo, vol.26, no.2, pp.165–68. 11 Koepnick, L. (2016) ‘Gesamtkunstwerk’, in Vincent Sherry (ed.) The Cambridge History of Modernism, Cambridge and New York, Cambridge University Press. 12 Moholy, L. (2020) ‘Questions of interpretation’ (trans. Eva Richter and Alba Norman), October, no.172, pp.125–34. 11 Koepnick, L. (2016) ‘Gesamtkunstwerk’, in Vincent Sherry (ed.) The Cambridge History of Modernism, 11 Koepnick, L. (2016) ‘Gesamtkunstwerk’, in Vincent Sherry (ed.) The Cambridge History of Modernism, Cambridge and New York, Cambridge University Press. 12 Moholy, L. (2020) ‘Questions of interpretation’ (trans. Eva Richter and Alba Norman), October, no.172, pp.125–34. 12 Moholy, L. (2020) ‘Questions of interpretation’ (trans. Eva Richter and Alba Norman), October, no.172, pp.125–34. 13 Moholy-Nagy, L. (1947) The New Vision and Abstract of an Artist, (4th edn), New York, Wittenborn, Schultz. 13 Moholy-Nagy, L. (1947) The New Vision and Abstract of an Artist, (4th edn), New York, Wittenborn, Schultz. 14 Moholy-Nagy, L. (1974) ‘Letter of resignation from the Bauhaus’ in Stephen Bann (ed.) The Tradition of Constructivism, Da Capo. 14 Moholy-Nagy, L. (1974) ‘Letter of resignation from the Bauhaus’ in Stephen Bann (ed.) The Tradition of Constructivism, Da Capo. 15 Müller, U. (2015) Bauhaus Women: Art, Handicraft, Design, Paris, Flammarion. 16 Otto, E. and Rössler, P. (2019) Bauhaus Women: A Global Perspective, London, Bloomsbury. 17 Rancière, J. (2013) Aisthesis: Scenes from the Aesthetic Regime of Art, translated from French by Zakir Paul, London and New York, Verso. 18 Roberts, J. (2007) The Intangibilities of Form: Skill and Deskilling in Art after the Readymade, London and New York, Verso. OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 ISSN 2050 www.openartsjournal.org ISSN 2050-3679 56 19 Roberts, D. (2011) The Total Work of Art in European Modernism, Ithaca, New York, Cornell University Press and Cornell University Library. 20 Rowland, A. (1988) ‘Business Management at the Weimar Bauhaus’, Journal of Design History, vol.1, no.3/4, pp.153–75. 21 Ruskin, J. (1997) ‘The nature of the Gothic’ in Clive Wilmer (ed.) Unto this Last and Other Writings, London, Penguin, pp.77–110. 22 Smith, T. (2014) Bauhaus Weaving Theory: From Feminine Craft to Mode of Design, Minneapolis and London, University of Minnesota Press. 23 Sparke, P. (2013) An Introduction to Design and Culture: 1900 to the Present (4th edn), Oxford, Routledge. 24 Tafuri, M. and Dal Co, F. (1976) Modern Architecture 1 (trans. Robert Erich Wolf), London, Faber and Faber and Electra. 25 Tafuri, M. (1976) Architecture and Utopia: Design and Capitalist Development (trans. Barbara Luigia La Penta), Cambridge, Mass., MIT Press. 26 Tonkinwise, C. (2014) ‘Design’s (dis)orders’, unpublished paper, https://medium.com/@camerontw/ designs-dis-orderstransition-design-cd53c3ad7d35, accessed 3.10.2020. 27 Wick, R.K. (2000) Teaching at the Bauhaus (trans. Stephen Mason and Simon Lèbe), Ostfildern-Ruit, Hatje Cantz Verlag. OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 www.openartsjournal.org ISSN 2050-3679 OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 ISSN 2050-3679 OPEN ARTS JOURNAL, ISSUE 9, WINTER 2020–1 www.openartsjournal.org
https://openalex.org/W2969290044	https://rrs.scholasticahq.com/article/10040.pdf	English	null	Drought Status, Price, and the Effectiveness of Water Use Restrictions in Pennsylvania	The review of regional studies/The Review of regional studies	2,019	cc-by	10,054	Drought Status, Price, and the Eﬀectiveness of Water Use Restrictions in Pennsylvania∗ Gregory Alan Krohn Department of Economics, Bucknell University, USA Department of Economics, Bucknell University, USA Abstract: During droughts, governments and water suppliers typically implement non-price policies to encourage water conservation. The state of Pennsylvania requests voluntary reductions in residential water use during moderate droughts and imposes mandatory restrictions during drought emergencies. This study utilizes data on household water consumption to measure the eﬀectiveness of the water use restrictions in Pennsylvania during the moderate drought years 2015-2017. Results suggest that voluntary water use restrictions have smaller than desired eﬀects and that the eﬀects are larger the higher the marginal price of water, perhaps reﬂecting a trade-oﬀbetween non-monetary beneﬁts and the welfare loss from reducing water usage. The eﬀectiveness of voluntary water use restrictions also is found to increase with the length of the drought. Keywords: drought, water conservation, conservation policy JEL Codes: Q25, Q28 Keywords: drought, water conservation, conservation policy JEL Codes: Q25, Q28 Keywords: drought, water conservation, conservation policy JEL Codes: Q25, Q28 (2019) 49, 222-243 (2019) 49, 222-243 Gregory Krohn is an Associate Professor of Economics at Bucknell University. Corr Gregory Krohn, e-mail: krohn@bucknell.edu. (c) Southern Regional Science Association 2019 ISSN 1553-0892, 0048-49X (online) www.srsa.org/rrs Gregory Krohn, e-mail: krohn@bucknell.edu. 1. INTRODUCTION It is widely expected that freshwater scarcity will become more common in regions around the world due to population growth, rising consumer demand for water-intensive products and services, environmental demands, pollution, and climate change. During droughts, gov- ernment agencies and municipal water suppliers typically implement non-price policies to encourage water conservation. The non-price conservation policies include water use re- strictions, information campaigns, social comparisons, and ﬁnancial incentives for installing water-saving devices (Wichman et al., 1992). Although the state of Pennsylvania typically receives ample precipitation, drought condi- tions sometimes occur. The state requests voluntary reductions in water use during moderate droughts and imposes mandatory restrictions during more serious droughts. Little is known, ∗The author thanks Chris Magee and the participants in the Department of Economics Research Seminar at Bucknell University for helpful comments, Kyle Adams for research assistance, Janine Glather for map creation, and the water companies which supplied data for the study. , p pp y Gregory Krohn is an Associate Professor of Economics at Bucknell University. Corresponding Author: Gregory Krohn, e-mail: krohn@bucknell.edu. (c) Southern Regional Science Association 2019 ISSN 1553-0892, 0048-49X (online) www.srsa.org/rrs KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS 223 however, about the eﬀectiveness of these Pennsylvania policies. If projections of increased frequency and intensity of drought bear out, knowledge of the eﬀectiveness of existing policies could enhanced drought management. however, about the eﬀectiveness of these Pennsylvania policies. If projections of increased frequency and intensity of drought bear out, knowledge of the eﬀectiveness of existing policies could enhanced drought management. Studies of droughts in other regions have shown varying results from water use restrictions depending on the location, the policies adopted, and the type of data analyzed. The present study contributes to this literature by analyzing data on household water consumption in Pennsylvania to measure the eﬀectiveness of water use restriction in counties experiencing moderate drought conditions during the years 2015-2017. Another contribution is the the- oretical and empirical examination of the conjecture that the higher the price of water, the more responsive residential water users are to requests for voluntary reductions in water use. Results suggest that the voluntary water use restrictions have a smaller than desired eﬀect and that the eﬀects are larger the higher the marginal price of water. Furthermore, the ﬁndings show that the eﬀectiveness of voluntary water use restrictions increases with the length of the drought. 1. INTRODUCTION The following section discusses how droughts are managed in Pennsylvania. Section 3 reviews the literature on the eﬀectiveness of water use restrictions. Section 4 considers various non-monetary motivations for complying with voluntary water use restrictions. It also examines how the welfare loss from a percentage reduction in water usage varies with the price of water. Section 5 describes the data used in the study, and Section 6 presents the empirical model and discusses the estimation method and results. Section 7 concludes. c⃝Southern Regional Science Association 2019. 2. DROUGHT MANAGEMENT IN PENNSYLVANIA There is no single, universally accepted deﬁnition of drought. Wilhite and Pulwarty (2018) deﬁne drought as a “natural hazard that results from a deﬁciency of precipitation from av- erage or ‘normal’ that, when extended over a season or longer, results in water supplies that are insuﬃcient to meet the demands of human activities and the environment.” All droughts begin with a deﬁciency of precipitation (meteorological drought) that can be aggravated by high temperatures, low humidity, and wind. When the deﬁciency of precipitation continues, it aﬀects agricultural production through reductions in soil moisture (agricultural drought). Persistent drought conditions reduce stream ﬂow, reservoir and lake levels, snowpack, and groundwater levels (hydrological drought), with signiﬁcant impacts on ecosystems. Socioeco- nomic drought associates human activity with elements of meteorological, agricultural, and hydrological drought. This type of drought may result from factors aﬀecting goods depen- dent on precipitation, such as hydroelectric power. Socioeconomic drought may also result from the diﬀerential impact of drought on groups of people. The impacts of drought depend on the timing, intensity, duration, and spatial extent of drought (Wilhite and Pulwarty, 2018). The Northeast region of the United States experienced a serious drought in 2016. In October, most of New England and New York experienced moderate or severe drought, and some areas experienced extreme drought (Figure 1). Portions of Pennsylvania also were aﬀected. In the state of Pennsylvania (PA), managing water resources during droughts is the re- 224 The Review of Regional Studies 49(2) R f R g S 9( ) Figure 1: U.S. Drought Monitor: Northeast, October 18, 2016 sponsibility of the Pennsylvania Emergency Management Agency (PEMA), with support from the Pennsylvania Department of Environmental Protection (PA DEP). The PA DEP regularly monitors precipitation, stream ﬂow, groundwater level, soil moisture, and reservoir levels, generally on a county basis. Every month, the PA DEP assigns a drought status (nor- mal, watch, warning, or emergency) to each of the drought indicators. Precipitation deﬁcits are the earliest indicators of a potential drought, because precipitation provides the basis for both surface water and groundwater resources. Precipitation levels over the past three months that are 25 percent below normal indicate a drought watch; 35 percent below nor- mal indicate a drought warning; and 45 percent below normal indicate a drought emergency (Commonwealth of Pennsylvania, Department of Environmental Protection, 2016). c⃝Southern Regional Science Association 2019. of Pennsylvania, Department of Environmental Protection, 2016). of Pennsylvania, Department of Environmental Protection, 2016). A similar process is followed when three or more of the indicators signal a drought warning. In this case, citizens are asked to reduce water use voluntarily by 10-15 percent. When three or more of the indicators signal a drought emergency, and upon recommendation of the Commonwealth Drought Task Force, PEMA convenes a meeting of the Emergency Management Council. Upon recommendation of the Council, the Governor may issue a proclamation of drought emergency, imposing mandatory restrictions on nonessential uses of water designed to achieve a reduction in overall water use of up to 25 percent. These restric- tions generally apply to watering of lawns, gardens, and shrubs; washing vehicles or paved surfaces; ﬁlling swimming pools; and use of water for ornamental purposes (Commonwealth of Pennsylvania, Department of Environmental Protection, 2016). A public water supplier’s drought contingency plan describes stages of drought for its particular sources of water. The water supplier may request voluntary or mandatory water use restrictions in advance of any state announcements or declarations. In some cases, water use restrictions may be insuﬃcient to protect the supplies of an individual public water supplier. When depleted supplies threaten health and safety, the state may approve the water supplier’s request to ration water within its service area. Rationing water is a more severe measure than banning nonessential uses, and generally involves allotting a given amount of water based on a percentage of previous usage or a speciﬁc quantity per household. In extreme droughts, large industrial and commercial water users may be required to sub- mit drought contingency plans, which identify actions that can be taken to reduce water use by various percentages. The Commonwealth Drought Coordinator will order such cutbacks only if conditions become so severe that health and safety are threatened. Pennsylvania has never experienced a drought so severe as to require industrial and commercial cutbacks (Commonwealth of Pennsylvania, Department of Environmental Protection, 2016). The last drought emergency declared in Pennsylvania was in 2002 (Schweiker, 2002), which followed the previous drought emergency in 1999 (Ridge, 1999). More recently, at the end of 2016 and into 2017, nearly one-half of Pennsylvania counties were under drought watches or warnings (Figure 2). Some counties experienced drought watches in 2015 as well. The eﬀectiveness of water use restrictions in Pennsylvania over the period 2015-2017 is the focus of the present study. c⃝Southern Regional Science Association 2019. 2. DROUGHT MANAGEMENT IN PENNSYLVANIA The Ap- pendix describes in detail how each of the drought indicators (precipitation deﬁcits, stream ﬂow, groundwater level, and soil moisture) is computed and assigned a drought status. Figure 1: U.S. Drought Monitor: Northeast, October 18, 2016 Figure 1: U.S. Drought Monitor: Northeast, October 18, 2016 sponsibility of the Pennsylvania Emergency Management Agency (PEMA), with support from the Pennsylvania Department of Environmental Protection (PA DEP). The PA DEP regularly monitors precipitation, stream ﬂow, groundwater level, soil moisture, and reservoir levels, generally on a county basis. Every month, the PA DEP assigns a drought status (nor- mal, watch, warning, or emergency) to each of the drought indicators. Precipitation deﬁcits are the earliest indicators of a potential drought, because precipitation provides the basis for both surface water and groundwater resources. Precipitation levels over the past three months that are 25 percent below normal indicate a drought watch; 35 percent below nor- mal indicate a drought warning; and 45 percent below normal indicate a drought emergency (Commonwealth of Pennsylvania, Department of Environmental Protection, 2016). The Ap- pendix describes in detail how each of the drought indicators (precipitation deﬁcits, stream ﬂow, groundwater level, and soil moisture) is computed and assigned a drought status. Generally, when three or more of the indicators are signaling a drought watch for a county or a group of counties, PA DEP will inform PEMA of the developing conditions and ask PEMA to convene a meeting of the Commonwealth Drought Task Force. The Commonwealth Drought Task Force includes membership from state, federal, and interstate agencies who may be impacted by drought or drought management operations. Upon recommendation of the Task Force and direction from the Governor, the PA DEP issues a drought watch. Press releases are issued to the media and letters are sent to all public water suppliers in the aﬀected area, notifying them to follow their drought contingency plans. The general goal is to reduce water use by 5-10 percent through voluntary water conservation (Commonwealth KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS 225 3. PREVIOUS LITERATURE A number of previous studies evaluate water use restrictions and other non-price demand management policies. The studies vary in their location (many are in the southwest U.S.), time period, measure of water use (daily, weekly, monthly, or quarterly observations on household, city per capita, or city per household water use), type of data (time series, cross section, or panel data), demand management policies considered, functional form of the water demand equation, and estimation methods. Considering just water use restrictions, the range of estimates is wide, but mandatory water use restrictions generally have larger eﬀects than voluntary ones. ated eﬀects of voluntary water use restrictions on average water consumption c⃝Southern Regional Science Association 2019. 226 The Review of Regional Studies 49(2) Figure 2: Drought Status by Pennsylvania County, December 22, 2016 Source: Pennsylvania Department of Environmental Protection and Ja- nine Glather. Note: Dots indicate locations of water companies supply- ing data for the study. Figure 2: Drought Status by Pennsylvania County, December 22, 2016 Figure 2: Drought Status by Pennsylvania County, December 22, 2016 Source: Pennsylvania Department of Environmental Protection and Ja- nine Glather. Note: Dots indicate locations of water companies supply- ing data for the study. typically fall in the range of -12 percent to 0 (Moncur, 1987; Shaw and Maidment, 1987, 1988; Nieswiadomy, 1992; Kenney et al., 2004; Coleman, 2009), although Shaw et al. (1992) found that voluntary water use restrictions in San Diego, California reduced water consumption by 27 percent in the summer of 1991. Two studies from the eastern U.S. are worth highlighting for comparison with the present study. Halich and Stephenson (2009) studied 21 locales in Virginia around the time of the 2002 drought. They found that the eﬀects of the voluntary water use restrictions vary between -7 percent and 0, the magnitudes of the reductions varying directly with the intensity of the public information campaigns conducted. Wichman et al. (1992) found that voluntary water use restrictions adopted in 6 North Carolina cities in 2007 and 2008 reduced water consumption on average by 8.5 percent. In a study of Cobb County, Georgia, Ferraro and Price (2013) considered the eﬀects of technical advice, prosocial appeals, and social comparisons on water conservation. Prosocial appeals ask households, for example, to conserve water in order to protect wildlife and human health. c⃝Southern Regional Science Association 2019. 3. PREVIOUS LITERATURE The social comparisons inform consumers of the percentage of similar households that they consumed more water than, an approach that has been tried with some eﬀect in electricity markets (Alcott, 2011). Ferraro and Price (2013) found that technical advice (“tip KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS 227 sheets”) reduce water use by less than 1 percent, prosocial appeals combined with technical advice reduce water use by 2.7 percent, and social comparisons combined with the other two approaches reduce water consumption by 4.8 percent on average. Brent et al. (2015) found similar eﬀects of social comparisons in two of three California water utilities studied; there is an insigniﬁcant eﬀect in the third company. Mandatory water use restrictions typically apply to outdoor water use, such as day of use restrictions, hand-watering only, or outright prohibitions. The range of estimates of the eﬀects of mandatory water use restrictions, -56 percent to -4 percent, is even wider than that of voluntary restrictions (Renwick and Archibald, 1998; Renwick and Green, 2000; Grafton and Ward, 2008; Castledine et al., 2014). It is notable that Halich and Stephenson (2009) found that the eﬀects of mandatory water use restrictions are larger in absolute value the greater the penalties for noncompliance and the stronger the enforcement eﬀorts. c⃝Southern Regional Science Association 2019. 4. NON-MONETARY MOTIVATIONS OF BEHAVIOR The ﬁndings that voluntary and mandatory water use restrictions aﬀect water use raise the question as to why. In standard consumer theory, demands for goods and services such as water depend on relative prices and income. If these factors and consumer preferences do not change, demands would not change. Alternatively, non-monetary motives of behavior could help explain why households comply with voluntary water use restrictions or mandatory restrictions with small penalties and weak enforcement mechanisms. In the context of recycling, Abbott et al. (2013) provide an informative summary of non-monetary motives of behavior found in the psychology, economics, sociology, and other literatures. They identify “warm glow” and social norms as the primary non-monetary motives of pro-environmental behavior. Warm glow can be deﬁned as the enjoyment derived from an activity independent of its outcome (Deci, 1971; Andreoni, 1990). It is an intrinsic motivation that reﬂects self-interest. Social norms are “shared understandings about actions that are obligatory, permitted, or forbidden” (Ostrom, 2000). Compliance with social norms depends on beliefs about the degree of conformity among the population (descriptive norms) and what other people expect (injunctive norms). Injunctive norms can inﬂuence behavior because of sanctions, such as shaming, which others can exert in the event of noncompliance. However, sanctions are not required for compliance if the social norms have become internalized (Abbott et al., 2013). Some writers link social norms and warm glow together. For example, Brekke et al. (2003), identify warm glow with an individual’s positive self-image, which depends on the degree to which their behavior is socially responsible. In light of these non-monetary motivations of behavior, households might comply with water use restrictions because of the warm glow they receive from doing so or because of a social norm they internalize or feel compelled to follow because of external sanctions. A tradeoﬀis the conventional loss of welfare from reducing consumption of water in the absence of a change in price. For example, Ferraro and Price (2013) incorporate this tradeoﬀ between consumption utility and the nonpecuniary payoﬀin their model of the channels through which technical information, prosocial messages, and social nudges might inﬂuence The Review of Regional Studies 49(2) 228 Figure 3: Welfare Loss from Water Use Restrictions Figure 3: Welfare Loss from Water Use Restrictions water consumption. c⃝Southern Regional Science Association 2019. 4. NON-MONETARY MOTIVATIONS OF BEHAVIOR Binder and Blankenberg (2017) note on page 307, “it is well-known from the environmental literature that people have problems with following through on certain behaviors if they are associated with additional costs (however small).” In the present context, the greater the welfare loss from reducing the consumption of water relative to the non-monetary beneﬁts, the lesser the extent of compliance with water use restrictions. water consumption. Binder and Blankenberg (2017) note on page 307, “it is well-known from the environmental literature that people have problems with following through on certain behaviors if they are associated with additional costs (however small).” In the present context, the greater the welfare loss from reducing the consumption of water relative to the non-monetary beneﬁts, the lesser the extent of compliance with water use restrictions. water consumption. Binder and Blankenberg (2017) note on page 307, “it is well-known from the environmental literature that people have problems with following through on certain behaviors if they are associated with additional costs (however small).” In the present context, the greater the welfare loss from reducing the consumption of water relative to the non-monetary beneﬁts, the lesser the extent of compliance with water use restrictions. Furthermore, the welfare loss from a given percentage reduction in water use might depend on the price of water. If so, then the price could aﬀect the extent to which consumers comply with voluntary water use restrictions expressed as a percentage of water use. This possibility does not appear to have been discussed in the literature. Consider the case of a linear demand function for water: Consider the case of a linear demand function for water: (1) q = a −bp + cy (1) where q is the quantity of water, p is the marginal price of water, y is income, and a, b, and c are positive parameters. Both p and y are deﬂated by an appropriate index of other prices. The initial price of water is denoted by p1 and the associated initial quantity of water by q1. Suppose that there is a voluntary 100r percent reduction of in the quantity of water consumed (0 < r < 1), so that q2 = (1 −r)q1. Using Marshallian consumer’s surplus as the measure of consumer welfare, the loss in welfare from the water restrictions (L) is given by: L = ( 1 2b)(q1q2)2 = ( 1 2b)r2q2 1. KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS 229 δL δp1 = −r2q1 < 0. (3) (3) This suggests that the higher the price of water, the smaller the welfare loss from a given percentage water use restriction, and the more likely a consumer is to comply with the restrictions, given the tradeoﬀbetween the welfare loss and the non-monetary beneﬁts of complying. This suggests that the higher the price of water, the smaller the welfare loss from a given percentage water use restriction, and the more likely a consumer is to comply with the restrictions, given the tradeoﬀbetween the welfare loss and the non-monetary beneﬁts of complying. Under certain conditions, Marshallian consumer’s surplus can be a good approximation of Hicksian compensating variation, an accurate measure of consumer welfare. Hausman (1981) shows, however, that the Marshallian measure of deadweight loss can be a poor approxima- tion of the compensated measure of deadweight loss. Nevertheless, with a linear demand function, the Hicksian measure of deadweight loss from water use restrictions decreases with the initial price, just as the Marshallian measure does. Following Hausman (1981), the linear demand function in Equation (1) has the following Hicksian compensated demand function: h(p, u1) = cu1ecp + b c (4) (4) where u1 is the initial utility level and u1 < 0 is necessary to satisfy the Slutsky condition in consumer theory. Let p2 denote the shadow price of water following the imposition of water use restrictions, such that q(p2) = q2 (point C in Figure 3). With q2 = (1 −r)q1 under the water restrictions, the compensating variation (CV) is given by: where u1 is the initial utility level and u1 < 0 is necessary to satisfy the Slutsky condition in consumer theory. Let p2 denote the shadow price of water following the imposition of water use restrictions, such that q(p2) = q2 (point C in Figure 3). With q2 = (1 −r)q1 under the water restrictions, the compensating variation (CV) is given by: CV (p1, p2, y1 = Z p2 p1 h(p, u1dp = 1 c[ec(p2−p1) −(1 −r)]q1 −b c2[ec(p2−p1)]. (5) (5) The compensated deadweight loss (CL) is: CL = CV −(p2 −p1)h(p2, u1). (6) (6) Substituting Equations (4) and (5) into Equation (6), it can be shown that the Hicksian compensated deadweight loss decreases with the initial price: δCL δp1 < 0. KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS (7) (7) In light of these theoretical results, one hypothesis tested in the empirical analysis below is that compliance with voluntary water use restrictions by consumers is greater the higher the price of water. c⃝Southern Regional Science Association 2019. KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS 229 KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS 4. NON-MONETARY MOTIVATIONS OF BEHAVIOR (2) (2) The Marshallian deadweight loss is shown by area ABC in Figure 3. The Marshallian deadweight loss is shown by area ABC in Figure 3. The Marshallian deadweight loss from a percentage reduction in water use is a decreasing function of the price: c⃝Southern Regional Science Association 2019. c⃝Southern Regional Science Association 2019. KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS 229 5. DATA The PA DEP deﬁnes a Community Water System (CWS) as a Public Water System which provides water to the same population year-round (Commonwealth of Pennsylvania, Depart- ment of Environmental Protection, 2017). There were 1,951 CWSs in Pennsylvania in 2016, c⃝Southern Regional Science Association 2019. The Review of Regional Studies 49(2) 230 Table 1: Community Water Systems in Pennsylvania Size Number Percent Pop Served Percent Served Small 1,619 83 950,753 8 Medium 299 14 3,935,318 35 Large 33 2 6,491,628 57 Total 1,951 100 11,377,699 100 serving 11.4 million of the state’s population of 12.8 million, but most of these systems are small, serving 3,300 or fewer people each. The 332 medium and large CWSs provided water to 92 percent of the population served (Table 1). Most of the systems are owned by munic- ipalities or regional authorities, but some are owned by private companies that operate one or more systems throughout the state. serving 11.4 million of the state’s population of 12.8 million, but most of these systems are small, serving 3,300 or fewer people each. The 332 medium and large CWSs provided water to 92 percent of the population served (Table 1). Most of the systems are owned by munic- ipalities or regional authorities, but some are owned by private companies that operate one or more systems throughout the state. To obtain the data used in the present study, requests were sent in October 2017 to 34 medium and large community water systems and to one small system that were identiﬁed as billing monthly and charging a uniform consumption rate (price) for water in addition to a ﬁxed (base) charge. The small system (Galeton Borough Water Authority) was selected because it was identiﬁed by in PA DEP drought reports as imposing water restrictions during 2016. Many Pennsylvania water companies bill only quarterly or bi-monthly. Companies that charge uniform rates for water were chosen because the price is administratively set and individuals take it as given and decide how much water to consume. Under increasing or decreasing block rate pricing structures, the marginal price of water depends on the amount consumed, making the price endogenous and creating econometric complexities. The 35 companies are located throughout Pennsylvania in counties with diﬀering histories of drought status over the years 2015-2017. c⃝Southern Regional Science Association 2019. 5. DATA Companies were requested to provide monthly water use data for each residential account from January 2015 to the most recent month in 2017, water rates charged over this period, and a description of measures taken to reduce water use during droughts. The request was supported by the Commonwealth Drought Coordinator and was accompanied by a letter of support from a Pennsylvania Representative. In the end, monthly account level data were provided by ﬁve water systems: the Milton facility of Pennsylvania American Water serving parts of Northumberland and Union Counties, the Borough of Hollidaysburg in Blair County, the Sunbury Municipal Authority in Northumberland County, the City of Bethlehem serving parts of Lehigh and Northampton Counties, and the Galeton Borough Authority in Potter County (see Figure 2). The primary source of water for each company is surface water (purchased surface water for Hollidaysburg). For the City of Bethlehem, less than 5 percent of the residential customers are billed monthly and included in the data provided, and many of those are in low-income housing and rental units, including multi-unit dwellings. In managing the data received, customers were dropped if there was information that identiﬁed them as institutions or commercial establishments. Observations were dropped if water use was missing or less than or equal to zero. Then observations were dropped if water use was greater than the mean plus four standard deviations by company in order to account for leaks or large, unidentiﬁed institutions. Finally, customers were dropped from KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS 231 Table 2: Summary Statistics by Water Company Milton Hollidbrg Sunbury Bethlhm Galeton Residential units 7,700 2,172 2,012 519 266 Observations 276,688 75,044 66,241 17,180 9,523 Mean water use (1000 gal/month) 3.3 3.3 4.4 10.0 3.0 Mean price (1000 gal, 2015$) 10.1 7.6 4.2 3.9 10.7 Mean base charge (2015$) 14.8 14.8 11.3 8.3 33.6 Table 2: Summary Statistics by Water Company the sample if they were missing two or more observations. the sample if they were missing two or more observations. Table 2 presents summary statistics for data used in the empirical analysis by water company. The number of residential units varies from a low of 266 in Galeton to a high of 7,700 in Milton. Mean monthly water use per residential unit in thousands of gallons over 2015-2017 ranges from 3.0 to 4.4 in Galeton, Milton, Hollidaysburg, and Sunbury, with Bethlehem averaging 10.0 per account. 5. DATA The latter value reﬂects the presence of multi-unit dwellings in Bethlehem’s small sample. The mean marginal price per thousand gallons, adjusted for inﬂation to 2015 dollars of purchasing power, varies from a low of $3.9 in Bethlehem to a high of $10.7 in Galeton. Galeton’s base charge of $33.6 also is relatively high, but it includes an allowance of 2,000 gallons of water. For 2,000 gallons or more of water used, a Galeton customer would pay an amount similar to a Milton customer. The pattern of average monthly water use by residential units over the years 2015 to 2017 for each of the ﬁve water companies is shown in Figure 4. There is noticeable variability month-to-month in average water use. This likely reﬂects variation in meter reading dates and the number of days of water use billed per month. In fact, the spike in average water use late in 2016 in Hollidaysburg can be traced to a substantial change in meter reading dates for a number of customers. The other noticeable outlier is the relatively low value of average water use in January 2015 in Bethlehem. There were many missing values for this month in the Bethlehem data, and apparently many of these were multi-unit dwellings. Although there is sizeable variation in the marginal price of water across water companies, there is less variation in the price over time within companies. As shown in Figure 5, there were slight step increases in the real price of water in Sunbury and Hollidaysburg, and a relatively large increase in 2017 in Galeton. The nominal price of water was unchanged over 2015-2017 in Milton, so the real price declines slightly due to the small rate of overall price inﬂation. Unusually, the marginal price of water took a sharp dip of over 1 dollar per 1000 gallons in 2017 in Bethlehem, only partially oﬀset by an increase of $0.61 in the base rate. Figure 6 shows the drought status of the counties that the water companies operated in. Over the 36-month period 2015-2017, Blair County was in drought watch status three months, Northampton and Northumberland Counties were in drought watch or warning status 10 months, and Union County was in drought watch or warning status 13 months. c⃝Southern Regional Science Association 2019. 5. DATA Potter County was in a drought watch for four months in 2015, and Galeton Borough was under mandatory water use restrictions for six months beginning in August 2016. Halich and Stephenson (2009) found that the eﬀects of the voluntary water use restrictions depend on the intensity of the public information campaigns that accompanies them. In light The Review of Regional Studies 49(2) 232 Figure 4: Average Water Use Figure 4: Average Water Use of this, is worth noting what the water companies in the sample do when their county is under a drought watch or warning, and the PA DEP requests voluntary reductions in water use. The drought contingency plans and practices of the ﬁve companies are summarized in Table 3. Based on information provided by the companies, when voluntary reductions in water use are called for, the company provides notices to customers and, perhaps, information to local news media. It is diﬃcult to judge the intensity of these eﬀorts from the descriptions provided, but the eﬀorts do not in general appear to be very vigorous in the event of a drought watch or warning. c⃝Southern Regional Science Association 2019. 6.1. Empirical Model Empirical models of residential water demand commonly include as explanatory variables some measure of the price of water, household income, household characteristics such as num- ber of residents, lot size, and landscape irrigation system, indicators of the season, weather variables, and policy variables such as voluntary and mandatory water use restrictions, and water technology rebates and subsidies (Olmstead, 2010). For the present study, however, data on household income or characteristics were not available, although an unobserved ﬁxed eﬀect represents them in the model. c⃝Southern Regional Science Association 2019. c⃝Southern Regional Science Association 2019. KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS 233 Figure 5: The Marginal Price of Water The empirical model estimated is as follows: Figure 5: The Marginal Price of Water The empirical model estimated is as follows: The empirical model estimated is as follows: ln(watergmt) = b0 + b1pricegt + b2watchgt + b3warninggt + b4mandatorygt + b5watchgt ∗pricegt + b6warninggt ∗pricegt + b7watchgt ∗durationgt + b8warninggt ∗durationgt + b9precipgt + b10tempgt + Ttb11 + hg + cgm + ugmt, (8) (8) where subscripts g, m, and t denote water company, residential unit, and time period, re- spectively; ln(water) is the natural logarithm of monthly water use in 1000 gallons, price is the marginal price per 1000 gallons of water in 2015 dollars, watch is a (0, 1) indicator variable for PA DEP drought watch status in the county, warning is an indicator variable for drought warning status in the county, mandatory is an indicator variable for manda- tory water restrictions in Galeton, duration gives the number of consecutive months in a drought (watch, warning, or mandatory restrictions), precip is monthly precipitation in inches recorded at the weather station nearest to the primary facility of the water company, temp is the monthly average of daily maximum temperature, T is a vector of period ﬁxed eﬀects, and hg, cgm, and ugmt are the unobserved group ﬁxed eﬀect, residential unit ﬁxed eﬀect, and idiosyncratic error, respectively. c⃝Southern Regional Science Association 2019. c⃝Southern Regional Science Association 2019. c⃝Southern Regional Science Association 2019. 6.1. Empirical Model The semilogarithmic functional form for water demand is adopted because with it, the percentage eﬀects of drought status are independent where subscripts g, m, and t denote water company, residential unit, and time period, re- spectively; ln(water) is the natural logarithm of monthly water use in 1000 gallons, price is the marginal price per 1000 gallons of water in 2015 dollars, watch is a (0, 1) indicator variable for PA DEP drought watch status in the county, warning is an indicator variable for drought warning status in the county, mandatory is an indicator variable for manda- tory water restrictions in Galeton, duration gives the number of consecutive months in a drought (watch, warning, or mandatory restrictions), precip is monthly precipitation in inches recorded at the weather station nearest to the primary facility of the water company, temp is the monthly average of daily maximum temperature, T is a vector of period ﬁxed eﬀects, and hg, cgm, and ugmt are the unobserved group ﬁxed eﬀect, residential unit ﬁxed eﬀect, and idiosyncratic error, respectively. The semilogarithmic functional form for water demand is adopted because with it, the percentage eﬀects of drought status are independent c⃝Southern Regional Science Association 2019. 234 The Review of Regional Studies 49(2) Figure 6: Draught Status Source: Consumer Expenditure Survey 2015. Figure 6: Draught Status Source: Consumer Expenditure Survey 2015. of water use, and the price elasticity of demand varies with the price. If the voluntary water use restrictions during drought watches and warnings are eﬀective, then the coeﬃcients on watch and warning will be negative. Similarly, if the eﬀectiveness of the voluntary restrictions rises with the price of water, then the coeﬃcients on the drought status interacted with price will be negative. If the eﬀectiveness of the voluntary restrictions increases the longer the drought lasts, then the coeﬃcients on the drought status interacted with duration will be negative as well. The marginal price of water in Galeton is zero up to 2000 gallons due to an allowance included with the base charge. This makes the marginal price endogenous, and results in a biased estimator of the coeﬃcient on the Galeton price variable. To address this issue, the instrumental variables technique is applied. The marginal price of water for Galeton households is estimated, with the base rate and the marginal price in excess of 2000 gallons KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS In the event of a declared drought emergency, mandatory restrictions on nonessential uses of water are imposed, and the company is authorized to collect ﬁnes for noncompliance. Hollidaysburg When the Blair County EMA issues a drought watch or a warning, notices are sent out to the aﬀected areas and water usage by customers is monitored. y the PA DEP’s declaration of a drought watch or warning, on is provided to news media. In the event of a drought warning, is informed about the potential for mandatory restrictions on Water conservation measures and use of water saving devices are In the event of a drought emergency, regulations on the ial uses of water are publicized and enforced in conjunction with enforcement agencies. KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS 235 Table 3: Drought Contingency Plans and Practices Milton Following PA DEP’s guidance for issuing conservation notices, a press release is sent to local media outlets and is posted on the company’s website and social media outlets. In the event of a declared drought emergency, mandatory restrictions on nonessential uses of water are imposed, and the company is authorized to collect ﬁnes for noncompliance. Hollidaysburg When the Blair County EMA issues a drought watch or a warning, notices are sent out to the aﬀected areas and water usage by customers is monitored. Sunbury Each year, a link is provided to our residential and commercial customers for the PA DEP’s Water Conservation Fact Sheet, which provides tips for conserving water and protecting water resources. The link is also posted on the company’s web site. In the event of a drought watch or warning in our local area, customers are informed of such on their monthly bills and are reminded to be conscious of their water usage, and to take steps to minimize it where they can. Bethlehem Following the PA DEP’s declaration of a drought watch or warning, information is provided to news media. In the event of a drought warning, the public is informed about the potential for mandatory restrictions on water use. Water conservation measures and use of water saving devices are promoted. In the event of a drought emergency, regulations on the nonessential uses of water are publicized and enforced in conjunction with local law enforcement agencies. Galeton In the event that the Galeton Borough Council declares a stage one drought, voluntary restrictions of nonessential are encouraged. In the event of the declaration of a stage two drought, mandatory restrictions of nonessential water use are implemented and the PA DEP is notiﬁed. In the event of a stage three drought, the local water rationing plan is implemented in addition to the ban on nonessential water use. Penalties for violations include a ﬁne not to exceed $1,000 and/or imprisonment for a term not to exceed 30 days. Table 3: Drought Contingency Plans and Practices Milton Following PA DEP’s guidance for issuing conservation notices, a press release is sent to local media outlets and is posted on the company’s website and social media outlets. Galeton In the event that the Galeton Borough Council declares a stage one drought, voluntary restrictions of nonessential are encouraged. In the event of the declaration of a stage two drought, mandatory restrictions of nonessential water use are implemented and the PA DEP is notiﬁed. In the event of a stage three drought, the local water rationing plan is implemented in addition to the ban on nonessential water use. Penalties for violations include a ﬁne not to exceed $1,000 and/or imprisonment for a term not to exceed 30 days. Source: Information provided by the water companies. used as instruments. The ﬁtted values from this equation are used as the water price variable for Galeton. Equation (8) is estimated by the ﬁxed eﬀects method using Stata, version 13.1. The ﬁxed eﬀects transformation, which subtracts the mean over time from each variable, eliminates the group and residential unit ﬁxed eﬀects as well as the cross section variation of the averages of the other variables such as the price. Note, however, that the ﬁxed eﬀects method will not eliminate all of the cross section variation in the interaction terms between price and drought status. For example, if a drought watch were in eﬀect in 25 percent of the months, then ﬁxed eﬀects estimation would leave about 75 percent of the price in the transformed interaction term between the price and the drought watch indicator. The Review of Regional Studies 49(2) 236 Table 4: Regression Results by Water Company Variable Coeﬃcient (standard error) Milton Hollidaysbrg Sunbury Bethlehem Galeton (1) (2) (3) (4) (5) price a 1.3223 a 1.8442* -0.0312* (1.0211) (0.0509) (0.0072) watch -0.0074 -0.0631 -0.0262* -2.0870* -0.0605 (0.0047) (0.0308) (0.0102) (0.0058) (0.0288) warning -0.0023 1.6167* (0.0076) (0.0459) mandatory -0.0485 (0.0309) precip -0.0145* -0.0016 -0.0073* 0.5473* -0.0312 (0.0008) (0.0029) (0.0003) (0.0003) (0.0072) temp 0.0013* 0.0012* 0.0023* 0.0480* 0.0010* (0.0001) (0.0006) (.0001) (.0012) (.0006) Observations 276,668 75,044 66,241 17,180 9,523 R2 0.011 0.007 0.006 0.013 0.023 Notes: Dependent variable is ln(water). Fixed eﬀects model with robust standard errors clustered at the residential unit level. , , and indicate statistical signiﬁcance at the 10, 5 and 1 percent levels, respectively. The letter “a” indicates omitted due to collinearity. Table 4: Regression Results by Water Company c⃝Southern Regional Science Association 2019. 6.2. Regression Results Results from estimating Equation (8) without interaction terms separately for each water company are reported in Table 4. Two observations are noteworthy. First, the price variable is eliminated in the Milton and Sunbury regressions due to collinearity. The collinearity is with the period ﬁxed eﬀects, and reﬂects the small extent of variation over time of the prices of water for these two companies in the sample. Second, the coeﬃcient estimates for Bethlehem are highly implausible. The data for the City of Bethlehem are unrepresentative, coming from less than 5 percent of its residences, mostly low-income housing and rental units, including multi-unit dwellings. In view of this, Bethlehem is excluded from the remainder of the analysis. Table 5 reports summary statistics for variables in the combined sample excluding Beth- lehem. Note that the average monthly water use in the sample is 3,460 gallons, the average marginal price per 1000 gallons of water in 2015 dollars is 8.7, drought watch status is present in 25 percent of the observations, but only 2 percent of the observations have a drought warning and less than 1 percent have mandatory restrictions. The sample mean of duration is 1.0 months, but conditional on there being a drought, the mean duration is 5.1 months (see Figure 6). Results from estimating Equation (8) with the combined sample are presented in Table 6. Column (1) shows coeﬃcient estimates when the interactive terms involving drought status are excluded. The coeﬃcient on price is negative and signiﬁcant, and implies a price KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS 237 Table 5: Summary Statistics Mean Variable Deﬁnition (Std. Dev.) water Monthly water use per residential unit (1000 gallons) 3.46 (2.18) price Marginal price per 1000 gallons of water in 2015 dollars 8.68 (2.15) watch =1 if county drought status ”Watch” for 1/2month 0.25 (0.43) warning =1 if county drought status ”Warning” for 1/2month 0.02 (0.14) mandatory =1 if mandatory water restrictions in place (Galeton) 0.004 (0.066) duration Consecutive months under a drought (0, 1, 2, ...) 1.00 (2.07) precip Monthly precipitation in inches 3.28 (1.82) temp Monthly average of daily maximum temperature (F) 61.2 (17.7) Note: Bethlehem is excluded. Table 5: Summary Statistics Note: Bethlehem is excluded. elasticity of water demand of -0.23 at the sample mean. The coeﬃcients on watch and warning are negative but statistically insigniﬁcant. c⃝Southern Regional Science Association 2019. g = exp(b2 + b5price + b7duration) −1, (9) 6.2. Regression Results The coeﬃcient on the mandatory water restrictions in Galeton, however, is negative and signiﬁcant. The weather coeﬃcients have the anticipated signs and are statistically signiﬁcant. Column (2) of Table 6 shows the estimates when the interaction term watch ∗price is included. Interaction terms involving warning and mandatory are excluded because there is no cross section variability, there are relatively few observations on them, and when included the estimated coeﬃcients (not shown) are implausible. Noteworthy is the negative and signiﬁcant coeﬃcient on watch ∗price. This result is consistent with the notion that the higher the price of water, the more likely is compliance with voluntary water use restrictions. The estimates when watch ∗duration is included are reported in column (3) of Table 6. With this speciﬁcation, all of the coeﬃcients have the anticipated signs and are statistically signiﬁcant, including the coeﬃcients on the drought warning indicator and the interaction terms. The higher the price of water and the longer a drought lasts, the greater the voluntary reduction in water use during a drought watch. The estimated price elasticity of demand evaluated at the sample mean is -0.33 during a normal period. This elasticity estimate is similar to the mean price elasticity of -0.41 in the meta-analysis of Dalhuisen et al. (2003), although it is sensitive to the inclusion of the watch ∗duration interaction term. Halversen and Palmquist (1980) showed that the coeﬃcient on a dummy variable in a semilogarithmic regression equation, multiplied by 100, does not give the percentage eﬀect of that variable on the variable being explained. This is because the dummy variable is a discrete variable, rather than a continuous one. In the present setting, the relative eﬀect on monthly residential water use (g) of being in a drought watch is given by: g = exp(b2 + b5price + b7duration) −1, (9) The Review of Regional Studies 49(2) 238 Table 6: Regression Results Variable Coeﬃcient (standard error) (1) (2) (3) price -0.0263* -0.0283* -0.0384* (0.0085) (0.0085) (0.0087) watch -0.0037 0.0289* 0.0459* (0.0027) (0.0061) (0.0063) warning -0.0070 -0.0089 -0.0200* (0.0060) (0.0060) (0.0069) mandatory -0.0498* -0.0498* -0.0482* (0.0158) (0.0158) (0.0158) watchprice -0.0038 -0.0035* (0.0006) (0.0006) watchduration -0.0072 (0.0010) precip -0.0048* -0.0047* -0.0046* (0.0007) (0.0007) (0.0007) temp 0.0017* 0.0019* 0.0019*** (0.0005) (0.0005) (.0005) Observations 424,700 424,700 424,700 R2 0.025 0.025 0.025 Notes: Dependent variable is ln(water). Bethlehem is excluded. Fixed eﬀects model with robust standard errors clustered at the residential unit level. c⃝Southern Regional Science Association 2019. 6.2. Regression Results , , and ** indicate statistical signiﬁcance at the 10, 5 and 1 percent levels, respectively. Notes: Calculated from estimates in Table 6, column (3). Standard errors are in parentheses. , , and ** indicate statistical signiﬁcance at the 10, 5 and 1 percent levels, respectively. water use restrictions during a drought watch is shown in the middle section of Table 7. When the price is one standard deviation below the mean (6.5), the relative eﬀect of being in a drought watch is -0.0128, or -1.3 percent. At one standard deviation above the mean (10.9), the relative eﬀect of being in a drought watch is -0.0277, or -2.8 percent. In other words, increasing the marginal price of water by 50 percent more than doubles the response to a drought watch, although the impact is only 1.5 percentage points of water use. The longer a drought lasts, the greater the estimated reduction in water use under a drought watch is. In the third month of a drought watch, the relative change in water use is estimated to be -0.0063, or -0.6 percent (Table 7, bottom section). By the seventh month, the relative change in water use is -0.0343, or -3.4 percent. In light of the limited lengths of the moderate droughts in the sample, however, care should be taken when extrapolating the eﬀects of drought duration. The estimates of the eﬀects of water use restrictions are conditional on the weather vari- ables. Water use is found to decrease with precipitation and to increase with temperature. Periods of droughts are drier and perhaps hotter than normal, so water use would be higher during droughts absent any policy actions. The estimated eﬀects of voluntary and manda- tory water use restrictions are relative to what water use would have been under drought conditions without such restrictions. Any reduction in water use relative to normal water use would be smaller than the estimated eﬀects. c⃝Southern Regional Science Association 2019. 6.2. Regression Results , , and indicate statistical signiﬁcance at the 10, 5 and 1 percent levels, respectively. Table 6: Regression Results and the percentage eﬀect on water use is 100g. The relative eﬀect of being in a drought watch is evaluated at particular values of price and duration because of the interaction terms. Similar expressions without the interaction terms can be given for the relative eﬀects of a drought warning or mandatory water restrictions. Replacing the coeﬃcients in Equation (9) with consistent estimates of them will provide a consistent estimate of g (Kennedy, 1981). Estimates of the relative eﬀects of drought status on water use are reported in Table 7. These eﬀects are based on the results in column (3) of Table 6. The ﬁrst section shows that the estimated relative eﬀect of being in a drought watch is -0.0204, or -2.0 percent, when evaluated at the sample mean price (8.7) and the average duration of a drought episode (5). The estimated relative eﬀect of being in a drought warning is essentially the same (-0.0198, or -2.0 percent). Both of these estimated eﬀects are smaller than the eﬀects of drought watches and warnings desired by the PA DEP (5-10 percent and 10-15 percent, respectively), but are within the range Halich and Stephenson (2009) found in Virginia. The relative eﬀect of the mandatory water use restrictions in Galeton is found to be -0.0471, or -4.7 percent. This falls near the bottom of the range of estimates in previous studies, but indicates that mandatory water use restrictions reduce residential water use more than voluntary restrictions. The quantitative importance of the marginal price of water to the eﬀects of voluntary The quantitative importance of the marginal price of water to the eﬀects c⃝Southern Regional Science Association 2019. KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS 239 Table 7: Relative Eﬀects of Drought Status on Water Use Variable Coeﬃcient (standard error) Price Duration Watch Warning Mandatory Mean (8.7) 5 months -0.0204 -0.0198* -0.0471* (0.0037) (0.0068) (0.0151) Mean+ (10.9) 5 months -0.0277* (0.0038) Mean- (6.5) 5 months -0.0128* (0.0041) Mean (8.7) 3 months -0.0063 (0.0027) Mean (8.7) 7 months -0.0343* (0.0052) Notes: Calculated from estimates in Table 6, column (3). Standard errors are in parentheses. , , and ** indicate statistical signiﬁcance at the 10, 5 and 1 percent levels, respectively. Notes: Calculated from estimates in Table 6, column (3). Standard errors are in parentheses. 7. CONCLUSION This study analyzes data on monthly household water use from four Pennsylvania water suppliers to assess the eﬀectiveness of water use restrictions over the period 2015-2017. Voluntary water use restrictions during drought watches are found to have signiﬁcant eﬀects, The Review of Regional Studies 49(2) 240 but smaller than desired by regulators. The reduction in water use during drought watches is found to be greater the higher the price of water. This is consistent with the notion that the higher the price of water, the smaller the welfare loss from some percentage reduction in water use, and the more likely is compliance with water use restrictions. Another ﬁnding is that the eﬀectiveness of voluntary water restrictions during drought watches increases with the length of the drought. There is some evidence that residential water use declines during drought warnings, but this result is sensitive to the speciﬁcation of the model, and the sparse number of time periods with drought warnings make any conclusions about them tenuous. The mandatory water use restrictions implemented by one small water supplier are found to reduce residential water demand by a slightly larger percentage than voluntary water use restrictions, consistent with the ﬁndings of previous studies. Based on the results of the present study, water suppliers in Pennsylvania may wish to reconsider the publicity that accompanies drought watches and warnings in order to increase their eﬀectiveness. For their part, state oﬃcials may wish to reconsider the importance of their goals for reductions in water use during drought watches and warnings and how to help water suppliers meet those goals. One problem is the revenue lost by water suppliers when water use restrictions are eﬀective. Lost revenue weakens the incentives water suppliers have to reduce water use during droughts. Addressing this problem satisfactorily might increase the eﬀectiveness of water use restrictions. Given the limitations of the data available for the present study, further investigation of the eﬀectiveness of water use restrictions during droughts in Pennsylvania and the impact of the price of water is warranted. Researchers, water suppliers, and state oﬃcials may wish to plan for a thorough randomized study of Pennsylvania households following the next period of widespread moderate and severe drought to inform and guide drought management policies and practices. KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS 241 Choose: Promoting Conservation by Relaxing Outdoor Watering Restriction Choose: Promoting Conservation by Relaxing Outdoor Watering Restrictions,” Journal of Economic Behavior and Organization, 107, 324–343. Choose: Promoting Conservation by Relaxing Outdoor Watering Restrictions,” Journal of Economic Behavior and Organization, 107, 324–343. C l E i A (2009) “A C i f D d Sid W t M t St t i g y g g , f Economic Behavior and Organization, 107, 324–343. Coleman, Eric A.. (2009) “A Comparison of Demand-Side Water Management Strategies Coleman, Eric A.. (2009) “A Comparison of Demand-Side Water Management Strategies Using Disaggregate Data,” Public Works Management Policy, 13(3), 215–223. Using Disaggregate Data,” Public Works Management Policy, 13(3), 215–223. Commonwealth of Pennsylvania, Department of Environmental Protection. (2016) Drought M t i P l i A il bl li i J l 2019 t htt // d / Commonwealth of Pennsylvania, Department of Environmental Protection. (2016) Drought Management in Pennsylvania. Available online in July 2019 at http://www.dep.pa.gov/ Business/Water/PlanningConservation/Drought/. Management in Pennsylvania. Available online in July 2019 at http://www.dep.pa.gov/ Business/Water/PlanningConservation/Drought/. Commonwealth of Pennsylvania, Department of Environmental Protection. (2017) Penn- sylvania Public Water System Compliance Report for 2016. Available online in July 2019 at http://www.dep.pa.gov/Business/Water/BureauSafeDrinkingWater/ DrinkingWaterMgmt/Pages/Program-Activities-Reports.aspx. Dalhuisen, Jaspar, Raymond Florax, Henri de Groot, and Peter Nijkamp. (2003) “Price and Income Elasticities of Residential Water Demand: A Meta-Analysis ” Land Economics Commonwealth of Pennsylvania, Department of Environmental Protection. (2017) Penn- sylvania Public Water System Compliance Report for 2016. Available online in July 2019 at http://www.dep.pa.gov/Business/Water/BureauSafeDrinkingWater/ DrinkingWaterMgmt/Pages/Program-Activities-Reports.aspx. Dalhuisen, Jaspar, Raymond Florax, Henri de Groot, and Peter Nijkamp. 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REFERENCES Abbott, Andrew, Shasikanta Nandeibaum, and Lucy O’Shea. (2013) “Recycling: Social Norms and Warm-Glow Revisited,” Ecological Economics, 90, 10–18. Alcott, Hunt. (2011) “Social Norms and Energy Conservation,” Journal of Public Economics, 95(10–11), 1082–1095. Andreoni, James. (1990) “Impure Altruism and Donations to Public Goods: A Theory of Warm-Glow Giving,” The Economic Journal, 100(4), 464–477. Binder, Martin and Ann-Kathrin Blankenberg. (2017) “Green Lifestyles and Subjective Well- being: More about Self-Image than Actual Behavior?,” Journal of Economic Behavior and Organization, 137, 304–323. Brekke, Kjell Arne, Snorre Kverndokk, and Karine Nyborg. (2003) “An Economic Model of Moral Motivation,” Journal of Public Economics, 87(9–10), 1967–1983. Brent, Daniel A., Joseph H. Cook, and Skylar Olsen. (2015) “Social Comparisons, Household Water Use, and Participation in Utility Conservation Programs: Evidence from Three Ran- domized Trials,” Journal of the Association of Environmental and Resource Economics, 2(4), 597–627. Castledine, A., Klaus Moeltner, Michael K. Price, and Shawn S. Stoddard. (2014) “Free to c⃝Southern Regional Science Association 2019. KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS (1981) “Exact Consumer’s Surplus and Deadweight Loss,” The American Economic Review, 71(4), 662–676. Kennedy, Peter E. (1981) “Estimation with Correctly Interpreted Dummy Variables in Semilogarithmic Equations,” The American Economic Review, 71(4), 201. Kenney, Douglas S., Roberta A. Klein, and Martyn P. Clark. (2004) “Use and Eﬀectiveness of Municipal Water Restrictions During Drought in Colorado,” Journal of the American Water Resources Association, 40(1), 77–87. ( ) Moncur, James E.T. (1987) “Urban Water Pricing and Drought Management,” Water Re- sources Research, 23(3), 393–398. Nieswiadomy, Michael L. (1992) “Estimating Urban Residential Water Demand: Eﬀects of Price Structure, Conservation, and Education,” Water Resources Research, 28(3), 609– 615. Olmstead, Sheila M. (2010) “The Economics of Managing Scarce Water Resources,” Review of Environmental Economics and Policy, 4(2), 179–198. Ostrom, Elinor. (2000) “Collective Action and the Evolution of Social Norms,” Journal of Economic Perspectives, 14(3), 137–158. Renwick, Mary E. and Sandra O Archibald. (1998) “Demand Side Management Policies for Residential Water Use: Who Bears the Conservation Burden?,” Land Economics, 74(3), 343–359. Renwick, Mary E. and Richard D. Green. (2000) “Do Residential Water Demand Side Man- c⃝Southern Regional Science Association 2019. c⃝Southern Regional Science Association 2019. The Review of Regional Studies 49(2) 242 agement Policies Measure Up? An Analysis of Eight California Water Agencies,” Journal of Environmental Economics and Management, 40(1), 37–55. agement Policies Measure Up? An Analysis of Eight California Water Agencies,” Journal of Environmental Economics and Management, 40(1), 37–55. Ridge, Tom. (1999) “Proclamation Declaration of Drought Emergency in the Commonwealth and Implementing Measures,” Pennsylvania Bulletin, 29(31), 4045–40049. Schweiker, Mark S. (2002) “Proclamation of Drought and Water Shortage Emergency,” Pennsylvania Bulletin, 32(8), 1035–1037. Shaw, Douglas T., R. Todd Henderson, and Martha E. Cardona. (1992) “Urban Drought Response in Southern California: 1990-91,” Journal – American Water Works Association, 84(10), 34–41. ( ) Shaw, Douglas T. and David R. Maidment. (1987) “Intervention Analysis of Water Use Restrictions, Austin, Texas,” Water Resources Bulletin, 23(6), 1037–1046. Shaw, Douglas T. and David R. Maidment. (1988) “Eﬀects of Conservation on Daily Water Use,” Journal – American Water Works Association, 80(9), 71–77. Wichman, Casey J., Laura O. Taylor, and Roger H. von Haefen. (1992) “Conservation Poli- cies: Who Responds to Price and Who Responds to Prescription?,” Journal of Environ- mental Economics and Management, 79, 114–134. Wilhite, Donald A and Roger S Pulwarty. c⃝Southern Regional Science Association 2019. KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS (2018) “Drought as Hazard: Understanding the Natural and Social Context,” In Wilhite, Donald A and Roger S Pulwarty, eds., Drought and Water Crises: Integrating Science, Management, and Policy, Second Edition. CRC Press: Boca Raton, FL. c⃝Southern Regional Science Association 2019. KROHN: DROUGHT STATUS, PRICE, AND WATER USE RESTRICTIONS 243 Stream Flows Stream Flows Every day, the average stream ﬂow of the preceding 30 days is computed from the United States Geological Survey (USGS) stream-gage records. This stream ﬂow statistic is compared with a ﬂow percentile that indicates the percent of the time on that date throughout the historical record that the ﬂow has been equal to or below that value. An average stream ﬂow over the last 30 days falling into a percentile range of 10 to 25 indicates a drought watch; a percentile range of 5 to 10 indicates a drought warning; and a percentile range of 0 to 5 indicates a drought emergency. APPENDIX: PENNSYLVANIA DROUGHT INDICATORS Precipitation Deﬁcits Precipitation Deﬁcits Every month in each county, total cumulative precipitation over periods ranging from three to 12 months are compared with the normal (average) values. Totals that are less than the normal values represent deﬁcits, which are converted to percentages of the normal values. Drought conditions are indicated by various precipitation deﬁcit percentages in relation to the duration of the deﬁcits. Table 8: Precipitation Deﬁcit Percentages Duration (months) Drought Watch Drought Warning Drought Emergency 3 25 35 45 4-6 20 30 40 7 18.5 28.5 38.5 8 17.5 27.5 37.5 9 16.5 26.5 36.5 10-12 15 25 35 Table 8: Precipitation Deﬁcit Percentages Groundwater Levels Every day, an average groundwater level in USGS observation wells over the preceding 30 days is computed. This groundwater statistic is compared with a groundwater percentile that indicates the percent of the time on that date throughout the historical record that the groundwater level has been equal to or below that value. As with stream ﬂows, an average groundwater level over the last 30 days falling into a percentile range of 10 to 25 indicates a drought watch; a percentile range of 5 to 10 indicates a drought warning; and a percentile range of 0 to 5 indicates a drought emergency. Soil moisture is measured by the Palmer Drought Severity Index, computed weekly by the National Weather Service for the 10 Palmer regions of the state. Palmer index values of -2.00 to -2.99 indicate a drought watch; values of -3.00 to 3.99 indicate a drought warning; and values of -4.00 and less indicate a drought emergency. Source: Pennsylvania Department of Environmental Protection, 2016.
https://openalex.org/W4387816735	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/E64EF389CF243BFDFFE00A2362701196/S0022377823000983a.pdf/div-class-title-non-thermal-particle-acceleration-and-power-law-tails-via-relaxation-to-universal-lynden-bell-equilibria-div.pdf	English	null	Non-thermal particle acceleration and power-law tails via relaxation to universal Lynden-Bell equilibria	Journal of plasma physics	2,023	cc-by	19,432	1 1 Non-thermal particle acceleration and power-law tails via relaxation to universal Lynden-Bell equilibria R.J. Ewart 1,2,†, M.L. Nastac 1,3 and A.A. Schekochihin 1,4 1Rudolf Peierls Centre for Theoretical Physics, University of Oxford, Oxford, OX1 3PU, UK 2Balliol College, Oxford, OX1 3BJ, UK 3St John’s College, Oxford, OX1 3JP, UK 4Merton College, Oxford, OX1 4JD, UK (Received 7 April 2023; revised 2 September 2023; accepted 4 September 2023) R.J. Ewart 1,2,†, M.L. Nastac 1,3 and A.A. Schekochihin 1,4 1Rudolf Peierls Centre for Theoretical Physics, University of Oxford, Oxford, OX1 3PU, UK 2Balliol College, Oxford, OX1 3BJ, UK 3St John’s College, Oxford, OX1 3JP, UK 4Merton College, Oxford, OX1 4JD, UK (Received 7 April 2023; revised 2 September 2023; accepted 4 September 2023) (Received 7 April 2023; revised 2 September 2023; accepted 4 September 2023) Collisionless and weakly collisional plasmas often exhibit non-thermal quasi-equilibria. Among these quasi-equilibria, distributions with power-law tails are ubiquitous. It is shown that the statistical-mechanical approach originally suggested by Lynden-Bell (Mon. Not. R. Astron. Soc., vol. 136, 1967, p. 101) can easily recover such power-law tails. Moreover, we show that, despite the apparent diversity of Lynden-Bell equilibria, a generic form of the equilibrium distribution at high energies is a ‘hard’ power-law tail ∝ε−2, where ε is the particle energy. The shape of the ‘core’ of the distribution, located at low energies, retains some dependence on the initial condition but it is the tail (or ‘halo’) that contains most of the energy. Thus, a degree of universality exists in collisionless plasmas. Key words: astrophysical plasmas, plasma nonlinear phenomena, space plasma physics † Email address for correspondence: robert.ewart@balliol.ox.ac.uk 1. Introduction It is well known that the ultimate fate of a homogeneous collisional plasma is to become a Maxwellian. This result was ﬁrst inferred for neutral particles by Maxwell (1860) on statistical grounds and given solid dynamical foundation by Boltzmann (1896) with his collision integral. Plasma physics was to wait for Landau (1936) and later Balescu (1960) and Lenard (1960) to be equipped with its own collision integral, and the resulting universality. Nevertheless, distributions with power-law tails, a far cry from Maxwellian equilibria, are observed in a myriad of plasma systems including cosmic rays (Becker Tjus & Merten 2020; Amato & Casanova 2021), the solar corona and solar ﬂares (Dudík et al. 2017; Oka et al. 2018), the solar wind (Gloeckler et al. 2008; Fisk & Gloeckler 2014; Livadiotis, Desai & Wilson 2018; Moncuquet et al. 2020), the Earth’s magnetosheath † Email address for correspondence: robert.ewart@balliol.ox.ac.uk https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press R.J. Ewart and others 2 (Birn et al. 2012; Ergun et al. 2020), and laser plasmas (Cruz et al. 2018; Hartouni et al. 2022). (Birn et al. 2012; Ergun et al. 2020), and laser plasmas (Cruz et al. 2018; Hartouni et al. 2022). That such non-Maxwellian distributions emerge should perhaps come as no surprise. In a plasma, the time scale of relaxation to Maxwellian equilibrium is associated with two-body Coulomb collisions, but, due to the long-range nature of the forces involved, the plasma may evolve, exchanging energy between ﬁelds and particles, on much shorter time scales. Indeed, in the absolute absence of collisions, the Vlasov equation has an inﬁnite set of nonlinearly stable equilibria: all distributions that are monotonically decreasing functions of particle energy are certainly stable (Gardner 1963). However, the set of all monotonically decreasing functions of energy is very large, and is certainly not exhausted by the Maxwellian equilibrium, which depends on only two parameters. It is, therefore, an outstanding challenge to determine whether any of these stable collisionless equilibria are naturally favoured by the dynamics of the system in a way that is not sensitive to initial conditions, i.e. whether a degree of universality exists in collisionless plasmas. 1. Introduction It is certainly the case that nature appears to prefer distributions with power-law tails, and direct numerical simulations have indicated that power-law tails are the natural result of a number of dynamical processes including relativistic and non-relativistic shocks (Sironi & Spitkovsky 2010; Caprioli & Spitkovsky 2014; Crumley et al. 2019), magnetic reconnection (Sironi & Spitkovsky 2014; Werner & Uzdensky 2021; Uzdensky 2022), and various types of plasma turbulence (Kunz, Stone & Quataert 2016; Zhdankin et al. 2017, 2019; Comisso & Sironi 2018, 2022; Zhdankin 2021, 2022b). In addition to suggesting dynamical paths towards distributions with power-law tails, there have been multiple attempts to justify the ubiquity of such distributions from a thermodynamic point of view. This is, however, entangled with the question of whether the standard Gibbs–Shannon entropy is applicable to systems with long-range interactions and, if not, then what entropy should be used. Naturally, many entropies have emerged to ﬁll this niche. A popular contender is the Tsallis (1988) entropy (or α-structural entropy: see Havrda & Charvát 1967). The Tsallis entropy was designed to be a non-additive version of the Gibbs–Shannon entropy as a way to model systems with correlations that therefore should be non-extensive (see, e.g. Livadiotis & McComas (2009), Pierrard & Lazar (2010), and references therein). While this model produces good ﬁts to observed distributions, it has a free parameter that is needed to quantify the degree of the non-extensivity and cannot be determined without ﬁtting data, or additional input of physics currently lacking (note some recent progress suggesting that this additional physics might be deducible from free-energy considerations: Zhdankin 2022a,b). gy ) An early attempt to tackle the question of entropy in collisionless systems was made by Lynden-Bell (1967). Let us consider a system of N particles with canonical positions ri and momenta pi that evolve subject to a Hamiltonian H(ri, pi). Such a system can be said to be ‘collisionless’ if the evolution equation for the single-particle distribution function f(r, p) is well approximated by an effective Hamiltonian Heff(r, p) acting on a single particle (i.e. if the mean-ﬁeld dynamics are a sufﬁciently good approximation to the true dynamics), viz., ∂f ∂t + ∂Heff ∂p · ∂f ∂r −∂Heff ∂r · ∂f ∂p = 0. (1.1) (1.1) In his original treatment, Lynden-Bell focused on relaxation of stellar systems, but the spirit of his statistical mechanics is the same for all collisionless systems, including plasmas. 1. Introduction While keeping the calculations as general as possible, one can think of (1.1) as the collisionless Vlasov equation for a plasma, which could be electrostatic or electromagnetic in a non-relativistic or relativistic regime. The collisionless dynamics described by (1.1) In his original treatment, Lynden-Bell focused on relaxation of stellar systems, but the spirit of his statistical mechanics is the same for all collisionless systems, including plasmas. While keeping the calculations as general as possible, one can think of (1.1) as the collisionless Vlasov equation for a plasma, which could be electrostatic or electromagnetic in a non-relativistic or relativistic regime. The collisionless dynamics described by (1.1) https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press Universal Lynden-Bell equilibria 3 conserve an inﬁnite number of invariants, equivalent to conserving the volume of level sets of the distribution function f(r, p) in phase space. Thus, the dynamics can be viewed as an extremely complicated rearranging of the elements of phase space, which, however much they are distorted and stirred, will keep the same level sets (often referred to as ‘waterbags’, in analogy with parcels of incompressible ﬂuid). Lynden-Bell posited that, after a short time, the exact phase-space density f(r, p) would become so chaotic that it could be treated as a random ﬁeld and that any measurement of it – in practice, of a coarse-grained version of it – was in fact a measurement of the mean phase-space density. This allowed the construction of a statistical mechanics, with an entropy closely related to the Gibbs–Shannon entropy, that encoded an inﬁnite number of invariants and thus predicted the steady states from a given initial condition. These steady states are the Lynden-Bell equilibria. y q Since its genesis, Lynden-Bell’s theory (often referred to as the theory of ‘violent relaxation’) has received continued attention both thermodynamically (Chavanis, Sommeria & Robert 1996; Arad & Johansson 2005; Chavanis 2006a,b; Levin, Pakter & Teles 2008; Levin et al. 2014) and dynamically, viz., effective ‘collisionless collision integrals’ have been proposed that recovered Lynden-Bell equilibria as their ﬁxed points (Kadomtsev & Pogutse 1970; Severne & Luwel 1980; Chavanis 2004, 2022; Ewart et al. 2022). However, the main strength of the theory is also its weakness. Unlike in the non-extensive entropy formulations, there is no ad hoc parameter in the Lynden-Bell theory: equilibria are uniquely determined by the ‘waterbag content’ of the initial conditions. 1This takes a surprising amount of work to show formally: see Appendix A. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 1. Introduction When the system is in its minimum-energy (ground) state, the Gardner distribution, the competition for phase volume is the overpowering factor, giving the distribution a highly non-universal shape. However, when the energy of the system is increased, more of the phase space becomes accessible, so, as in Fermi–Dirac statistics, the competition for the same phase volume becomes weaker. For sufﬁciently large energies, the competition for any volume of phase space is minuscule. In this limit, each waterbag will form its own Maxwellian distribution, in thermal equilibrium with all other waterbags. However, despite these Maxwellian equilibria having the same effective temperature, they will have different thermal spreads because waterbags of larger phase-space density ‘cost’ more energy to be placed at a given momentum p in phase space. The true distribution function is recovered by summing up (in the limit of many waterbags, integrating) the contributions from each of these Maxwellians to the mean phase-space density. This procedure naturally gives rise to a power-law tail that depends on the relative weighting for each Maxwellian (this is qualitatively similar to the formalism of ‘superstatistics’; cf. Beck & Cohen 2003; Chavanis 2006a; Davis et al. 2023). This weighting turns out to depend only weakly on the level sets of the initial condition. For a wide class of initial conditions, the resulting Lynden-Bell equilibria turn out to have the same universal power-law tail, ε−2.2 The rest of this paper is organised as follows. In § 2, we will review brieﬂy the Lynden-Bell formalism, to state the problem and establish notation. We will then proceed to perform a systematic exploration of the nature of the Lynden-Bell equilibria. In § 3.1, we will argue that to each initial condition, one can uniquely assign a Gardner distribution function with the same Casimir invariants (waterbag content). All Lynden-Bell equilibria can thus be viewed as the result of adding some amount of energy to a Gardner distribution with the same waterbag content and letting it reach a maximum-entropy state (one can think of this approach as describing how adding energy to a population of collisionless particles causes them to form a ‘non-thermal’ distribution with a tail). In § 3.2, we will show that the function describing the waterbag content of a large class of Gardner distributions has a relatively generic form, which will contribute to the universality of the resulting equilibria. 1. Introduction However, this necessarily means that the equilibria depend (seemingly, in a complicated way) on an inﬁnite family of invariants (sometimes referred to as ‘Casimirs’). This has limited any actual calculations with Lynden-Bell equilibria to simpliﬁed situations with only a small number of level sets (in practice, between one and three, e.g. Assllani et al. 2012). At any rate, the intricate dependence on an inﬁnite family of invariants might not appear to be a step towards general power-law tails or any other meaningful form of universality. To see just how non-universal Lynden-Bell equilibria can be, one only needs to consider the relation between the Lynden-Bell equilibria and the aforementioned Gardner distributions. Should the initial distribution be a monotonically decreasing function of particle energy, then it is a Gardner distribution and there are no possible rearrangements of the phase volume that do not increase energy. Hence, the only state available via collisionless dynamics is this Gardner distribution, which must therefore be its own Lynden-Bell equilibrium.1 But since any monotonically decreasing function of energy is a Gardner distribution, these minimum-energy states are clearly highly non-universal. However, this is only a good intuition for systems where the number of level sets is small or where phase-volume conservation conspires with energy conservation to render much of the phase space inaccessible to the system (as is the case for Gardner distributions). In this paper, by solving for the full Lynden-Bell equilibria numerically (as well as analytically, in a tractable limit), we will show that most Lynden-Bell equilibria are much more generic. Namely, we will show that, in the limit of a continuum of level sets, and for energies sufﬁciently greater than the ground-state energy (the energy of the corresponding Gardner distribution), the Lynden-Bell equilibria exhibit power-law tails at high energies, typically with a scaling of ε−2, where ε is the particle energy. The physical argument for these power-law tails is as follows. Phase-volume conservation effectively makes the particles occupying each waterbag (level set of the distribution) behave as if they were members of a separate species, which can https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 4 R.J. Ewart and others communicate with the other waterbags only via the equilibration of some effective ‘temperature’ subject to competition for the same volumes of phase space. In essence, this turns the system into a ensemble of many different fermionic species, all of which exclude each other. 2This is similar to how Zipf’s law arises in systems where one marginalises over a ‘hidden variable’ (cf. Mora & Bialek 2011; Schwab, Nemenman & Mehta 2014; Aitchison, Corradi & Latham 2016). 1. Introduction In § 3.3, we will solve for the Lynden-Bell equilibria in the limit where the energy of the system far exceeds the energy of the corresponding Gardner distribution. This will ensure that competition for volumes of phase space can be neglected. This makes the problem analytically tractable, and the resulting analytical solution will exhibit the universal power-law tail ∝ε−2 at high energies. In § 4, by solving for the Lynden-Bell equilibria numerically, we will show that the qualitative features of this analytical solution are retained even for energies that are of the same order as the energy of the Gardner distribution. Therefore, a large class of Lynden-Bell equilibria display a universal power-law tail. This tail contains much of the distribution’s energy, whereas the low-energy ‘core’ retains some dependence on the initial conditions. In § 5, we summarise our ﬁndings and discuss their implications for real (observed) plasmas. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 2. Lynden-Bell’s statistical mechanics In this section, we present a brief re-derivation of Lynden-Bell’s equilibria as applied to a homogeneous system. We begin with the collisionless Vlasov equation (1.1) evolving a https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press Universal Lynden-Bell equilibria 5 5 Universal Lynden-Bell equilibria single species of particles. As well as particle number, momentum, and total energy (i.e. of ﬁelds and particles), (1.1) conserves an inﬁnite number of ‘Casimir’ invariants, e.g. the volume of phase space where the exact phase-space density is greater than a given value: single species of particles. As well as particle number, momentum, and total energy (i.e. of ﬁelds and particles), (1.1) conserves an inﬁnite number of ‘Casimir’ invariants, e.g. the volume of phase space where the exact phase-space density is greater than a given value: (η) = dr dpH( f(r, p) −η) = const., (2.1) (2.1) where H(x) is the Heaviside function (unity for x > 0 and zero otherwise). As discussed above, despite the existence of these invariants, the system’s evolution can still be highly chaotic, which prompted Lynden-Bell to consider the exact phase-space density f(r, p) as a random ﬁeld. Therefore, one may introduce the probability density P(r, p, η) for the exact phase-space density f(r, p) to take the value η at position (r, p) (Robert & Sommeria 1991; Chavanis et al. 1996). The distinct values of η will be referred to as ‘waterbags’ since this term conjures up the correct mental image: parcels of phase space of a certain density that can be distorted and moved, but not rareﬁed, compressed, or superimposed. The mean phase-space density is then ⟨f⟩(p) = dηηP(p, η). (2.2) (2.2) Here, we have applied the intuition that the steady-state distribution function P(p, η) will be homogeneous in space (this contrasts with Lynden-Bell’s original treatment, which focused on gravitationally bound, and therefore inhomogeneous, systems). Lynden-Bell’s statistical mechanics amounts to positing that, before the onset of ‘true’ collisions, P(p, η) will maximise the Gibbs–Shannon entropy S = − dp dηP(p, η) ln P(p, η). (2.3) (2.3) Note that the integral in η must run over all the possible values, including η = 0 (the empty waterbag). Equipped with an entropy ripe for maximisation, we must decide upon a set of reasonable constraints under which to maximise it. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 2. Lynden-Bell’s statistical mechanics Naturally, since P(p, η) is a probability-density function in η at a given p, its integral in η at any p must equal unity: dηP(p, η) = 1. (2.4) (2.4) As well as this, we ﬁx the energy density of the system: As well as this, we ﬁx the energy density of the system: dp dηε(p)ηP(p, η) = E = const., (2.5) (2.5) where ε(p) is the energy of a particle as a function of its momentum p. Note that, within this formalism, one could include the interaction energy of particles with ﬁelds (electromagnetic, gravitational, etc.), so that in its most general form ε would be a function of both position and momentum, which could need to be solved self-consistently with P. Here, we will neglect this rich complexity, assuming instead that, in the relaxed state, the energy of the ﬁelds has decayed to a negligible fraction of the total energy and, in the process of decaying, has mediated the relaxation of the distribution function. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 6 R.J. Ewart and others Next, we enforce the conservation of the Casimir invariants (2.1) by requiring that the volume-integrated probability of each waterbag stays constant, viz., dpP(p, η) = ρ(η) = const. (2.6) (2.6) The function ρ(η) will be referred to as the ‘waterbag content’ and is determined by initial conditions. The waterbag content of the initial condition can be read off by integrating over all portions of phase space where the initial exact phase-space density is equal to a particular value, viz., The function ρ(η) will be referred to as the ‘waterbag content’ and is determined by initial conditions. The waterbag content of the initial condition can be read off by integrating over all portions of phase space where the initial exact phase-space density is equal to a particular value, viz., ρ(η) = 1 V dr dpδ(η −f(r, p, t = 0)) = −1 V d dη , (2.7) (2.7) where V is the system’s spatial volume. A priori, in Lynden-Bell’s statistical mechanics, the degree of universality of the equilibrium distribution is determined by ρ(η): all initial conditions with the same waterbag content and energy lead to the same equilibrium. 3We note that, while we have endowed the invariants (2.4)–(2.6) with special signiﬁcance as constraints, there may be situations where additional invariants are necessary. For instance, in strongly magnetised plasmas, relaxation may occur before the conservation of particles’ magnetic moments are broken. In such cases, further invariants would be necessary and would alter the character of the solution (cf. Helander 2017). Here, we shall consider only systems where the ﬁelds driving the relaxation may be arbitrary, but the only quantities conserved on the relaxation time scale are (2.4)–(2.6). 4Indeed, the Fermi–Dirac distribution can be thought of as the special case of a two-level-set system, which further reduces to the Maxwell–Boltzmann distribution when degeneracy is neglected (see, e.g. Chavanis (2006a) and Ewart et al. (2022) for details). https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 3.1. Lynden-Bell equilibria as excited Gardner distributions 3.1. Lynden-Bell equilibria as excited Gardner distributions Just as it is only meaningful to consider a Maxwellian with a positive energy, it is only meaningful to solve the Lynden-Bell equilibria (2.9) subject to reasonable choices of the constraints (2.5) and (2.6). It is therefore instructive to understand the properties of the waterbag-content function (2.6). To get a feel for waterbag contents of typical initial conditions, one could compute the integral (2.7) for a range of examples (this is relatively simple due to the presence of the delta function). One quickly discovers that many different initial conditions have similar waterbag contents, just as many different initial conditions can have the same energy. To see this, we note that, from (2.7), any volume-preserving transformation of the coordinates (r, p), including those transformations that ‘splice’ the phase space discontinuously, will leave the waterbag content unchanged. This is unsurprising because the true, incompressible, ﬂow of probability in phase space is precisely one such volume-preserving transformation. It is this freedom that implies that vastly different initial conditions can possess identical, or similar, waterbag contents. A cartoon illustrating this is given in ﬁgure 1, showing how seemingly complex distributions have the same waterbag content as very simple distributions. There will be families of initial conditions that have the same waterbag content, but different energies. For every family of initial conditions possessing the same waterbag content, there will be a unique distribution function that has that waterbag content but is a monotonically decreasing function of energy and, therefore, has the minimum possible energy associated with that waterbag content. Such a distribution function, for which the exact phase-space density satisﬁes f(r, p) = fG(ε(p)), is known as the Gardner distribution (Gardner 1963; Helander 2017). The sequence of deformations of the distribution function to map an initial condition to its Gardner distribution function is often referred to as a ‘restacking’, as it amounts to a reordering of phase-space elements into their minimum-energy conﬁguration (Dodin & Fisch 2005; Kolmes & Fisch 2020; Kolmes, Helander & Fisch 2020). Gardner distributions can be viewed as ‘ground states’ associated with a given waterbag content (e.g. Helander 2017), since no more energy can be extracted from such a distribution without violating phase-volume conservation. This fact intuitively guarantees that any initial condition that is a Gardner distribution is its own Lynden-Bell equilibrium since no other states are available to the system. 2. Lynden-Bell’s statistical mechanics Maximising the entropy (2.3) subject to the constraints (2.4)–(2.6)3 is equivalent to maximising, unconditionally, the functional S[P(p, η)] − dpλ(p) dηP(p, η) −1 −β dp dηε(p)ηP(p, η) −E + β dηημ(η) dpP(p, η) −ρ(η) , (2.8) (2.8) where λ(p), β and −βημ(η) are Lagrange multipliers. By analogy with textbook statistical mechanics, we will sometimes refer to μ(η) as the ‘chemical potential’ (which it is, being the Lagrange multiplier that ﬁxes the number of particles in waterbag η). Doing so, we ﬁnd the Lynden-Bell equilibria P(p, η) = e−βη[ε(p)−μ(η)] dη′ e−βη′[ε(p)−μ(η′)] , (2.9) (2.9) where λ(p) has been computed explicitly to arrange for the correct normalisation (2.4), whereas β and μ(η) must be chosen in such a way as to satisfy the constraints (2.5) and (2.6). We note that, despite (2.6), the mean phase-space density ⟨f⟩, given by (2.2), will, in general, not have the same level sets (2.1) as the exact one f (since ⟨f⟩is an averaged quantity). The equilibria (2.9) are both homogeneous and isotropic: an inevitable consequence of the system having no preferred position or direction. The similarity between the Lynden-Bell equilibria (2.9) and the Fermi–Dirac distribution is immediately apparent.4 This should come as no surprise, because phase-volume conservation functions analogously to Pauli’s exclusion principle: pieces of https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press Universal Lynden-Bell equilibria 7 the same waterbag, or different waterbags, cannot cohabit in phase space. The equilibria, therefore, have degeneracy effects incorporated within them. The prescription for computing Lynden-Bell equilibria is now clear: given an initial condition, with the initial energy density E and waterbag content ρ(η) (determined by (2.7)), solve two coupled integral equations (2.5) and (2.6) with P(p, η) given by (2.9), determine β and μ(η), and substitute back into (2.9). Before considering the numerical solutions of this problem in § 4, we will ﬁrst seek to understand the system analytically. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 3.1. Lynden-Bell equilibria as excited Gardner distributions Indeed, one can show that any Gardner distribution can be reconstructed from (2.9) for a particular choice of β and μ(η), although the proof is technical and left to Appendix A. A generic initial condition can then be viewed as equivalent to taking some Gardner distribution and driving it out of equilibrium by the injection of some energy without changing the waterbag content. The Lynden-Bell equilibria are then simply the collisionless, phase-volume preserving, entropy-maximising equilibria of these higher-energy states, making them the natural excited states of Gardner distributions. Therefore, to capture the set of all possible waterbag contents, we need only study the set of all these ‘ground states’, to which we would then add energy – the ﬁrst step in R.J. Ewart and others 8 8 (a) (b) (c) FIGURE 1. A cartoon contour plot in phase space of three possible distribution functions, all of which possess identical waterbag contents. Panel (a) shows the Gardner distribution function corresponding to this waterbag content. Panels (b) and (c) show distributions, at different (higher) energies, which can be reduced to the Gardner distribution by deforming and splicing the phase space incompressibly. A small patch of phase space is highlighted in red between plots to show the effect of the deformation. b) (a) (b) (c) (a) ) FIGURE 1. A cartoon contour plot in phase space of three possible distribution functions, all of which possess identical waterbag contents. Panel (a) shows the Gardner distribution function corresponding to this waterbag content. Panels (b) and (c) show distributions, at different (higher) energies, which can be reduced to the Gardner distribution by deforming and splicing the phase space incompressibly. A small patch of phase space is highlighted in red between plots to show the effect of the deformation. the direction of universal outcomes. Physically, this approach is equivalent to asking to what distribution a population of collisionless particles will relax once a certain amount of energy is injected into it – in a manner of speaking, a ‘thermodynamical’ approach to ‘non-thermal’ particle acceleration. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 3.2. Waterbag content of Gardner distributions Having stated the problem in this way, we now consider the waterbag content associated with Gardner distributions. In what follows, we will consider Gardner distribution functions that are truncated at some minimum phase-space density ηmin. This mathematical convenience will turn out to be a physical necessity. Thankfully, while it is mathematically and physically important that the cutoff ηmin be ﬁnite, it will only appear logarithmically in the outcomes of our calculations, making them highly insensitive to its actual value – yet another theory where the need for a cutoff is unavoidable but non-lethal. y y As a prototypical example, we compute ρ(η) for a particular Gardner distribution: a truncated Maxwellian, viz., fG(p) = ⎧ ⎪⎨ ⎪⎩ ηmax e−ε(p)/ε0 for ε(p) < ε0 ln ηmax ηmin , 0 for ε(p) > ε0 ln ηmax ηmin . (3.1) (3.1) Besides ηmin, the parameters of this distribution are the energy scale ε0 and the maximum phase-space density ηmax. The latter is straightforwardly related to the particle’s spatial density, e.g. n0 = ηmax(2πmε0)3/2 in the limit ηmin/ηmax →0 for a three-dimensional, non-relativistic plasma, where ε(p) = p2/2m. The waterbag content of the Gardner Besides ηmin, the parameters of this distribution are the energy scale ε0 and the maximum phase-space density ηmax. The latter is straightforwardly related to the particle’s spatial density, e.g. n0 = ηmax(2πmε0)3/2 in the limit ηmin/ηmax →0 for a three-dimensional, non-relativistic plasma, where ε(p) = p2/2m. The waterbag content of the Gardner oi.org/10.1017/S0022377823000983 Published online by Cambridge University Press https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press Universal Lynden-Bell equilibria 9 Universal Lynden-Bell equilibria 9 Universal Lynden-Bell equilibria 9 Universal Lynden-Bell equilibria 9 distribution for such a plasma is then, from (2.7), distribution for such a plasma is then, from (2.7), ρ(η) = freeδ(η) + ⎧ ⎨ ⎩ 2n0 √πηmaxη ln ηmax η 1/2 for ηmin < η < ηmax, 0 otherwise, (3.2) (3.2) where free is the total volume of the momentum space that is unoccupied (i.e. where the exact phase-space density is zero). Of course, in reality, momentum space is unbounded and so free is inﬁnite. Formally, we are solving for the waterbag content and Lynden-Bell equilibrium in a momentum space of large, but ﬁnite, volume and will take this volume to inﬁnity at the end of the calculation – of course, nothing physical will depend on free as it becomes large. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 3.2. Waterbag content of Gardner distributions In terms of this inverse, one can explicitly express the R.J. Ewart and others 10 (a) (b) FIGURE 2. (a) Three example Gardner distribution functions (the phase-space density here is plotted as a function of energy) and (b) their corresponding waterbag contents. All three distributions were chosen to have the same particle density n0 and energy density EG. The maximum phase-space density ηmax of the distribution sets the upper cutoff of the waterbag content in η, shown by the dashed vertical lines in (b). The lower cutoff ηmin is justiﬁed in § 3.5. We see that large differences at low ε only change the behaviour of ρ(η) signiﬁcantly at η ∼ηmax. For η ≪ηmax, all three waterbag contents asymptote to a universal η−1 scaling. (a) (b) (b) (a) FIGURE 2. (a) Three example Gardner distribution functions (the phase-space density here is plotted as a function of energy) and (b) their corresponding waterbag contents. All three distributions were chosen to have the same particle density n0 and energy density EG. The maximum phase-space density ηmax of the distribution sets the upper cutoff of the waterbag content in η, shown by the dashed vertical lines in (b). The lower cutoff ηmin is justiﬁed in § 3.5. We see that large differences at low ε only change the behaviour of ρ(η) signiﬁcantly at η ∼ηmax. For η ≪ηmax, all three waterbag contents asymptote to a universal η−1 scaling. waterbag content (2.7) as waterbag content (2.7) as bag content (2.7) as ρ(η) = dεg(ε)δ(η −fG(ε)) = −g( f −1 G (η))df −1 G dη =⇒dfG dε = − g(ε) ρ( fG(ε)), (3.5) (3.5) where g(ε) is the density of states in energy, deﬁned by the equation dp(· · · ) = dεg(ε)(· · · ). (3.6) (3.6) The ﬁrst equality in (3.5) is a straightforward way to calculate the waterbag content of a given Gardner distribution, while the second is an equation from which the Gardner distribution can be constructed given knowledge of the system’s waterbag content (cf. Dodin & Fisch 2005; Helander 2017). It is immediately clear why the η−1 scaling should arise in ρ(η). For any exponentially decaying fG(ε), the inverse function will be some logarithmic function of η, which, after differentiation in (3.5), will give an η−1 asymptotic multiplied by some logarithmic function of η. 3.2. Waterbag content of Gardner distributions The presence of a δ(η) term in the expression for ρ(η) is a generic feature, not restricted to the speciﬁc example (3.2). When it comes to solving (2.6) with P(p, η) given by (2.9), in order to ﬁnd μ(η), this delta function can be accommodated by writing the chemical potential as eβημ(η) = ηrefδ(η) + ηrefF(η) for ηmin < η < ηmax, 0 otherwise, (3.3) (3.3) where ηref is some reference constant that must have dimensions of phase-space density. Its value is unimportant because eβημ(η) can always be rescaled by a constant without changing the Lynden-Bell equilibrium (2.9) – in essence, ηref is a gauge choice for the function μ(η). By analogy to textbook statistical mechanics, the function F(η) will be referred to as the ‘fugacity’ of the distribution. The form (3.3) results in the following expression for the Lynden-Bell equilibrium (2.9): P(p, η) = δ(η) + e−βηε(p)F(η) 1 + ηmax ηmin dη′ e−βη′ε(p)F(η′) . (3.4) (3.4) In (3.4), the ﬁrst, δ(η), term in the numerator accounts for the probability density of ﬁnding phase space to be empty at a given location (this part of the phase space is referred to, aptly, as the ‘vacuum’ by Chavanis 2006a), whereas the second term accounts for non-empty waterbags. Already free has dropped out of the calculation, as it must, and it is safe to let free →∞. Likewise, it is immediately obvious that the reference phase-space density ηref has cancelled, as it also must. Thus, the Lynden-Bell equilibrium distribution (3.4) depends only on the Lagrange multiplier β and the fugacity F(η) (which themselves depend on ρ(η) for η > 0 and the energy density E). p ρ η η gy y The key feature of the example (3.2) is the η−1 power-law behaviour. While the speciﬁc form of the logarithmic factor in (3.2) was set by our (non-universal) choice of a Maxwellian Gardner distribution, the η−1 behaviour at small η is relatively universal. For any exponentially decaying Gardner distribution, i.e. for any distribution that at large energies can be approximated by ∝exp[−(ε/ε0)σ], for some σ > 0 (or indeed can be bounded between two such functions), one will ﬁnd a waterbag content with an η−1 asymptotic at η ≪ηmax. To see this, we note that, since fG(ε) is monotonically decreasing with energy, it has a well-deﬁned inverse f −1 G (η). https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 3.2. Waterbag content of Gardner distributions However, for γ = 0, (3.7) becomes the (spatially averaged) momentum-space volume occupied by the truncated distribution: its support. For exponentially decaying distributions, which do not have compact support without truncation, this quantity will continue to grow without bound as ηmin is decreased. Therefore, ρ(η) integrated with no powers of η must diverge as ηmin →0. It is obvious that η−1 is a function that has all these properties, but, more generally, ρ(η) could be any function of the form where we have used (2.7), and the phase-space integral is taken over the volumes where η > ηmin. We have also used the property that the process of varying ηmin while otherwise leaving f(r, p) unchanged only changes the integration limits of ρ(η), without changing ρ(η) itself. The idea now is to vary the values of ηmin and γ and use what we know about f(r, p) to deduce the form of ρ(η). Clearly, for γ = 1, (3.7) is the particle density of the truncated f(r, p), which must be ﬁnite. This tells us that ρ(η) must integrate to a ﬁnite value when multiplied by η. Furthermore, should f be any exponentially decaying function, then there would be a characteristic momentum scale above which the distribution is suppressed. This means that, as ηmin is taken to zero, both sides of (3.7) must converge for γ = 1. This is effectively a statement that the amount of probability contained beyond a few standard deviations is small. However, for an exponentially decaying phase-space density f and any positive power γ > 0, f γ is also exponentially decaying, so the same argument applies. Therefore, for exponentially decaying phase-space densities, ρ(η) must be such that, when multiplied by any positive power of η, it integrates to some ﬁnite value and is largely independent of the choice of lower cutoff ηmin of the integral. However, for γ = 0, (3.7) becomes the (spatially averaged) momentum-space volume occupied by the truncated distribution: its support. For exponentially decaying distributions, which do not have compact support without truncation, this quantity will continue to grow without bound as ηmin is decreased. Therefore, ρ(η) integrated with no powers of η must diverge as ηmin →0. 3.2. Waterbag content of Gardner distributions To illustrate this, in ﬁgure 2, we give three examples of starkly different Gardner distributions that, despite their differences, all possess waterbag contents which scale as η−1 at low η. To convince a doubtful reader, we consider an alternative argument in support of the η−1 scaling of ρ(η). First, let us imagine a system in which we are allowed to vary ηmin freely while leaving the exact phase-space density f(v) otherwise unchanged, as if there were some true distribution that had ηmin = 0 and we were examining successive approximations to it (e.g. the difference between a truncated Maxwellian (3.1) and a true Maxwellian). Let us consider the following integrals of such distribution functions (similar Universal Lynden-Bell equilibria 11 Universal Lynden-Bell equilibria 11 Universal Lynden-Bell equilibria 11 to the Casimir invariants considered by Zhdankin 2022a): to the Casimir invariants considered by Zhdankin 2022a): 1 V f(r,p)>ηmin dr dp[ f(r, p)]γ = ηmax ηmin dηηγρ(η), (3.7) (3.7) where we have used (2.7), and the phase-space integral is taken over the volumes where η > ηmin. We have also used the property that the process of varying ηmin while otherwise leaving f(r, p) unchanged only changes the integration limits of ρ(η), without changing ρ(η) itself. The idea now is to vary the values of ηmin and γ and use what we know about f(r, p) to deduce the form of ρ(η). Clearly, for γ = 1, (3.7) is the particle density of the truncated f(r, p), which must be ﬁnite. This tells us that ρ(η) must integrate to a ﬁnite value when multiplied by η. Furthermore, should f be any exponentially decaying function, then there would be a characteristic momentum scale above which the distribution is suppressed. This means that, as ηmin is taken to zero, both sides of (3.7) must converge for γ = 1. This is effectively a statement that the amount of probability contained beyond a few standard deviations is small. However, for an exponentially decaying phase-space density f and any positive power γ > 0, f γ is also exponentially decaying, so the same argument applies. Therefore, for exponentially decaying phase-space densities, ρ(η) must be such that, when multiplied by any positive power of η, it integrates to some ﬁnite value and is largely independent of the choice of lower cutoff ηmin of the integral. 3.2. Waterbag content of Gardner distributions To enable an explicit calculation, we will assume henceforth that the density of states g(ε) is related to energy by a simple power law, viz., g(ε) = Aεa, (3.11) (3.11) where A is an appropriate constant with dimensions [n0/ηmaxε1+a], and a is a real number. The assumption (3.11) is not too restrictive as it can capture both non-relativistic and ultra-relativistic systems of any dimensionality. With the assumption (3.11), we may now use (3.5) to link the value of δ to the high-energy asymptotic of the Gardner distribution. If the Gardner distribution has the power-law tail fG(p) ∝ε(p)−(χ+a) at high energies, then, via (3.5), one ﬁnds δ = 1 + (a + 1)/(a + χ). If, instead, the Gardner distribution goes to zero at some ﬁnite energy εmax so that fG(p) ∝[εmax −ε(p)]χ near ε = εmax, then δ = 1 −1/χ. Having catalogued the possible Gardner distributions and their corresponding ρ(η), an important open question is now what Gardner distributions are the most common. Within the domain of numerical experiments, this is clearly decided by the whims of the numerical experimenter. However, it would seem reasonable to conjecture that in nature, the most common Gardner distributions should be ones with exponential tails. The reason for this is that the only processes that can change the Gardner distribution are, by deﬁnition, collisional, and collisional processes naturally relax the system to a Maxwell–Boltzmann distribution. More concretely, one often thinks of the sources of energy for violent relaxation as being large-scale inhomogeneities (e.g. counter-propagating ﬂows, collapsing distributions of matter, etc.) of a system that is locally collisionally relaxed, and, therefore, has an exponential Gardner distribution. We are now safe in the knowledge that the exponentially decaying Gardner distributions have ρ(η) ∝η−1, or, in more exotic cases, ρ(η) ∝η−δ. We may now return to the task of solving (2.5) and (2.6) in light of these facts. 3.2. Waterbag content of Gardner distributions It is obvious that η−1 is a function that has all these properties, but, more generally, ρ(η) could be any function of the form ρ(η) = n0 ηmaxηG η ηmax for ηmin < η < ηmax, (3.8) (3.8) where G(x) is a dimensionless function whose dependence on x is weaker than any power law, viz., lim x→0 xγ −1G(x) = 0 if γ > 1, ∞ if γ < 1. (3.9) (3.9) Note that the exact limit at γ = 1 cannot be determined by this argument; it is, in fact, dependent on the exact details of the exponential decay, but we will not require it for any calculations. This concludes the argument that Gardner distributions with exponential tails have waterbag contents with a universal low-η asymptotic. It is a straightforward extension of this argument to work out what the waterbag content will be for Gardner distributions with non-exponential tails at high energies (low η). Suppose that, instead of being exponentially decaying, the Gardner distribution behaves as a power law at large momenta. Then there is a choice of γ > 0 for which f γ , multiplied by the density of states in \|p\|, decays slower than \|p\|−1, implying that the integral (3.7) will diverge as ηmin is decreased. This means that ρ(η) multiplied by ηγ for some γ > 0 must still have a divergent integral, so it must take the form ρ(η) = n0 η2−δ maxηδ G η ηmax for ηmin < η < ηmax, (3.10) (3.10) https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 12 R.J. Ewart and others with some δ > 1. It turns out that (3.10) also holds, but with δ < 1, for phase-space densities that go to zero algebraically even when ηmin = 0.5 This is because the integral (3.7) will be over a ﬁnite momentum-space volume even as ηmin is taken to zero, so, while γ < 0 will make f γ diverge in this ﬁnite domain, that divergence will still be integrable if γ is chosen sufﬁciently small and negative. The explicit value of δ can be found from (3.5). 5To apply this argument more generally to functions that have compact support in momentum space but do not go to zero algebraically (e.g. step or bump functions), one can represent them as the limit of a sequence of functions that do go to zero algebraically. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 3.3. Non-degenerate Lynden-Bell equilibria Taking inspiration from the similarity between Fermi–Dirac and Lynden-Bell statistics, we may expect that there are two analytically tractable limits: degenerate (‘cold’) and non-degenerate (‘hot’). In this section, we will explore the latter limit, which will turn out to be far more useful than the former (which is, nevertheless, also treated, for completeness, in Appendix A). We deﬁne the non-degenerate limit as one in which the probability of ﬁnding the exact phase-space density to be non-zero is small, viz., D(ε) = ηmax ηmin dη e−βηεF(η) ≪1. (3.12) (3.12) We shall call D(ε) the ‘degeneracy parameter’ since, from (3.4), the probability that a position in phase space with a given energy is non-empty is given by the We shall call D(ε) the ‘degeneracy parameter’ since, from (3.4), the probability that a position in phase space with a given energy is non-empty is given by the 5To apply this argument more generally to functions that have compact support in momentum space but do not go to zero algebraically (e.g. step or bump functions), one can represent them as the limit of a sequence of functions that do go to zero algebraically. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press Universal Lynden-Bell equilibria Universal Lynden-Bell equilibria 13 quotient D(ε)/[1 + D(ε)]. In the limit (3.12), the distribution function (3.4) can be approximated by quotient D(ε)/[1 + D(ε)]. In the limit (3.12), the distribution function (3.4) can be approximated by P(p, η) ≃δ(η)[1 −D(ε(p))] + e−βηε(p)F(η). (3.13) (3.13) The effect of this simpliﬁcation is that the competition for any particular sub-volume of phase space is so weak that the waterbags are free to arrange themselves as Maxwellians η by η. The waterbags with lower η cost less energy to be placed at larger momenta – therefore, they have a larger thermal spread. In the non-relativistic limit, this is equivalent to the intuition that particles belonging to the waterbags with higher phase-space densities behave as though they have larger masses. The approximate form (3.13) of the Lynden-Bell distribution makes computing the momentum-space integral in (2.6) and determining the fugacity F(η) a simple matter. Substituting (3.13) into (2.6) and using the explicit form (3.11) of the density of states, we ﬁnd the fugacity in the non-degenerate limit to be F(η) = (βη)1+a AΓ (a + 1)ρ(η), ηmin ≤η ≤ηmax, (3.14) (3.14) where Γ (a + 1) is the gamma function. S0022377823000983 Published online by Cambridge University Press 3.3. Non-degenerate Lynden-Bell equilibria Substituting (3.14) back into (3.13) and using (2.2) ﬁnally gives us an expression for the mean phase-space density (although still in terms of the as yet unspeciﬁed parameter β): where Γ (a + 1) is the gamma function. Substituting (3.14) back into (3.13) and using (2.2) ﬁnally gives us an expression for the mean phase-space density (although still in terms of the as yet unspeciﬁed parameter β): ⟨f⟩(p) = β1+a AΓ (a + 1) ηmax ηmin dηη2+aρ(η) e−βηε(p). (3.15) (3.15) From (3.15), it might seem as though, by diverse choices of ρ(η), a wide variety of distribution functions ⟨f⟩can be obtained. However, as we showed in § 3.2, a diversity of choices of waterbag contents is exactly what we do not have. Instead, fairly generic Gardner distributions with any form of exponential tails possess waterbag contents that are highly universal at low η. Using (3.8), we can make a convenient change of variables in (3.15), x = βηε(p), to ﬁnd ⟨f⟩(p) = n0 AβΓ (a + 1)ηmaxε(p)2+a βηmaxε(p) βηminε(p) dxx1+aG x βηmaxε(p) e−x. (3.16) (3.16) This form exposes the fact that there is a natural power-law behaviour at energies such that This form exposes the fact that there is a natural power-law behaviour at energies such that 1 βηmax ≪ε(p) ≪ 1 βηmin . (3.17) (3.17) This corresponds to the range of energies that are well within the thermal spread of the least dense waterbags, but far outside the thermal spread of the densest ones. At ε(p) ≫1/βηmin, the lower limit of the integral in (3.16) imposes an exponential cutoff on ⟨f⟩. The distribution function N(ε) of particle energies corresponding to (3.16) is obtained by multiplying the mean phase-space density ⟨f⟩(p) by the density of states: N(ε) = g(ε)⟨f⟩(p) = n0 βΓ (a + 1)ηmaxε2 βηmaxε βηminε dxx1+aG x βηmaxε e−x. (3.18) (3.18) This shows that the non-degenerate Lynden-Bell equilibria express a natural power-law tail and, furthermore, that this power law is independent of the type of plasma system https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 14 R.J. Ewart and others under consideration. Note that the origin of the ε−2 scaling found here is entirely different than the ε−2 arising from particle acceleration in shocks (Bell 1978). Consider now what happens if the Gardner distribution does not have an exponential tail. Then ρ(η) can be written as (3.10). 3.3. Non-degenerate Lynden-Bell equilibria Following all the same steps as before from (3.15) onwards, but using (3.10) in place of (3.8), one arrives at N(ε) = n0 β2−δΓ (a + 1)η2−δ maxε3−δ βηmaxε βηminε dxx2+a−δG x βηmaxε e−x. (3.19) (3.19) Thus, the resulting Lynden-Bell equilibrium again displays a power-law tail. The power law’s exponent is set by the particular value of δ in (3.10), which is related to the Gardner distribution of that Lynden-Bell equilibrium via (3.5), as explained at the end of § 3.2. For Gardner distributions that already have power-law tails, the resulting Lynden-Bell equilibria have shallower (ultra-‘hard’) power-law tails that strongly diverge in energy, giving the cutoff ηmin pivotal importance. For Gardner distributions that decay faster than any exponential, the Lynden-Bell equilibria have ‘soft’ power-law tails, with total energy depending only very weakly on the cutoff.6 p g y y y Presently, however, we shall return to the Lynden-Bell equilibria (3.18) arising from exponential Gardner distributions, for which we complete the calculation. 6The fact that certain choices of fugacity F(η) give rise to Lynden-Bell equilibria that have power laws with various exponents was ﬁrst noted, in connection to superstatistics, by Chavanis (2006a). https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 3.4. Calculation of β and inevitability of partial degeneracy Both (3.17) and (3.18) still depend on the as yet unknown parameter β, which, as well as ﬁxing the energy, will determine the accuracy of the non-degeneracy approximation (3.12). To ﬁnd β, we must compute the energy of our Lynden-Bell equilibrium (3.13) according to (2.5). Equivalently, from (3.15), E = dpε(p)⟨f⟩(p) = a + 1 β ηmax ηmin dηρ(η). (3.20) (3.20) Therefore, in the non-degenerate limit, Therefore, in the non-degenerate limit, β = a + 1 E ηmax ηmin dηρ(η). (3.21) (3.21) We see that β decreases with increasing total energy of the distribution function; this is natural if β is viewed as an inverse thermodynamic temperature. To see how this affects the underlying assumption (3.12) of non-degeneracy, we evaluate D(ε) at ε →0, where the degeneracy effect is strongest, since D(ε) ≤D(0). Requiring D(0) ≪1 gives the following condition on the total energy of the distribution, via (3.12), (3.14) and (3.21): E ≫(a + 1) ηmax ηmin dη ηa+1ρ(η) AΓ (a + 1) 1/(a+1) ηmax ηmin dηρ(η). (3.22) (3.22) Since ρ(η) is a function only of the Gardner distribution, the right-hand side of (3.22) must scale with the energy density EG of the Gardner distribution that has the same waterbag content ρ(η), but will always have EG ≤E. For example, for the Gardner distribution (3.1), the right-hand side of (3.22) should be proportional to n0ε0. Thus, just like in https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press Universal Lynden-Bell equilibria 15 Fermi–Dirac statistics, the non-degeneracy approximation becomes more accurate as the distribution’s energy density E begins to dwarf the energy density EG of the ground state. Note, however, that (3.22) contains an integral of the waterbag content ρ(η) with no weighting by η, which, by (2.4) and (2.6), is the total phase volume occupied by non-empty waterbags. Since ρ(η) ∝η−1 at small η, this will be large, depending, albeit logarithmically, on the minimum waterbag density ηmin. Indeed, for our example (3.1), (3.22) becomes E ≫ 4 3√πn0ε0 ln ηmax ηmin 3/2 1 + O ηmin ηmax . (3.23) (3.23) This means that the condition (3.22) requires the energy of the distribution to be much greater not just than the energy of the corresponding Gardner distribution, but than the Gardner energy multiplied by a polylogarithmic function of ηmin. 3.4. Calculation of β and inevitability of partial degeneracy This is a manifestation of the fact that, as ηmin is taken to zero, more of the phase space is pervaded by low-density waterbags, making true non-degeneracy harder to achieve. It is thus impossible to achieve a non-degenerate limit unless ηmin is kept ﬁnite. This is not the only place where the ﬁniteness of the cutoff ηmin has raged against the dying of the light. The same effect is manifest in the power law of ε−2 appearing in (3.18), which would have led to a logarithmically divergent mean particle energy were it not for the exponential cutoff at ε ∼1/βηmin. This is obvious in (3.20), where the integral of ρ(η) has the same logarithmic divergence with ηmin →0 as it did in the right-hand side of (3.22). This then makes its way into the expression (3.21) for β, so, formally in the limit ηmin →0, β →∞always! Since we must keep ηmin ﬁnite, it is of substantial importance to understand the physical signiﬁcance of it and the extent to which one should be prepared to accept one’s equilibrium’s dependence on its value. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 3.5. Physical meaning of minimum waterbag density A Lynden-Bell equilibrium is essentially the thermal equilibrium of a collection of correlated blobs in phase space. This is to say that, inherent to the idea of computing the mean phase-space density, we have assumed that an exact phase-space density, i.e. a ﬁnite value of η, is a meaningful concept. But, of course, an exact phase-space density is a ﬁction, since a plasma is composed of many discrete particles, and a phase-space density is only an average occupation number of particles’ positions in phase space. The only sense in which an exact, continuous, phase-space density can be meaningful in a collisionless plasma then is if, within a small enough phase-space volume , many particles can be considered to move as a collective entity: a waterbag. Then, on the scale of , the system is composed of many waterbags with some ‘exact’ phase-space density, whereas on scales much larger than , the system can attain a mean phase-space density. This ‘correlation volume’ provides a natural way to introduce the minimum non-zero phase-space density: clearly that should correspond to a single particle sitting in , giving ηmin = −1. y p g p g g g Determining the value of , is, however, a non-trivial challenge (see, e.g. discussions in Kadomtsev & Pogutse 1970; Chavanis 2022; Ewart et al. 2022). Ewart et al. (2022) argued, on the grounds that any meaningful collisionless-relaxation rate must be smaller than the plasma frequency but larger than the rate at which collisions break phase-volume conservation, that a reasonable constraint to place on the correlation volume is ∼1 ηeff (n0λ3 D)α, 2 3 < α < 1, (3.24) (3.24) https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 16 R.J. Ewart and others where ηeff is some typical phase-space density, which we can here estimate by ηmax, and λD is the Debye length. This gives us an estimate for the minimum waterbag density where ηeff is some typical phase-space density, which we can here estimate by ηmax, and λD is the Debye length. This gives us an estimate for the minimum waterbag density ηmin = −1 ∼ηmax(n0λ3 D)−α. (3.25) (3.25) ηmin = −1 ∼ηmax(n0λ3 D)−α. Since the typical number of particles in a Debye sphere can be as large as 106 to 108 in collisionless plasma environments (such as the solar wind or interstellar gas: see, e.g. 3.5. Physical meaning of minimum waterbag density Ferrière 2019; Verscharen, Klein & Maruca 2019), the estimate (3.25) might seem damningly small. It is, in fact, ideal. The existence of a broad power-law tail in (3.18) required a scale separation between ηmax and ηmin. The estimate (3.25) certainly provides this separation, tied to the plasma parameter. As for the breakdown of the non-degenerate approximation and the marginal divergence of the mean particle energy of a distribution with an ε−2 power law, we are saved by the fact that only the logarithm of the ratio ηmax/ηmin will appear, which, while large, can only ever be in the range of 10–30. This is somewhat reminiscent of the situations in the conventional theory of Coulomb collisions in plasmas, where forcible introduction of a phase-space cutoff into the collision integral results in only a weak dependence on the value of this cutoff, via the so-called Coulomb logarithm (see, e.g. Helander & Sigmar 2005). Nevertheless, the presence of the logarithmic divergence with ηmin in the expression for β, signposted at the end of § 3.4, will make it difﬁcult to satisfy the non-degeneracy approximation.7 There is good reason to suppose, however, that its breakdown may only be partial. We note that evaluating (3.12) at ε = 0 is tantamount to requesting non-degeneracy everywhere in the distribution. Since the degeneracy parameter D(ε) decreases with increasing energy, it is reasonable to expect (and indeed we will see) some degeneracy at low energies, which gives way to non-degeneracy at higher energies, where our power-law tails will be recovered. As ever, the true solution lies on the cusp of asymptotic theory, and to go any further we must resort to numerical computation. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 4. Numerical results: partially degenerate equilibria In this section, we shall recover the analytically predicted power-law tail in (3.18) by solving the constraint equations (2.5) and (2.6) for the Lynden-Bell equilibria (3.4). The numerical scheme for this is documented in detail in Appendix B, amounting to an iterative method coupled to a one-dimensional root ﬁnder. For these numerical results, we have restricted ourselves to a three-dimensional, non-relativistic plasma with ε(p) = p2/2m, although we anticipate from § 3 that these results extend, qualitatively, to general regimes. To capture a broad range of initial conditions, we consider a family of waterbag contents deﬁned by ρ(η) = 4πp3 th,σ ση ln ηmax,σ η (3−σ)/σ , ηmin < η < ηmax,σ, (4.1) (4.1) with σ > 0. This deﬁnes the family of Gardner distributions fG,σ(p) = ⎧ ⎪⎪⎨ ⎪⎪⎩ ηmax,σ e−( p/pth,σ )σ for p < pth,σ ln ηmax,σ ηmin 1/σ , 0 for p > pth,σ ln ηmax,σ ηmin 1/σ , (4.2) (4.2) 7Although, notably, the fully non-degenerate limit can be naturally recovered by numerical noise in particle-in-cell (PIC) codes: see Appendix C. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press shed online by Cambridge University Press https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press Universal Lynden-Bell equilibria 17 (a) (b) FIGURE 3. Numerically computed Lynden-Bell equilibria for a range of ηmax,σ/ηmin and with ρ(η) given by (4.1) with σ = 2. The energy density is equal to 10EG in all cases. (a) The numerically computed fugacity F(η) (solid lines) compared with the analytical solution (3.14) obtained in the non-degenerate limit (dashed lines). (b) The resulting distributions N(ε) of particle energies, with the universal power law ∝ε−2 shown for reference, cf. (3.18). Overplotted in solid colour (with the value range shown on the right) is the level of degeneracy D(ε)/[1 + D(ε)] (the probability that a given energy is occupied by a non-empty waterbag) as a function of energy; D(ε) is deﬁned in (3.12). (a) (b) (b) (a) FIGURE 3. Numerically computed Lynden-Bell equilibria for a range of ηmax,σ/ηmin and with ρ(η) given by (4.1) with σ = 2. The energy density is equal to 10EG in all cases. (a) The numerically computed fugacity F(η) (solid lines) compared with the analytical solution (3.14) obtained in the non-degenerate limit (dashed lines). (b) The resulting distributions N(ε) of particle energies, with the universal power law ∝ε−2 shown for reference, cf. (3.18). 4. Numerical results: partially degenerate equilibria Overplotted in solid colour (with the value range shown on the right) is the level of degeneracy D(ε)/[1 + D(ε)] (the probability that a given energy is occupied by a non-empty waterbag) as a function of energy; D(ε) is deﬁned in (3.12). which, in the limit of ηmin/ηmax,σ →0, have particle densities n0 and energy densities EG that satisfy n0 = 4π σ Γ 3 σ p3 th,σηmax,σ, EG = Γ (5/σ) Γ (3/σ)n0 p2 th,σ 2m . (4.3a,b) (4.3a,b) Since these Gardner distributions represent minimum-energy states, we will be able to scan in the energy density of the system for all E ≥EG, imagining some initial distribution of particles, with waterbag content ρ(η) and energy density EG, being accelerated to the energy density E, and then seeking a maximum-entropy state (see § 3.1). We can also vary σ in order to see the effects of the shape of the underlying Gardner distribution on the resulting Lynden-Bell equilibria. Since these Gardner distributions represent minimum-energy states, we will be able to scan in the energy density of the system for all E ≥EG, imagining some initial distribution of particles, with waterbag content ρ(η) and energy density EG, being accelerated to the energy density E, and then seeking a maximum-entropy state (see § 3.1). We can also vary σ in order to see the effects of the shape of the underlying Gardner distribution on the resulting Lynden-Bell equilibria. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 4.1. Degrees of degeneracy Let us ﬁrst scan in ηmax,σ/ηmin in order to show that we can indeed recover the fully non-degenerate limit solved in § 3. Figure 3 shows the result of such a scan for σ = 2 and E = 10EG. By comparing the exact (numerically calculated) fugacity with the theoretical prediction (3.14) obtained in the absence of phase-space degeneracy, we see that the agreement is nearly perfect when ηmax is close to ηmin, e.g. when ηmax/ηmin = 10. This is as expected, since the non-degenerate limit is valid when (3.22) holds, which it does, as can be conﬁrmed from the solid red colour in panel (b), showing the probability D(ε)/[1 + D(ε)] (with D(ε) deﬁned in (3.12)) that a region of phase space is occupied. However, at ηmax/ηmin = 10, there is an insufﬁcient range of waterbag levels to achieve the scale separation (3.17) necessary to resolve a power-law tail in energies. To R.J. Ewart and others 18 (a) (b) (a) (b) FIGURE 4. Numerically computed Lynden-Bell equilibria for a range of energy densities E multiples of the energy density EG of the underlying Gardner distribution) with ρ(η) given (4.1) with σ = 2 and ηmax/ηmin = 106. In each plot, the dashed line is the mean phase-sp density, while the underplotted solid lines are the contributions from four distinct ranges of ex phase-space density as functions of energy. Note that, while the exact phase-space densi have been grouped into four, this is not the same as solving for a four-waterbag Lynden-B equilibrium, as each grouping is still composed of a continuum of waterbags. (a) (b) (a) (b) (a) (a) (a) ( ) FIGURE 4. Numerically computed Lynden-Bell equilibria for a range of energy densities E (in multiples of the energy density EG of the underlying Gardner distribution) with ρ(η) given by (4.1) with σ = 2 and ηmax/ηmin = 106. In each plot, the dashed line is the mean phase-space density, while the underplotted solid lines are the contributions from four distinct ranges of exact phase-space density as functions of energy. Note that, while the exact phase-space densities have been grouped into four, this is not the same as solving for a four-waterbag Lynden-Bell equilibrium, as each grouping is still composed of a continuum of waterbags. 4.1. Degrees of degeneracy see a power-law tail, one must increase ηmax/ηmin to higher values, but this comes at the price of increasing the degeneracy of the system and, hence, undermining the asymptotic regime in which the tail was derived in the ﬁrst place. For the values of ηmax/ηmin that we argued in § 3.5 to be realistic, the system becomes strongly degenerate at low energies, which can again be seen from the solid colours in panel (b). In spite of this, at high energies, the degeneracy falls away, and, correspondingly, F(η) at low η still agrees well with the non-degenerate approximation (3.14). All this conspires to ensure that, even formally outside the non-degenerate limit, the power-law tail N(ε) ∝ε−2 is still manifestly present. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press Universal Lynden-Bell equilibria 19 (a) (b) FIGURE 5. Numerically computed Lynden-Bell equilibria with waterbag content given by the σ = 2 (top) and σ = 8 (bottom) cases of (4.1), ηmax,σ/ηmin = 106. (a) The phase-space densities shown in linear scale, (b) the corresponding distributions of particle energies in logarithmic scale, for a range of ratios of E/EG. The small deviations from the ε−2 tail can be attributed to the logarithmic corrections arising from the x integral in (3.18). (b) (a) FIGURE 5. Numerically computed Lynden-Bell equilibria with waterbag content given by the σ = 2 (top) and σ = 8 (bottom) cases of (4.1), ηmax,σ/ηmin = 106. (a) The phase-space densities shown in linear scale, (b) the corresponding distributions of particle energies in logarithmic scale, for a range of ratios of E/EG. The small deviations from the ε−2 tail can be attributed to the logarithmic corrections arising from the x integral in (3.18). 4.2. Energisation of particles and power-law tails Let us now scan in the energies densities E of the distribution and again look for power-law tails and assess the effect of degeneracy. Figure 4 shows the results of such a scan, again with ρ(η) speciﬁed by (4.1) with σ = 2. Plotted underneath the mean phase-space densities are the contributions from a number of ﬁnite ranges of exact phase-space density deﬁned by ⟨f⟩η1<η<η2(p) = η2 η1 dηηP(p, η). (4.4) (4.4) As one would anticipate, when E is only slightly larger than EG, the effects of phase-space degeneracy are most prominent: the densest portions of the phase space clog up the lowest energies, forcing less dense portions to higher energies. As the total energy density is increased, we see that the contribution from each range of waterbags spreads out. This is because the increased energy allows dense portions of phase space to be promoted to As one would anticipate, when E is only slightly larger than EG, the effects of phase-space degeneracy are most prominent: the densest portions of the phase space clog up the lowest energies, forcing less dense portions to higher energies. As the total energy density is increased, we see that the contribution from each range of waterbags spreads out. This is because the increased energy allows dense portions of phase space to be promoted to https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 20 R.J. Ewart and others larger energies, making room at lower energies for less dense portions of phase space to ﬁll. larger energies, making room at lower energies for less dense portions of phase space to ﬁll. While the solutions plotted in ﬁgure 4 might appear qualitatively similar to the Lynden-Bell equilibria obtained in numerical experiments with a small discrete number of level sets (see, e.g. Assllani et al. 2012; Ewart et al. 2022), this hides key universal features of systems with a continuum of level sets. To showcase this universality, in ﬁgure 5, we plot the Lynden-Bell equilibria for two different waterbag contents, σ = 2 and σ = 8 in (4.1), and a range of energy densities E. The ε−2 power-law tails of these equilibria are immediately apparent, as predicted in (3.18). Figure 5 shows how these power-law tails become more prominent as one adds more energy to the Gardner distribution. At E = EG, one has a highly non-universal Gardner equilibrium. 4.2. Energisation of particles and power-law tails As a small amount of energy E −EG ≪EG is added to EG, the mean phase-space density at low energies is largely unaffected, while the power-law tail grows from the lowest-density waterbags. Thus, for energies close to the energy of the underlying Gardner distribution, the Lynden-Bell equilibria have a ‘core-halo’ structure: the ‘core’, which has energy density ∼EG, is comprised of dense waterbags, which the system does not have sufﬁcient energy to excite, whereas the ‘halo’ is the tail comprised of those less dense waterbags that are capable of sampling a larger portion of phase space and thus arrange themselves into a universal ε−2 power law, containing the excess energy density ∼E −EG. As the energy of the distribution is further increased, more waterbags have sufﬁcient energy to sample a larger range of phase space, the halo continues to eat into the core, but both thermally broaden. At E ≫EG, the asymptotically non-degenerate solution (3.18) with a power law in the energy range (3.17) suggests that the system strives for a state in which the halo has much more energy than the core. In this limit, one expects the transition between the core and halo to occur at ε ∼1/βηmax. Since the halo should be exponentially suppressed at ε ≳1/βηmin, one can compute the ratio of the core energy to the halo energy. Owing to the ε−2 tail, this ratio will be proportional to ln(ηmax/ηmin) raised to some power, which depends on the speciﬁc functional form of the Gardner distribution. Whatever this power is, the vast majority of the total energy will be contained in the universal power-law tail for any system whose energy is much larger than that of its Gardner state. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 5.1. Summary The Lynden-Bell (1967) equilibria are the natural maximum-entropy states for systems, such as a plasma described by the collisionless Vlasov equation (1.1), which conserve not only density, momentum, and energy, but also an inﬁnite family of further invariants (2.1). These additional invariants are due to the conservation of phase volume, encoded by the ‘waterbag content’ function ρ(η) given by (2.7) (equivalent to the Casimir invariants (3.7)), which measures the amount of phase volume where the exact phase-space density takes the value η (a ‘waterbag’), per unit η. Maximising entropy subject to all these conservation laws then gives the mean phase-space density (2.2) in the form of the Lynden-Bell equilibrium (2.9) coupled with the constraints (2.5) and (2.6). In this paper, we have solved these constraint equations numerically in the general case, as well as analytically in a tractable limit (which turned out to be the practically relevant one). We have been able to show that, despite their apparent dependence on non-universal initial conditions, Lynden-Bell equilibria generically exhibit power-law tails, and, in particular, that a broad class of initial conditions will give rise to the distribution of particles’ energies scaling as ε−2 at high ε. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 21 Universal Lynden-Bell equilibria To study the Lynden-Bell equilibria systematically, we ﬁrst considered what values the invariants of the system, the energy density E and waterbag content ρ(η), could take. This led us to the concept of a Gardner distribution function, which is any monotonically decreasing function of the particle’s energy. In § 3.1, we argued that to each possible initial condition one could assign a unique Gardner distribution, with the same waterbag content (and, therefore, the same Casimir invariants) as the initial condition, but a different energy, the Gardner distribution having, by deﬁnition, the lowest possible energy of all distributions with a given waterbag content ρ(η). The Lynden-Bell equilibria at higher energies and the same ρ(η) can, therefore, be thought of as excited states of this Gardner distribution. In § 3.2, we argued that the typical ρ(η) would have a fairly generic power-law form at low η, and, in particular, that it would scale as η−1 for a wide class of initial conditions (see (3.8)). In § 3.3, we were able to ﬁnd the Lynden-Bell equilibria analytically provided the energy of the system was sufﬁciently large for the competition of waterbags for phase space to be ignorable. 5.1. Summary In this ‘non-degenerate’ limit, particles belonging to each waterbag arrange themselves into a separate Maxwellian equilibrium with an effective ‘temperature’ inversely proportional to the phase-space density of that waterbag: denser portions of phase space are energetically costlier to move to higher energies. The resulting mean phase-space density (3.15) was found by integrating the contributions of all waterbags, each with their own Maxwellian distribution weighted by the amount of phase space which that waterbag occupied. Since the amount of each waterbag had a universal form (3.8), this gave rise to a universal power-law tail (3.18) scaling as ε−2 at high particle energies. However, the non-degenerate limit required the system’s energy to be asymptotically larger than the energy of the corresponding Gardner distribution. Formally, this turned out to be a very stringent limitation, and indeed one that could hardly ever be strictly fulﬁlled. Our analytical results were rescued by the argument, conﬁrmed by the numerical solutions presented in § 4, that the effects of phase-space degeneracy were conﬁned to the low-energy part of the distribution. The universal ε−2 tail was numerically conﬁrmed to be a robust feature of the generic Lynden-Bell equilibria. As well as ascertaining that a range of different initial conditions (4.1) gave rise to the same power-law tail, the numerical solution also showed how this power-law tail formed. We found (ﬁgure 5) that at energies comparable to the Gardner energy, the Lynden-Bell equilibria had a ‘core-halo’ structure. The halo, consisting of the ε−2 tail, was formed from low-density waterbags, which had sufﬁcient energy to explore large portions of phase space, while the non-universal core was made up of denser waterbags, which did not have sufﬁcient energies to be excited. As the energy of the Lynden-Bell equilibrium was increased, the halo ate its way into the core, making the distribution less and less degenerate and more universal in its shape. This behaviour is perhaps reminiscent of the measurements of the ‘non-thermal fraction’ of particles in the solar wind (see, e.g. Pierrard & Lazar 2010; Oka et al. 2015). https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 5.2. Limitations and applications The Lynden-Bell statistical mechanics presents an attractive scenario for the universal generation of power-law tails, which could perhaps offer insight into the distribution of particles in such astrophysical systems as cosmic rays and the solar wind. Indeed, a power law of ε−2 in energy is roughly consistent with the observed power laws typical in the quiet-time solar wind (e.g. Gloeckler et al. 2008; Fisk & Gloeckler 2014; Yang et al. 2020) and close to, but distinct from, the inferred value for cosmic-ray sources (e.g. Ormes & Freier 1978; Reichherzer et al. 2021). However, the universality of this predicted power law fails to capture the wide range of power laws seen both in numerical 22 R.J. Ewart and others simulations (e.g. Sironi & Spitkovsky 2014; Werner & Uzdensky 2017; Zhdankin et al. 2017) and observationally nearer to the Sun (see Oka et al. (2018) and references therein). Such systems are usually turbulent, possibly inhomogeneous, and invariably magnetised. In contrast, the theory that we have proposed for a universal power-law tail assumes a homogeneous system, in which all the ﬂuctuating ﬁeld’s energy has decayed to a negligible fraction of the total energy. It is therefore an intriguing question whether our theory can be adjusted to apply to these cases. It is this question that we will address in this section, speculatively. p y One common limitation in the applicability of Lynden-Bell’s theory is that the ﬂuctuating ﬁelds may decay away faster than the equilibrium state is reached – this is a well-understood feature in galactic dynamics, where it is referred to as ‘incomplete relaxation’ (see Chavanis (2006b) and references therein). Here we have ignored such a possibility, assuming effectively that there will always be a sufﬁcient amount of ﬂuctuations to see the plasma through to its maximum-entropy state. Perhaps an even more pressing concern is the possible reliance of the theory on precise phase-volume conservation. The validity of the Lynden-Bell statistics rests on the assumption that such an equilibrium can be reached long before the conservation of phase volume is broken. Conventional (linear) estimates for the time scale on which it would be broken, scaling as an inverse fractional power of the true Coulomb-collision frequency (Su & Oberman 1968), indicate that such a relaxation should be possible. However, recent progress (Beraldo e Silva et al. 2017; Zhdankin 2022a; Nastac et al. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 5.2. Limitations and applications This implies a new conserved function 2π V dr dv∥B(r)δ(η −f(r, v∥, μb)) = ρ(η, μb), (5.1) (5.1) which would supersede the conservation of the now mundane ρ(η). While some Gardner distributions have been studied for such systems (see Helander 2020; Mackenbach, Proll & Helander 2022), the Lynden-Bell equilibria in them are unexplored and may contain a wealth of interesting physics. This said, in turbulent systems, the conservation law (5.1) may be just as fragile as the conservation of phase volume. Indeed it has been suggested that the breaking of adiabatic invariance may be essential to understanding the transport properties of non-thermal particles (see Ruszkowski & Pfrommer (2023) and references therein). From the previous discussion it becomes clear that perhaps the most relevant limitation of Lynden-Bell’s statistical mechanics – or indeed of any equilibrium statistical mechanics – in application to observed plasma phenomena is that much of real plasma dynamics are out of equilibrium in a physically essential way: any local relaxation processes, collisional or collisionless, tend to have to be taken into account alongside various ‘sources’ and ‘sinks’ of particles and/or energy, e.g. the energisation and escape of cosmic rays (Schlickeiser 1989; Chandran 2000; Becker Tjus & Merten 2020; Hopkins et al. 2022; Kempski & Quataert 2022), the turbulent heating and radiative cooling of the intracluster medium (e.g. Zhuravleva et al. (2014) and references therein) or accretion-disc plasmas (e.g. Lesur (2021), Kawazura et al. (2022) and references therein), a veritable zoo of such processes in the solar wind (e.g. Verscharen et al. 2019; Chen et al. 2020) and the Earth’s magnetosphere (e.g. Lucek et al. 2005), the birth of energetic α-particles in fusion reactions and their subsequent slowing down and escape from conﬁned plasmas (e.g. Helander & Sigmar 2005; Mailloux et al. 2022), etc. In plasmas where Coulomb collisions can be assumed to relax the particle distribution quickly to a local Maxwellian, we have a robust analytical framework for handling all these non-equilibrium processes in terms of the evolution of the density, momentum and temperature of that Maxwellian and a separation of the dynamics into that evolution plus the turbulence of small ﬂuctuations around the local equilibrium (e.g. Schekochihin et al. 2009; Abel et al. 2013). 5.2. Limitations and applications In collisionless plasmas, such a framework is lacking as both the turbulence and the nature of the underlying equilibrium are mysterious and indeed it is not even guaranteed that they can be understood without detailed reference to each system’s particular initial circumstances. If Lynden-Bell’s statistical mechanics proves to be a viable collisionless substitute for Maxwell’s, a path could be charted towards a theory of the dynamics and thermodynamics of collisionless plasmas possessing a modicum of universality. Acknowledgements We would like to thank G. Acton, M. Barnes, A. Bott, A. Brown, S. Cowley, J.-B. Fouvry, C. Hamilton, P. Helander, D. Hosking, M. Kunz, A. Louis, R. Mackenbach, I. Nemenman, S. Parameswaran, P. Reichherzer, J. Ruiz Ruiz and L. Silva for illuminating discussions. The paper has also been improved by the recommendations of two anonymous reviewers. We would like to thank G. Acton, M. Barnes, A. Bott, A. Brown, S. Cowley, J.-B. Fouvry, C. Hamilton, P. Helander, D. Hosking, M. Kunz, A. Louis, R. Mackenbach, I. Nemenman, S. Parameswaran, P. Reichherzer, J. Ruiz Ruiz and L. Silva for illuminating discussions. The paper has also been improved by the recommendations of two anonymous reviewers. Editor Per Helander thanks the referees for their advice in evaluating this article. 5.2. Limitations and applications 2023) has shown that in turbulent systems, the conservation of phase volume may be broken on fast, collision-frequency-independent time scales. While it is possible that this is damning evidence against the existence (or persistence) of Lynden-Bell equilibria, it is also possible that it points to interesting interplay between the Lynden-Bell relaxation and collisional effects. For instance, one can imagine the possibility that the effect of collisions is to evolve ρ(η) without immediately pinning the system to a Maxwellian equilibrium. This can happen if collisions are already acting to erase small-scale phase-space structure of the exact phase-space density f but not yet to change its mean ⟨f⟩directly. In such a situation, one’s aim would be to compute the evolution of ρ(η) or, equivalently, of the underlying Gardner ground state fG(ε). In particular, one can imagine a regime in which the underlying Gardner distribution evolves slower than the Lynden-Bell equilibrium is reached, causing the distribution to go through a sequence of Lynden-Bell equilibria. If the effect of collisions is to smooth the ﬂuctuations f −⟨f⟩diffusively, it is clear that this can only cause the energy EG of the Gardner distribution to increase or remain constant (cf. Kolmes & Fisch 2020). One would therefore expect that, as the system evolves through a sequence of Lynden-Bell equilibria, it will steadily become more degenerate as EG approaches the system’s energy E, with the core of the Lynden-Bell distribution eating into its tail (halo; see § 4.2). This partially collisionless evolution would ﬁnally freeze once all ﬂuctuations are diffused away, leaving a degenerate Lynden-Bell equilibrium doomed to further gradual erosion by weak Coulomb collisions on ⟨f⟩. Since we have argued that an ε−2 tail is generic for any Lynden-Bell equilibrium whose underlying Gardner distribution has an exponential tail, this scenario suggests that the universal power-law tail could persist as long as EG < E, despite the breaking of phase-volume conservation due to weak collisionality. Finally, there is the question of whether any of this formalism can be ported smoothly to magnetised equilibria. Here, the most obvious straw to grasp at is that there is no guarantee that the invariants (2.5) and (2.6) are the only invariants respected on the relaxation time scale. As previously mentioned, in drift-kinetic plasmas, each particle conserves its https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press Universal Lynden-Bell equilibria 23 Universal Lynden-Bell equilibria 23 Universal Lynden-Bell equilibria 23 magnetic moment μb. R.J. Ewart and others R.J. Ewart and others Funding R.J.E.’s work was supported by a UK EPSRC studentship. M.L.N. was supported by a Clarendon Scholarship. The work of A.A.S. was supported in part by grants from STFC (ST/W000903/1) and EPSRC (EP/R034737/1), as well as by the Simons Foundations via a Simons Investigator award. Appendix A. The degenerate limit of Lynden-Bell’s statistics In the main text, we have made use of the claim that, given the waterbag content ρ(η), the Gardner distribution with the same waterbag content represents the ground state of all possible Lynden-Bell equilibria given by (3.4) that have this waterbag content. This is intuitive: should the initial condition of the system be a Gardner distribution, then that is, by deﬁnition, the only state available to the system, so it must also be the maximum-entropy state for that choice of ρ(η). However, for completeness, and as a test of the Lynden-Bell formalism, it is prudent to check that the Gardner distribution can be recovered for some choice of the fugacity F(η) and β. This is the aim of this appendix: to solve explicitly for β and F(η) in (3.4) when E = EG, where EG is the energy of a given Gardner distribution fG, and to show that the mean phase-space density obtained by maximising Lynden-Bell’s entropy is ⟨f⟩= fG. To understand how the Gardner distribution will be recovered, we appeal to the familiar Fermi–Dirac distribution fFD(ε) = ηmax e−β(ε−μ) 1 + e−β(ε−μ) . (A1) (A1) To work out what this is in the degenerate limit, every textbook notes that, when β is very large, the numerator (and second term in the denominator) is either very small for ε > μ, making the expression approximately zero, or very large for ε < μ, making the exponentials in the numerator and denominator approximately cancel to give ηmax. Borrowing this intuition, we anticipate that our solution should have β →∞. It is clear what kind of solution one must search for: the degeneracy parameter D(ε) deﬁned by (3.12) must be large wherever fG(ε) ̸= 0 and zero wherever fG(ε) = 0. Furthermore, the dominant contribution to D(ε) in the integral (3.12) must come from η = fG(ε). This is the Lynden-Bell version of the statement that the phase space is completely ﬁlled up below the Fermi energy. To see how this works in practice, let us posit the fugacity in the form F(η) = 1 ¯η exp β η ηmin dη′f −1 G (η′) , (A2) (A2) and prove that, via (3.4) and (2.6), it recovers the Gardner distribution with the correct waterbag content ρ(η) when β →∞, for a suitable choice of the dimensional constant ¯η. Declaration of interest The authors report no conﬂict of interest. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 24 https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press Appendix A. The degenerate limit of Lynden-Bell’s statistics In the denominator of (3.4), we must evaluate the integral g ρ η β In the denominator of (3.4), we must evaluate the integral D(ε) = 1 ¯η ηmax ηmin dη e−βηε+ln ¯ηF(η). (A3) (A3) Since we are working in the limit of large β, this integral will be dominated by the contribution near the maximum (in η at ﬁxed ε) of the exponent and thus can be evaluated by the method of steepest descent (Bender & Orszag 1978). The location of the maximum Since we are working in the limit of large β, this integral will be dominated by the contribution near the maximum (in η at ﬁxed ε) of the exponent and thus can be evaluated by the method of steepest descent (Bender & Orszag 1978). The location of the maximum https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press Universal Lynden-Bell equilibria 25 Universal Lynden-Bell equilibria 25 25 of the exponent, which we denote by ηstat(ε), is given by the solution to β d dη ηε − η ηmin f −1 G (η′)dη′ = 0 =⇒ηstat(ε) = fG(ε), (A4) (A4) as we anticipated above. We may now expand the exponent of the integrand in (A4) around this maximum to approximate the integral by D(ε) ≃ ∞ −∞ dη exp −βfG(ε)ε + β 2 df −1 G dη η=fG(ε) [η −fG(ε)]2 F( fG(ε)) = e−βfG(ε)εF( fG(ε)) 2π β −df −1 G dη η=fG(ε) −1/2 . (A5 D(ε) ≃ ∞ −∞ dη exp −βfG(ε)ε + β 2 df −1 G dη η=fG(ε) [η −fG(ε)]2 F( fG(ε)) = e−βfG(ε)εF( fG(ε)) 2π β −df −1 G dη η=fG(ε) −1/2 . (A5) (A5) As is customary, we have neglected the contributions from higher derivatives of the exponent in the knowledge that they will contribute terms that are smaller by O(1/β). We have also replaced the upper and lower limits of integration by ±∞, assuming that the exponential decays sufﬁciently fast for the presence of integration limits not to be noticed by the integral. This will not be accurate near ε = 0 and ε = f −1 G (ηmin), where the dominant contribution comes precisely from the limit of integration. This is, however, ﬁne for β →∞because the intervals in ε where this approximation is bad shrink as O(β−1/2). https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press Appendix A. The degenerate limit of Lynden-Bell’s statistics We can again expand the exponent around this stationary point and neglect O(1/β) terms, giving us Again using the method of steepest descent, we observe that the exponent has its stationary point, this time in ε at ﬁxed η, at ε = f −1 G (η), just as we should expect. We can again expand the exponent around this stationary point and neglect O(1/β) terms, giving us ρ(η) ≃ ∞ −∞ dεg( f −1 G (η)) exp β 2 dfG dε ε=f −1 G (η) [ε −f −1 G (η)]2 β 2π −df −1 G dη 1/2 = −g( f −1 G (η))df −1 G dη . (A10) (A10) This is exactly the expression (3.5) that we desire. This completes the proof that any Gardner distribution can be written as the β →∞limit of the Lynden-Bell statistics, implying that one could, in principle, have discovered Gardner restacking just by analysing the Lynden-Bell equilibria at β →∞. By retaining terms that are small in O(1/β), it is possible to analyse the Lynden-Bell equilibria analytically for energies very close but slightly above EG. However, in analogy to Fermi–Dirac statistics, this should just amount to broadening the ‘step’ in the Gardner distribution around ε = f −1 G (ηmin), which limits the validity of this expansion to a very small range of energies, and thus makes such an expansion an exercise of inﬁnitesimal utility. Appendix A. The degenerate limit of Lynden-Bell’s statistics If we further demand that the constant ¯η in (A2) is chosen so that 1 ¯η e−βηminf −1 G (ηmin) 2π β −df −1 G dη η=ηmin −1/2 = 1, (A6) (A6) then, neglecting terms of O(1/β), D(ε) satisﬁes then, neglecting terms of O(1/β), D(ε) satisﬁes then, neglecting terms of O(1/β), D(ε) satisﬁes 1 + D(ε) ≃ D(ε) for ε < f −1 G (ηmin), 1 for ε > f −1 G (ηmin). (A7) (A7) Calculating the mean phase-space density ⟨f⟩(p) from (2.2) and (3.4), we ﬁnd ⟨f⟩(ε) ≃ ⎧ ⎨ ⎩ 1 ¯ηD(ε( p)) ηmax ηmin dηη e−βηε+ln ¯ηF(η) for ε < f −1 G (ηmin), 0 for ε > f −1 G (ηmin). (A8) (A8) Let us prove that ⟨f⟩(p) = fG(ε(p)). The integral in (A8) can again be computed by the method of steepest descent. The exponent is the same as in (A3) and so will have the same maximum, at ηstat = fG(ε) (up to a small O(1/β) correction due to the factor of η in (A8)). Expanding the integral around this maximum and neglecting all terms that are O(1/β) causes the factor of η in the integral to be replaced by ηstat(ε). After this, the remainder of the integral has the same form as (A5), which cancels with the denominator of (A8), leaving only the factor of ηstat = fG(ε). Q.E.D. Thus, the fugacity (A2) correctly recovers the Gardner distribution fG(ε) in the limit of β →∞. However, it is possible, in principle, to have accidentally chosen a fugacity which, while recovering the correct distribution, has an incorrect waterbag content. To R.J. Ewart and others R.J. Ewart and others R.J. Ewart and others 26 complete the proof, we compute the waterbag content (2.6) for the fugacity (A2), to show that it is the same waterbag content as that of: ρ(η) ≃ ∞ 0 dεg(ε) exp −βε[η −fG(ε)] + β η fG(ε) f −1 G (η) dη · β 2π −df −1 G dη η=fG(ε) 1/2 . (A9) (A9) Again using the method of steepest descent, we observe that the exponent has its stationary point, this time in ε at ﬁxed η, at ε = f −1 G (η), just as we should expect. Appendix B. Numerical method for solving for Lynden-Bell equilibria An initial guess is chosen for the fugacity and thermodynamic beta, denoted F(0)(ηj) d β(0) i l h i l l i h i § 4 h i i i l where the number q > 1 is chosen depending on the waterbag content: for (4.1), q = 3 was used for σ ≤3 and q = 2 for σ > 3, to compromise on the resolution near η = ηmax. where the number q > 1 is chosen depending on the waterbag content: for (4.1), q = 3 was used for σ ≤3 and q = 2 for σ > 3, to compromise on the resolution near η = ηmax. An initial guess is chosen for the fugacity and thermodynamic beta, denoted F(0)(ηj) and β(0), respectively. For the numerical solutions shown in § 4, the initial guess was set to the analytical solutions (3.14) and (3.21) obtained in the non-degenerate limit; other arbitrary choices were tested, all of which converged, albeit usually taking more iterations An initial guess is chosen for the fugacity and thermodynamic beta, denoted F(0)(ηj) and β(0), respectively. For the numerical solutions shown in § 4, the initial guess was set to the analytical solutions (3.14) and (3.21) obtained in the non-degenerate limit; other arbitrary choices were tested, all of which converged, albeit usually taking more iterations to do so. At each further iteration, the fugacity is updated according to (B1). The η′ integral in (B1) is computed by a second-order midpoint method, interpolating the fugacity linearly between the neighbouring grid points. To compute the ε integral, a preliminary scan is ﬁrst conducted to ﬁnd the energy εupper at which the degeneracy parameter (3.12) becomes smaller than 10−5. This allows the energy integral in (B1) to be split into two parts. The ﬁrst of them, over energies below εupper, must be computed numerically, while the second, over energies above εupper, can be approximated by an analytically calculable function of εupper and η. In the region where the integral must be computed numerically, the integration is carried out assuming the denominator to be piecewise linear on a momentum grid (rather than an energy grid, although this distinction is unimportant) linearly spaced with a spacing of 10−3 in units such that EG = 1. The fugacity can thus be iterated at ﬁxed thermodynamic beta. Appendix B. Numerical method for solving for Lynden-Bell equilibria In this appendix, we detail the numerical method by which we solve for the Lynden-Bell equilibria. To reiterate, the objective is, for a given waterbag content ρ(η) and energy density E, to compute the function F(η) and the parameter β such that, with the waterbag distribution given by (3.4), the constraints (2.5) and (2.6) are satisﬁed to sufﬁcient accuracy. For the numerical solutions given in § 4, the numerical method was tailored to a three-dimensional, non-relativistic, system where ε = p2/2m, but the method can be easily extended to any systems with a speciﬁed density of states. The formula (3.4) for the distribution function can be rewritten in such a way as to lend itself naturally to an iterative scheme. Namely, if one denotes the fugacity and the thermodynamic beta at the nth iteration by F(n) and β(n), respectively, then the natural iteration for the fugacity is F(n+1)(η) = ρ(η) ⎡ ⎢⎢⎣2π(2m)3/2 ∞ 0 dεε1/2 e−β(n)ηε 1 + ηmax ηmin dη′ e−β(n)η′εF(n)(η′) ⎤ ⎥⎥⎦ −1 . (B1) (B1) 3000983 Published online by Cambridge University Press https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press Universal Lynden-Bell equilibria 27 Ignoring for a moment how one iterates β(n), we note that if the iteration (B1) converges, then by deﬁnition (2.6) is satisﬁed. This means that a solution with the correct waterbag content has been found, but it may have an incorrect energy, since it does not necessarily satisfy (2.5). However, by converging to a correct fugacity for a given β(n), the problem is essentially reduced to a one-dimensional root-ﬁnding problem: ﬁnding β such that the energy takes the desired value, for which numerous numerical methods exist. This is the basis for our numerical algorithm, of which we will now give the speciﬁc details. The η domain is discretised into Nη = 10 000 points in preparation for future integration. To ensure that the lowest-density waterbags are well resolved without wasting resolution on the highest-density ones, a non-uniform discretisation in η is used: the jth grid point is given by ηj = ηmin + j Nη q (ηmax −ηmin), (B2) (B2) where the number q > 1 is chosen depending on the waterbag content: for (4.1), q = 3 was used for σ ≤3 and q = 2 for σ > 3, to compromise on the resolution near η = ηmax. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press Appendix C. Lynden-Bell equilibria and PIC plasmas While we have shown numerically that the non-degenerate approximation of Lynden-Bell equilibria taken in § 3.3 is qualitatively accurate even in systems that are nowhere near complete non-degeneracy, there is one (admittedly contrived) case where it is not just approximately true but represents an exact result. In this appendix, we show that non-degenerate Lynden-Bell equilibria are the natural long-time equilibria of a plasma which is evolved using the PIC algorithm (PIC plasma) in which any given true species is represented by PIC particles with multiple different weights. The intuitive reason for this is that PIC particles behave in a manner analogous to the ‘waterbags’ central to the idea of Lynden-Bell relaxation. Waterbags are parcels of phase space, therefore containing some inherent number of true particles that move as a collective entity. PIC particles are hard wired to represent such collections of true particles. To make this comparison more concrete, we will map a ‘collisionless collision operator’, that describes the relaxation of a system to a Lynden-Bell equilibrium (Ewart et al. 2022) onto a numerical collision operator describing relaxation in a PIC plasma (Touati et al. 2022) – by mapping the physical picture of waterbags onto that of PIC particles. The collisionless collision operator relaxes the probability density Pα(v, η) of species α as follows: ∂Pα ∂t = α′ q2 αq2 α′ mα ∂ ∂v · dv′Q(v, v′) · dη′η′ α′ mα [η′ −fα′(v′)]Pα′(v′, η′) ∂Pα ∂v η −α mα′ [η −fα(v)]Pα(v, η) ∂Pα′ ∂v′ η′ , (C1) (C1) where α is the typical volume over which a ﬂuctuation in phase space is correlated (for details, see the discussion in § 3.5 or in Ewart et al. 2022) and Q(v, v′) is a tensor containing information about the interaction potential, whose explicit form we will not need here. The derivation of collision operators such as (C1) is, naturally, subject to a number of approximations and caveats. Chief amongst these is the assumption of an electrostatic, quasilinear system in which phase volume is conserved. A full derivation and discussion of such collision operators can be found in, e.g. Chavanis (2022) or Ewart et al. (2022). In a PIC simulation, a given true species of particle may be represented by a number of different macroparticles that have different ‘weights’ – what this means quantitatively in our language, we shall explain shortly. Appendix B. Numerical method for solving for Lynden-Bell equilibria Once the integrated root-mean-square relative change in the fugacity over a single iteration is ϵF = ⎡ ⎣ 1 ηmax −ηmin ηj (ηj+1 −ηj) F(n+1) F(n) −1 2⎤ ⎦ 1/2 < 10−3, (B3) (B3) the energy of the resulting mean distribution can be computed. The root-ﬁnding method that we then use to determine β is an extremely primitive one: interval halving. The energy of the mean phase-space density with the fugacity resulting from above is computed, and β(n+1) is then increased or decreased depending on whether the computed energy is too high or too low, respectively. The initial step size in β is β(0)/2. If the iteration in β passes over the root (i.e. in going from the nth iteration to the (n + 1)st, the energy goes from above the correct energy to below it, or vice versa), then the step size in β is halved, so that it eventually homes in on the root correctly. The step size is also halved if the step would otherwise result in a negative value for β(n+1). If the computed energy is within a tolerance of 10−3 in units where EG = 1, then the iteration in fugacity is allowed https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 28 R.J. Ewart and others to proceed until ϵF ﬁnally falls below 10−5, at which point the solution is considered converged. to proceed until ϵF ﬁnally falls below 10−5, at which point the solution is considered converged. https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press Appendix C. Lynden-Bell equilibria and PIC plasmas To describe such a system, we set the distribution Pα to be discrete in η, the latter taking values ηα,a corresponding to macroparticle ‘species’: Pα(v, η) = a Pα,a(v)δ(η −ηα,a). (C2) (C2) https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press Universal Lynden-Bell equilibria 29 Universal Lynden-Bell equilibria 29 The collision operator (C1) then becomes The collision operator (C1) then becomes The collision operator (C1) then becomes ∂Pα,a ∂t = α′ q2 αq2 α′ mα ∂ ∂v · dv′Q(v, v′) ∂Pα,a ∂t = α′ q2 αq2 α′ mα ∂ ∂v · dv′Q(v, v′) · a′ α′ηα′,a′ mα [ηα′,a′ −fα′(v′)]Pα′,a′(v′)∂Pα,a ∂v −αηα′,a′ mα′ [ηα,a −fα(v)]Pα,a(v)∂Pα′,a′ ∂v′ . (C3) · a′ α′ηα′,a′ mα [ηα′,a′ −fα′(v′)]Pα′,a′(v′)∂Pα,a ∂v −αηα′,a′ mα′ [ηα,a −fα(v)]Pα,a(v)∂Pα′,a′ ∂v′ . (C3) (C3) Here, to reiterate, fα(v) is the mean phase-space density of particles of species α, which can be written as the sum of the mean phase-space densities fα,a(v) of different macroparticle ‘species’: Here, to reiterate, fα(v) is the mean phase-space density of particles of species α, which can be written as the sum of the mean phase-space densities fα,a(v) of different macroparticle ‘species’: fα(v) = a fα,a(v) = a ηα,aPα,a(v). (C4) (C4) (C4) Next, one must note that PIC particles, like classical particles, occupy zero phase volume. Therefore, if all PIC particles are assumed decorrelated, α = 0. However, the phase-space densities ηα,a are then inﬁnite. Mathematically, this corresponds to taking the limit of ηα,a →∞and α →0 in (C3) and (C4) while holding the product αηα,a – the number of particles in a correlated volume – ﬁxed to the number of ‘true’ particles δNα,a contained in a macroparticle of species (α, a); the quantity δNα,a is what is usually called the ‘weight’ of the macroparticle in the PIC terminology. Clearly, as ηα,a is taken to inﬁnity, it is the mean phase-space density fα,a = ηα,aPα,a that remains ﬁnite. Making all these substitutions and taking the appropriate limit, one ﬁnds from (C3) that it relaxes according to ∂fα,a ∂t = α′,a′ q2 αq2 α′ mα ∂ ∂v · dv′Q(v, v′) · δNα′a′ mα fα′,a′ ∂fα,a ∂v −δNα,a mα′ fα,a ∂fα′,a′ ∂v′ . REFERENCES ABEL, I.G., PLUNK, G.G., WANG, E., BARNES, M., COWLEY, S.C., DORLAND, W. & SCHEKOCHIHIN, A.A. 2013 Multiscale gyrokinetics for rotating tokamak plasmas: ﬂuctuations, transport and energy ﬂows. Rep. Prog. Phys. 76, 116201. 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It is easy to show that, provided the tensor Q(v, v′) is positive deﬁnite and symmetric in (v, v′), the collision operator (C5) has an H-theorem with the entropy S = − α,a 1 δNα,a dvfα,a(v) ln fα,a(v), (C6) (C6) maximised by the equilibria fα,a = Nα,a e−βδNα,aε(v), (C7) (C7) where Nα,a is a normalisation constant set by the number of macroparticles of each weight (the fugacity of these macroparticles). The resulting distribution function of particles of true species α is simply a superposition of Maxwellians: fα(v) = a fα,a(v) = a Nα,a e−βδNα,aε(v), (C8) (C8) which is manifestly the discrete form of the non-degenerate Lynden-Bell equilibrium (3.15). which is manifestly the discrete form of the non-degenerate Lynden-Bell equilibrium (3.15). 983 Published online by Cambridge University Press https://doi.org/10.1017/S0022377823000983 Published online by Cambridge University Press 30 R.J. 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https://openalex.org/W3017039593	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/8C7065F2B489CFD317F796BDB632AF70/S0033291720000823a.pdf/div-class-title-it-is-a-family-affair-individual-experiences-and-sibling-exposure-to-emotional-physical-and-sexual-abuse-and-the-impact-on-adult-depressive-symptoms-div.pdf	English	null	It is a family affair: individual experiences and sibling exposure to emotional, physical and sexual abuse and the impact on adult depressive symptoms	Psychological medicine	2,020	cc-by	9,055	Psychological Medicine cambridge.org/psm Psychological Medicine It is a family affair: individual experiences and sibling exposure to emotional, physical and sexual abuse and the impact on adult depressive symptoms Abstract y Abuse; childhood maltreatment; depression; Family; multilevel; neglect; siblings Background. Childhood abuse and neglect often occurs within families and can have a large influence on mental well-being across the lifespan. However, the sibling concordance of emo- tional abuse and neglect (i.e. together referred to as emotional maltreatment; EM), physical abuse (PA) and sexual abuse (SA) and the long-term impact on the context of siblings’ mal- treatment experiences are unclear. To examine the influence of EM, PA and SA on adult depressive symptoms within the family framework we differentiate between (a) the family- wide (mean level of all siblings) effects and (b) the individual deviation from the mean family level of maltreatment. Author for correspondence: Marie-Louise Kullberg, E-mail: m.j.kullberg@ fsw.leidenuniv.nl Author for correspondence: Marie-Louise Kullberg, E-mail: m.j.kullberg@ fsw.leidenuniv.nl Methods. The sample (N = 636) consists of 256 families, including at least one lifetime depressed or anxious individual and their siblings. Multilevel modeling was used to examine the family-wide and relative individual effects of childhood maltreatment (CM). Results. (a) Siblings showed most similarity in their reports of EM followed by PA. SA was mostly reported by one person within a family. In line with these observations, the mean fam- ily levels of EM and PA, but not SA, were associated with more depressive symptoms. In add- ition, (b) depression levels were more elevated in individuals reporting more EM than the family mean. Conclusions. Particularly in the case of more visible forms of CM, siblings’ experiences of EM and PA are associated with the elevated levels of adult depressive symptoms. Findings implicate that in addition to individual maltreatment experiences, the context of siblings’ experiences is another crucial risk factor for an individuals’ adult depressive symptomatology. Original Article Marie-Louise Kullberg1 , Charlotte van Schie1,2,3 , Eleonore van Sprang4 , Dominique Maciejewski5 , Catharina A. Hartman6 , Bert van Hemert7 , Brenda W. J. H. Penninx4 and Bernet M. Elzinga1,2 Marie-Louise Kullberg1 , Charlotte van Schie1,2,3 , Eleonore van Sprang4 , Marie-Louise Kullberg1 , Charlotte van Schie1,2,3 , Eleonore van Sprang4 , Dominique Maciejewski5 , Catharina A. Hartman6 , Bert van Hemert7 , Brenda W. J. H. Penninx4 and Bernet M. Elzinga1,2 Cite this article: Kullberg M-L, van Schie C, van Sprang E, Maciejewski D, Hartman CA, van Hemert B, Penninx BWJH, Elzinga BM (2021). It is a family affair: individual experiences and sibling exposure to emotional, physical and sexual abuse and the impact on adult depressive symptoms. Psychological Medicine 51, 2063–2073. https://doi.org/10.1017/ S0033291720000823 1Institute of Clinical Psychology, Leiden University, Leiden, The Netherlands; 2Leiden Institute for Brain and Cognition (LIBC), Leiden University Medical Center, Leiden, The Netherlands; 3School of Psychology and Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, Australia; 4Amsterdam UMC, Vrije Universiteit, Psychiatry, Amsterdam Public Health Research Institute, Amsterdam, The Netherlands; 5Department of Developmental Psychopathology, Behavioral Science Institute, Radboud University, Nijmegen, The Netherlands; 6Department of Psychiatry, University of Groningen, University Medical Center Groningen, Interdisciplinary Center Psychopathology and Emotion Regulation, Groningen, The Netherlands and 7Department of Psychiatry, Leiden University Medical Center, Leiden, The Netherlands Received: 15 July 2019 Revised: 23 January 2020 Accepted: 19 March 2020 First published online: 20 April 2020 Received: 15 July 2019 Revised: 23 January 2020 Accepted: 19 March 2020 First published online: 20 April 2020 Introduction Depression is one of the most prevalent mental health problems worldwide (De Graaf, Ten Have, Van Gool, & Van Dorsselaer, 2012; World Health Organization, 2017) and accounted for 40.5% of disability-adjusted life years (Whiteford et al., 2013). As most psychiatric disor- ders, depression finds its roots in adverse childhood circumstances like parental abusive behav- ior (Kessler et al., 2010; Norman et al., 2012). Exposure to childhood maltreatment (CM) is associated with an increased risk of mood disorders (Norman et al., 2012) and in particular depression (Spinhoven et al., 2010), as opposed to other psychopathology. Epidemiological studies have shown that emotional abuse and neglect (emotional maltreatment; EM) strongly predict depression (Cecil, Viding, Fearon, Glaser, & McCrory, 2017; Gerke et al., 2018), fol- lowed by sexual abuse (SA) and physical abuse (PA; Infurna et al., 2016; Spinhoven et al., 2010). The high prevalence of abuse and neglect across the population (Stoltenborgh, Bakermans-Kranenburg, Alink, & van Ijzendoorn, 2015) and its effect on emotional disorders across the entire lifespan (Ege, Messias, Thapa, & Krain, 2015; Nanni, Uher, & Danese, 2012) contributes to the fact that CM has been identified as one of the greatest psychopathology risk factors (Sara & Lappin, 2017). © The Author(s), 2020. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Similarities in sibling experience of CM Studies on the impact of abuse and neglect and the association with psychopathology have typically focused on a variety of consequences of CM for individuals being at risk. However, CM typically involves families, and siblings of maltreated chil- dren are likely exposed to similar parental behaviors (Baldwin & Oliver, 1975; Hamilton-Giachritsis & Browne, 2005; Hines, Kantor, & Holt, 2006; Jean-Gilles & Crittenden, 1990). Studies have shown greatest overlap between siblings in reported emo- tional abuse and neglect, followed by PA and neglect, whereas SA seems to show the least overlap (MacMillan, Tanaka, Duku, Vaillancourt, & Boyle, 2013; Witte, Fegert, & Walper, 2018). The degree of concordance may be related to whether the perpetrator is a family member, since in the case of EM and PA the perpetrator is a parent in 80% of the cases, whereas in SA this is most often someone else (86%; Bifulco, Brown, Lillie, & Jarvis, 1997; Hovens et al. 2009). Although ∼90% of the Western individuals grow up with at least one sibling (Milevsky, 2013) and siblings are at increased risk for similar maltreating behavior, until now the influence of any CM type on adult functioning is mostly studied in unrelated individuals, not considering the experiences of siblings growing up in the same household. The present study High concordance, and moreover an impactful family level of CM would imply that CM is engrained in the family structure and could point out the potential harm of siblings’ childhood experi- ences of abuse and neglect on the long term. Studying the family- wide and relative effects of harsh parenting has shown to be insightful in children and adolescents (Jenkins et al., 2016). However, in adult siblings, the family context of CM and the long-term impact of EM, PA and SA on depressive symptomatol- ogy has, to the best of our knowledge, never been investigated. A within-family approach provides new insights into the family- wide impact of CM and the individual relative harm compared to other siblings that cannot be detected or understood with a between-subjects design, without the reports of siblings from the same household. To elucidate the sibling concordance of abuse and neglect and evaluate the potential harm of CM within a family frame- work, we aim to: (1) examine the extent to which the reports on the three types of CM are similar among siblings (i.e. sibling concordance), where we expect to find most similarity for EM, followed by PA and SA; (2a) elucidate whether the family level of EM, PA and SA is associated with adult depressive symptoms; (2b) and elucidate whether the experience of being maltreated more (EM, PA and SA) relative to the other siblings in the fam- ily is associated with more depressive symptoms, while account- ing for the family level of CM. Based on the abovementioned literature, it is hypothesized that family levels of CM are asso- ciated with individual depressive symptomatology and that the experience of being maltreated more than the other siblings is associated with more depressive symptoms with the strongest effects for EM. Whether and how siblings’ childhood experiences of abuse and neglect are related to depressive symptomatology of an individual on the long-term remains unclear. To unravel the psychological consequences of CM in the family context, a differentiation needs to be made between the impact of family-wide (e.g. an adverse family atmosphere) and the individual experiences con- trasting with those of other siblings (e.g. ‘being the black sheep’), within the family (Feaster, Brincks, Robbins, & Szapocznik, 2011; Steinglass, 1987). Method The present study is part of the Netherlands Study of Depression and Anxiety (NESDA), an ongoing longitudinal cohort study started in 2004, aiming to determine the long-term course and consequences of depression and anxiety. A detailed description of the NESDA study design can be found elsewhere (Penninx et al., 2008). The study protocol was approved by the Ethical Review Board of Amsterdam Medical Centre, location VUmc and by local review boards of each participating center. After full verbal and written information about the study, writ- ten informed consent was obtained from all participants. At the 9-year follow-up (wave 6, 2014–2017), 380 siblings from 256 participants with a lifetime anxiety and/or depressive disorder were interviewed to collect data on anxiety and depression, psy- chosocial functioning and health (behavior) to examine the family context of the development of depression and anxiety disorders in this cohort. For the current study, information on CM and depressive symptoms was collected using self-report measures between 2010 and 2013, 6 years after baseline in wave 4 (W4) and at wave 6 (W6), 9 years after baseline. Siblings participated in W6 only. In the analyses, data of the affected targets collected in W4 and at W6 and sibling data from W6 were used. https://doi.org/10.1017/S0033291720000823 Published online by Cambridge University Press 2064 Marie-Louise Kullberg et al. Marie-Louise Kullberg et al. Marie-Louise Kullberg et al. the same family (i.e. being the black sheep) is related to adult depressive symptomatology. Family-wide effects of CM Within a family context, when one sibling is exposed to abuse or neglect by a parent or other family members may also be at risk by being exposed to the same harmful behavior. In addition to direct exposure, emotional abuse (EA) such as criticism (e.g. ‘you are stupid or lazy, you are the most worthless child ever born’) or physical violence (e.g. getting hit or kicked by a parent), could also affect siblings indirect by being the spectator, i.e. the vicari- ous effect of abuse (Spano, 2018). Thus, a key question we aim to investigate is whether CM reported by siblings growing up together (i.e. the family level) relates to an individual’s adult depression. The impact of being the black sheep Research on negative parenting in children indicates that, next to CM experienced by all or several children in the family, receiving more parental negativity compared to other siblings (i.e. being the black sheep) is also associated with unfavorable mental health outcomes (Dunn, Stocker, & Plomin, 1990; Jenkins, McGowan, & Knafo-Noam, 2016; Meunier, Bisceglia, & Jenkins, 2012; Pike & Plomin, 1996). For example, children reporting less support from their parents relative to their sibling (less favored) reported more depressive symptoms in young adulthood (Jensen, Whiteman, Fingerman, & Birditt, 2013). Hence, the second ques- tion we aim to address is whether being more emotionally mal- treated or physically or sexually abused than other siblings from https://doi.org/10.1017/S0033291720000823 Published online by Cambridge University Press Psychological Medicine 2065 Table 1. Sample characteristics of 636 respondents from 256 families Childhood maltreatment Childhood Trauma Questionnaire-Short Form (CTQ-SF). The CTQ SF is a 25 item retrospective questionnaire assessing five Table 1. Sample characteristics of 636 respondents from 256 families Individual characteristics (N = 636) Female, N (%) 397 (62.4) Level of education Basic (lower vocational education), N (%) 14 (2.2) Intermediate (higher vocational education), N (%) 318 (50.0) High (college/university education), N (%) 304 (478) Age (years), M (S.D.) 49.68 (13.2) Lifetime anxiety disorder, N (%) 338 (53.1) Lifetime depression, N (%) 376 (59.1) Lifetime comorbid depression and anxiety, N (%) 267 (42.0) Current depression, N (%) 65 (10.2) Current anxiety disorder, N (%) 100 (15.7) Depressive symptoms, M (S.D.) 14.53(10.4) Childhood maltreatment total, M (S.D.) 37.88 (10.6) Emotional maltreatment, M (S.D.) 9.82 (3.6) Physical abuse, M (S.D.) 5.63 (1.8) Sexual abuse, M (S.D.) 5.78 (2.4) Family characteristics (n = 256) N (%) Number of participating individuals per family 2 168 (65.6) 3 61 (23.8) ⩾4 27 (10.5) Total family size 2 82 (32.0) 3 73 (28.5) 4 42 (16.4) 5 23 (9.0) 6 21 (8.2) ⩾7 15 (5.9) Sibling constellation Same sex, male 28 (10.9) Same sex, female 92 (35.9) Mixed sex 136 (53.1) Maximum age difference 0–5 years 147 (57.4) 6–9 years 75 (29.3) 10–19 years 34 (13.3) Sample The study sample consisted of 636 participants, within 256 unique families. In total, 380 siblings participated, aged 20–78 years, with and without depression and anxiety disorders, who were related to 256 original NESDA participants with a lifetime anxiety and/or depressive disorder diagnosis, i.e. the affected targets. Inclusion criteria for affected targets were: (1) a lifetime anxiety and/or depressive disorder diagnosis assessed based on the CIDI psychi- atric interview (see below) at least two time points during NESDA measurements; (2) 100% the same biological parents as their sib- lings; (3) participated in at least three out of four NESDA face-to-face interviews; (4) availability of genetic information; (5) approval of contacting siblings for research purposes and (6) participated in 9-year follow-up face-to-face interview. Inclusion criteria for the siblings were (1) currently living in the Netherlands; (2) aged between 18 and 78 years and (3) willing to participate in the 9-year follow-up (W6) face-to-face interview. Targets and siblings with a diagnosis of psychotic disorder, obses- sive–compulsive disorder, bipolar disorder or severe addiction disorder were excluded. A second exclusion criterion was not being fluent in Dutch. Individual and family characteristics are described in Table 1. Mean age of respondents was 49.7 years, 62% was female. Of all respondents, 74% reported EM and 9% reported any form of PA. For SA, 18% from the respondents reported any experience of SA before the age of 16 based on the Childhood Trauma Questionnaire (CTQ). For more detailed information on the prevalence of CM in this sample see Table A1 and Fig. A1 from Appendix 1 in the online Supplementary materials. From 380 siblings, 191 (50.3%) were lifetime affected with an anxiety and/or depressive disorder and 189 siblings (49.7%) did not have a lifetime anxiety and/or depression. https://doi.org/10.1017/S0033291720000823 Published online by Cambridge University Press Psychopathology Given the large overlap between EA and EN (r = 0.63, p < 0.001) we combined the EA and EN subscale into an EM subscale, in line with previous studies in the NESDA cohort by taking the average of the two subscales (see Van Der Werff et al. 2013; van Harmelen et al. 2010), for similar definition see the American Professional Society on the Abuse of Children (APSAC) and Glaser (2002). The Cronbach’s α for the combined emotional abuse and neglect scale was 0.89 in our sample. Subscales used in the analyses are EM, PA and SA. Scores on the CTQ subscales can be assorted into four categories: no mal- treatment (i.e. EM score 5–7, PA score 5–7, SA score 5), low mal- treatment (i.e. EM 8–10; PA 8–9; SA 6–7), moderate maltreatment (i.e. EM 11–14; PA 10–12; SA 8–12) and severe maltreatment (i.e. EM > 14; PA > 13, SA > 13; Bernstein and Fink, 1998). lme4-package version 1.1-17 (Bates, Mächler, Bolker, & Walker, 2014) was used with R version 3.5.0 (R Core Team, 2018). ) ( , ) To identify the family-wide (common across all siblings) and relative (individual deviation from the siblings mean) effects of EM, PA and SA on depressive symptoms, regression analyses using multilevel modeling with two-levels were performed with a family grouping variable as random effect. First, an uncondi- tional means model, without predictors, was built to calculate the ICC. In the baseline model age, gender and educational level were added as covariates. Model 1 includes the family means of the CTQ subscales as predictor variables to assess the contribution of the family level EM, PA and SA, common across all siblings, to individual depressive symptoms and examine between-family differences (2a). Next, in model 2, the individual deviations from the family mean were added to model 1 to iden- tify the effect of the relative level of EM, PA and SA on depressive symptoms and examine within-family differences (2b). A descrip- tion of the exact calculation of the predictors can be found in the online Supplementary materials, Appendix 3. Model fit was com- pared according to the methods for multilevel models with mul- tiple imputed data described by Li, Meng, Raghunathan, and Rubin (1991) using the F test. Multilevel regression analyses (lme4-package 1.1-17; Bates et al., 2014) were carried out with R version 3.5.0 (R Core Team, 2018). Sibling concordance of CM types y We first tested whether the previously found association between individual levels of EM, PA and SA and depressive symptoms (Hovens et al., 2012; Spinhoven et al., 2010) could be replicated in the current sample using a multilevel regression model with a grouping variable unique for each family as a random effect to control for the family structured data. Sum scores per CTQ subscale (EM, PA and SA) were taken as predictors, and IDS sum scores as outcome. Subsequently, to investigate the main research questions, first the sibling concordance of CM, i.e. the degree of similarity within the family for CM, was assessed using intraclass correlations (ICC) for EM, PA and SA (1). The ICC was calculated by dividing the total family variance by the between-family variance on the CTQ subscale, which gives an indication of the degree of sibling resemblance (Higgins & Keller, 1975; Shoukri & Ward, 1989). A high correlation within families is defined as a coefficient of 0.3 or higher (Donner, Eliasziw, & Shoukri, 1998). For the calculation of the ICC the To understand what types of CM are more family-wide and what types are more individual-specific, ICC were calculated. For EM the highest ICC (r = 0.37) was found, followed by PA (r = 0.21), indicating large and medium concordance among siblings, respectively (Donner et al., 1998). The ICC of SA was 0.04, which indicates almost no concordance of reports of SA within the family, meaning that siblings from the same family are not more alike in their reports of SA than random other individuals in the sample. Family levels and relative scores of CM with adult depressive symptoms Psychopathology To examine the level of mul- ticollinearity the variance inflation factor (VIF) score for the final models were inspected. The rule of thumb cut-off criterion of 2.5 for deciding if a given independent variable displays too much multicollinearity was used (O’Brien, 2007). The VIFs of the pre- dictors in models 1 and 2 were all <2.0, indicating that multicol- linearity was not a problem. The R code is available online (https://osf.io/g39yk/) to reproduce all analyses. Individual CM and adult depressive symptoms Individual CM and adult depressive symptoms Individual scores of EM (t = 6.92, p < 0.001) and SA (t = 2.09, p = 0.037), but not PA (t = 1.25, p = 0.212) were significantly asso- ciated with the severity of depressive symptoms, see Table 2 for all model statistics. Of the covariates, being female (t = 2.99, p = 0.003) and having low education (t = −2.55, p = 0.011), but not age (t = 0.51, p = 0.610) were associated to depressive symptoms. Psychopathology Psychopathology Inventory of Depressive Symptomatology-SR (IDS-SR): The Inventory of Depressive Symptomatology (IDS) is a self-report questionnaire designed to measure the number of depressive symptoms (Rush, Giles, & Schlesser, 1986). The questionnaire consists of 30 items, each with four answering options from 0 through 3. Sum scores on the items range from 0 to 84, with higher values indicating more symptoms of depression. The psy- chometric properties are acceptable; for instance, high correla- tions were found between the IDS and scores on the Hamilton Depression Rating Scale and Beck Depression Inventory (Rush, Gullion, Basco, Jarrett, & Trivedi, 1996). The IDS showed excel- lent internal consistency (α = 0.95) in the current sample. Depressive symptomatology was measured in W6 for all participants. Composite Interview Diagnostic Instrument (CIDI): The pres- ence (current and lifetime) of DSM-IV-TR (American Psychiatric Association, 2000) based depressive disorders (dys- thymia and major depressive disorder) and anxiety disorders (generalized anxiety disorder, social phobia, panic disorder with or without agoraphobia and agoraphobia) was established using Composite Interview Diagnostic Instrument (CIDI, version 2.1, WHO) in W6. The CIDI is used worldwide in clinical and epi- demiological studies (e.g. de Graaf et al. 2010; Kessler et al. 2010) and high validity for depressive and anxiety disorders were found (Wittchen, 1994). Childhood Trauma Questionnaire-Short Form (CTQ-SF). The CTQ-SF is a 25-item retrospective questionnaire assessing five types of CM before the age of 16: emotional abuse (EA), physical abuse (PA) and sexual abuse (SA), as well as emotional neglect (EN) and physical neglect (PN). Each scale consists of five items scored on a 5-point Likert scale ranging from never true to very often true. A sum score on the CTQ, ranging from 25 to 125, is calculated by adding the five subscales. Data were collected https://doi.org/10.1017/S0033291720000823 Published online by Cambridge University Press Marie-Louise Kullberg et al. 2066 during W4 for the targets and during W6 for the siblings. Psychometric properties were good (Bernstein et al., 2003; Spinhoven et al., 2014; Thombs, Bernstein, Lobbestael, & Arntz, 2009). The internal consistency of the CTQ (α = 0.88) and most subscales are excellent in the current sample (PA: α = 0.92; SA: α = 0.94; EA: α = 0.90; EN: α = 0.89). Because of the moderate internal consistency for PN (α = 0.45), this subscale is excluded from the analyses. Handling missing data g g Data cleaning, preparation and descriptive statistics were per- formed with IBM SPSS Statistics 23.0 (SPSS Inc., Chicago, Illinois). For the IDS, 1.6% and for the CTQ 2.8% was missing and Little’s Missing Completely at Random (MCAR) test indi- cated that data were not MCAR (χ2 = 16.23, df = 8, p = 0.039). As compared to those with complete data, those with missing data tend to be younger, t(634) = −3.2, p ⩽0.001. There were no differences regarding gender, educational level, CTQ and IDS scores between those with complete and those with missing data (all p’s > 0.05). To retain the sample size, missing data were handled using multiple imputations (mice-package version 3.3.0; Van Buuren and Groothuis-Oudshoorn, 2011), carried out with R version 3.5.0 (R Core Team, 2018). A detailed description of the procedure of imputation can be found in the online Supplementary materials, Appendix 2. The newly generated data- sets reflected the original means. The parameter estimates of all models were combined according to Rubin’s (1987) rules. Family levels and relative scores of CM with adult depressive symptoms Figure 1 illustrates the variation between and within families in CM and depressive symptom levels in six families, randomly drawn from the study sample, emphasizing the value of this https://doi.org/10.1017/S0033291720000823 Published online by Cambridge University Press Psychological Medicine 2067 sibling design to provide more accurate estimates regarding the impact of CM on depressive symptoms. Whereas for example in family 1 no one reports PA or SA, in family 5 both individuals report moderate to severe EM, PA and SA, illustrating the differ- ences between families. Within the families, some individuals report high levels of CM, whereas their sibling(s) do not, as is shown in families 2, 3 and 5. Table 2. Multilevel regression analyses on depressive symptoms: unconditional means model, baseline and individual model (N = 636) Unconditional means model Baseline model Individual model Estimate S.E. T p 95% CI Estimate S.E. T p 95% CI Estimate S.E. T p 95% CI Intercept 14.64 0.48 30.70 <0.001 13.70 15.57 14.56 3.05 4.79 <0.001 8.60 20.51 4.07 3.17 1.28 0.20 −2.15 10.29 EM 0.86 0.12 6.92 <0.001 0.62 1.10 PA 0.33 0.26 1.25 0.212 −0.19 0.84 SA 0.37 0.18 2.09 0.037 0.02 0.71 Covariates Age 0.02 0.04 0.51 0.610 −0.05 0.09 −0.02 0.03 −0.57 0.567 −0.08 0.05 Educational level −1.98 0.78 −2.55 0.011 −3.50 −0.46 −1.48 0.73 −2.02 0.043 −2.91 −0.05 Gender 2.49 0.83 2.99 0.003 0.80 4.07 1.64 0.79 2.06 0.039 0.08 3.19 Between-family variance IDS 21.28 20.97 17.06 Within-family variance IDS 86.42 84.09 73.69 ICC 0.20 0.20 0.19 F 5.29 27.70 p 0.001 <0.001 To test whether the family level of CM related to depressive symptoms, model 1 contained the family means of EM, PA and SA and showed significant improvement in model fit compared to the baseline model including covariates only (F(3, 36367) = 14.25, p < 0.001), see Table 3 for all model statistics. Compared to the baseline model, about 40% of the between-family variation in the IDS score was explained by the family levels of CM. The family mean of EM (t = 3.36, p < 0.001) and PA (t = 2.10, p = 0.036), predicted depressive symptoms, whereas for SA only a trend was found (t = 1.94, p = 0.052). p In order to test the hypothesis whether being relatively more maltreated in comparison with the other siblings related to depressive symptoms, model 2 included the individual deviations from the family mean, i.e. Family levels and relative scores of CM with adult depressive symptoms relative scores, which was a significant improvement to model 1, i.e. family means only, F(3, 42892) = 16.18, p < 0.001 (model 2). The relative EM score (t = 6.40, p < 0.001) was associated with depressive symptoms, whereas no association was found for relative PA score (t = −0.03, p = 0.977) and relative SA score (t = 0.90, p = 0.370). Compared to the baseline model, about 13% of the within-family variation in the IDS score was explained by the relative levels of CM. Post hoc specificity analyses Anxiety disorders were highly prevalent in the current sample (53.1%); therefore, we tested for specificity of our findings to depressive symptoms by rerunning the analyses with anxiety symptoms as assessed by the Beck Anxiety Inventory (BAI; Beck, Epstein, Brown, and Steer, 1988). Individual scores of EM (t = 3.95, p < 0.001), PA (t = 2.11, p = 0.035) and SA (t = 2.29, p = 0.022) were significantly associated with the severity of anxiety symptoms, see Table A2 from Appendix 4 in the online Supplementary materials for all model statistics. The family mean of PA (t = 2.14, p = 0.032) was associated with anxiety symptoms, whereas the family mean of EM (t = 1.83, p = 0.067) and SA (t = 1.83, p = 0.067) did not, see Table A3 from Appendix 4 in the online Supplementary materials for all model statistics. In line with our findings for depression, the relative EM score (t = 3.76, p < 0.001) was associated with anx- iety symptoms, whereas no association was found for relative PA score (t = 0.93, p = 0.352) and relative SA score (t = 1.29, p = 0.196). EM, emotional maltreatment; PA, physical abuse; SA, sexual abuse. Sex: 0 = male 1 = female Sibling concordance of CM types of CM a slightly different picture emerges. The family-wide effects of CM on an individual may be either direct, by being exposed to the same adverse parental behavior, or indirect, the so-called vic- arious effect of CM (Spano, 2018). For EM, both the family means and relative levels (i.e. reporting more abuse or neglect than sib- lings) were jointly related to more depressive symptoms in adult- hood. In addition to the family-wide impact, individuals who report more childhood EM than the family average also report more depressive symptoms. This may reflect the ‘Black Sheep Effect’. Previous studies in both children and adults indicate that differential parenting, e.g. the perception or actually receiving less affect and engagement from a parent than another child in the family (Boyle et al., 2004; Jenkins et al., 2016), are associated with negative outcomes such as depressive symptoms (Jensen et al., 2013; Meunier et al., 2012). Compared to EM, levels of reported PA varied more among siblings from the same family. Surprisingly, despite the variation of PA levels within the family, the family level of PA related to individual depression severity, whereas individual PA reports did not. It should be noted that, when CM types are examined in isolation, individual reports of PA do contribute to elevated depression levels. However, PA often occurs in the context of EM (Higgins & McCabe, 2001) and when modeled simultaneously, the effect of individual PA reports often diminishes or even disappears (Cecil et al., 2017; Spinhoven et al., 2010). In line with these observations, internal- izing psychopathology is often mostly explained by emotional forms of abuse and neglect over and above PA (Cecil et al., 2017). In the case of EM, negative schemas and negative self- perceptions are explicitly handed to the child by parental criticism and belittlement, hence inducing a cognitive vulnerability for depressive symptomatology (van Harmelen et al., 2010; Wright, Crawford, & Del Castillo, 2009). EM was most shared between siblings followed by PA, whereas SA was most individual specific, which was in line with our hypoth- esis and supporting previous studies (Bifulco et al., 1997; Hamilton-Giachritsis & Browne, 2005; Hines et al., 2006; Jean-Gilles & Crittenden, 1990; Witte et al., 2018). The levels of sibling concordance (i.e. Discussion This sibling study on the impact of CM on adult depression examined (1) the extent to which reports on EM (abuse and neg- lect), PA and SA were similar among siblings (i.e. sibling concord- ance), and the association of (2a) the family level and (2b) the differential experience of EM, PA and SA of siblings (e.g. ‘being the black sheep’) with siblings’ individual depressive symptoms in adulthood. In addition, associations with anxiety symptoms were tested to examine the specificity of our findings for depression. https://doi.org/10.1017/S0033291720000823 Published online by Cambridge University Press https://doi.org/10.1017/S0033291720000823 Published online by Cambridge University Press 2068 Marie-Louise Kullberg et al. Fig. 1. Childhood maltreatment (bars) and adult depressive symptoms (lines): An example of six random families from the sample Fig. 1. Childhood maltreatment (bars) and adult depressive symptoms (lines): An example of six random families from the sample Fig. 1. Childhood maltreatment (bars) and adult depressive symptoms (lines): An example of six random families from the sampl Fig. 1. Childhood maltreatment (bars) and adult depressive symptoms (lines): An example of six random families from the sample symptoms (lines): An example of six random families from the sam Sibling concordance of CM types highest for EM and lowest for SA) are in line with the findings that the majority of individuals reporting EM or PA indicated that a parent was the perpetrator, whereas the perpetrator of SA is often someone else rather than a first-degree relative (Hovens et al., 2009). The risk factors for EM described in the literature are mostly parental or environmental factors shared within a family (e.g. parental psychopathology and family struc- ture), resulting in an increased risk of CM for all siblings. In con- trast, risk factors for PA and SA appear to be predominantly individual characteristics (e.g. sex and child’s behavioral pro- blems, Hamilton-Giachritsis & Browne, 2005; Witte et al. 2018). To summarize, when an individual reports EM, there is an increased probability that a sibling experiences similar adversity. However, the other maltreatment types showed small to medium overlap within families (Donner et al., 1998), indicating that PA and SA experiences in childhood are quite unique to each individ- ual of the family. eans CM in the family context and adult depressive symptoms In line with earlier findings, our analyses showed that individual reports of EM and SA, but not PA, were simultaneously related to more depressive symptoms (Cecil et al., 2017; Infurna et al., 2016; Spinhoven et al., 2010). Results of our post hoc specificity analyses indicate that individual experiences of EM and SA are linked to elevated symptom levels of both depression and anxiety, whereas PA is only associated with anxiety, but not depression severity. However, when considering the family mean and relative level The family level’s impact of physical violence on adult depres- sion and anxiety, raises the question whether this is due to a vic- arious effect by witnessing that a sibling has been abused or https://doi.org/10.1017/S0033291720000823 Published online by Cambridge University Press ns of EM, PA and SA) and model 2 (relative EM, PA and SA) (N = 636) Model 2 p 95% CI Estimate S.E. T p <0.001 10.05 21.53 15.79 2.86 5.52 <0.001 10.18 0.001 0.25 0.95 0.59 0.18 3.34 0.001 0.25 1.12 0.18 6.40 <0.001 0.78 0.036 0.06 1.71 0.91 0.42 2.17 0.030 0.09 −0.01 0.33 −0.03 0.977 −0.65 0.052 −0.01 1.20 0.60 0.31 1.95 0.051 0.00 0.19 0.22 0.90 0.370 −0.23 0.643 −0.08 0.05 −0.01 0.03 −0.29 0.775 −0.07 0.038 −3.02 −0.09 −1.35 0.73 −1.86 0.063 −2.78 0.011 0.48 3.67 1.74 0.79 2.21 0.027 0.20 16.40 73.18 0.18 16.18 <0.001 10.1 0.2 0.7 0.0 −0.6 0.0 −0.2 −0.0 −2.7 0.2 10.1 0.2 0.7 0.0 −0.6 0.0 −0.2 −0.0 −2.7 0.2 Marie-Louise Kullberg et al. 2070 on the Childhood Trauma Interview 4 years earlier (W1) in NESDA (Spinhoven et al., 2014), which indicates that the CTQ shows adequate consistency across time, suggesting stability of retrospective CM reports. Hence, it can be assumed that the dif- ference of 5–8 years between data collection points of targets (W4) and siblings (W6) in retrospective recall does not signifi- cantly affect the reliability of the reports on childhood experi- ences. Third, the decomposed variables depend on the number of siblings participating within the family. That is, in large fam- ilies an extreme score of an individual, as may be the case for SA, has a smaller effect on mean family level than in small fam- ilies (Feaster et al., 2011). However, for EM and PA, i.e. abuse occurring within the household, differentiation between family and individual relative effects as risk factors for depression revealed new valuable information. Implications and future research The overall family-wide experience (common across siblings) of EM and PA and the relative experience (individual difference from the family mean; the Black Sheep Effect) of EM contribute to adult depression levels. Our findings underline that the context of siblings’ CM experiences play a vital role in an individuals’ adult depression and anxiety. Even though the environmental influence of CM on depression is substantial, it is important to bear in mind that shared-genetic factors partly determine and explain the presence of depressive symptoms within families (Smoller, 2016). Moreover, our results provide a clear image of sibling concordance with respect to the three CM types, which helps to understand to what extent adult siblings share their (per- ception on) childhood experiences (see Plomin, 2011; Plomin & Daniels, 1987; Turkheimer & Waldron, 2000). Although siblings report similar levels of CM experiences, substantial differences within families also remain. Further research should focus on the underlying determinants of CM within the family to better understand the processes of parental maltreatment and how one individual from the same family may feel more or less depressed than a sibling after similar adverse childhood experiences. Future studies addressing the impact of childhood family adversities, should address the sibling context to disentangle the effect of the family system. Agreement between concurrent and retrospect- ive reports of CM is low (Baldwin, Reuben, Newbury, & Danese, 2019). We therefore like to underline that our retrospective approach targets a specific group of adults reporting CM. Nevertheless, it should be acknowledged that both concurrent and retrospective reports of CM are linked to psychopathology (Newbury et al., 2018; Scott, McLaughlin, Smith, & Ellis, 2012). Observational and qualitative studies would be valuable to eluci- date the within-family differences in more detail to identify indi- viduals within the family at risk, and to advance the understanding of the influence of family context on adult depres- sion. In clinical practice, health professionals should be aware of the effect of CM not only on the targeted individual, but also on other siblings. Informing parents, community health services, general practitioners and schools could lead to better identifica- tion and understanding of the impact of (parental) abuse and neglect and vulnerability to adult depression and anxiety. Moreover, focusing on the improvement of the family environ- ment could potentially contribute to adequate prevention of both CM and psychopathology in long-term. CM in the family context and adult depressive symptoms maltreated (Gerke et al., 2018), or whether reported physical vio- lence is an indication of an adverse family environment in gen- eral, which in turn influences the development of later depressive and anxiety symptoms. The negative impact of the family level of EM and PA could thus also be an expression of a negative family atmosphere in which siblings grew up. Parental psychopathology, substance abuse or divorce, to which all family members are exposed, increase the risk of CM for all offspring (Witte et al., 2018) and also relate to adult depression (Lieb, Isensee, Höfler, Pfister, & Wittchen, 2002; Weissman et al., 2006). This interplay between family risk factors within the household should be further investigated. However, the simul- taneous association between the family level of EM and PA with depression suggests that both types of CM are unique risk factors for depressive symptoms, rather than the same underlying con- struct like an overall negative family atmosphere. The association of the family level of PA with anxiety and depressive symptom levels suggests that physical violence has a common effect on adult anxiety and depression, whereas the family level of EM has a specific effect on depression as compared to anxiety. Although the individual report of SA was associated with depres- sive and anxiety symptoms, the family and relative levels of SA were not. This may be related to the fact that the perpetrator is usually someone outside the core family and the fact that if it occurs within the family it happens mostly in secret, which both may greatly reduce the chance of a vicarious effect. A meth- odological explanation of these findings is that the family mean of SA can be inflated due to one extreme value within the family while the other siblings report no SA. Therefore, association of the SA family mean with depression may reflect the association between an individual report of SA and depression rather than the overall family context. Associations of maltreatment types and depressive and anxiety symptoms remain when controlling for the main effects of gender and educational levels. Conclusion de Graaf, R., Radovanovic, M., van Laar, M., Fairman, B., Degenhardt, L., Aguilar-Gaxiola, S., … Anthony, J. C. (2010). Early cannabis use and esti- mated risk of later onset of depression spells: Epidemiologic evidence from the population-based world health organization world mental health survey initiative. American Journal of Epidemiology, 172(2), 149–159. https://doi. org/10.1093/aje/kwq096 Altogether, the findings of this study illustrate that the childhood experiences of adult siblings are in part interdependent and, fur- thermore, suggest that in addition to individual experiences, mal- treatment experiences of a brother or sister may also have a long-term burden on an individual. Considering the experiences of multiple siblings from the same family and, moreover, differen- tiating between family and relative (within-family) level allows new insights into the individual and family-wide effects of CM on adult depression and anxiety. De Graaf, R., Ten Have, M., Van Gool, C., & Van Dorsselaer, S. (2012). Prevalence of mental disorders and trends from 1996 to 2009. Results from the Netherlands Mental Health Survey and Incidence Study-2. Social Psychiatry and Psychiatric Epidemiology, 47, 203–213. https://doi. org/10.1007/s00127-010-0334-8 Donner, A., Eliasziw, M., & Shoukri, M. (1998). Review of inference proce- dures for the interclass correlation coefficient with emphasis on applications to family studies. Genetic Epidemiology, 15(6), 627–646. https://doi.org/10. 1002/(SICI)1098-2272(1998)15:6<627::AID-GEPI6>3.0.CO;2-G Supplementary material. The supplementary material for this article can be found at https://doi.org/10.1017/S0033291720000823. Dunn, J., Stocker, C., & Plomin, R. (1990). Nonshared experiences within the family: Correlates of behavioral problems in middle childhood. Development and Psychopathology, 2(2), 113–126. https://doi.org/10.1017/ S0954579400000651 Acknowledgement. The infrastructure for the NESDA study (www.nesda. nl) is funded through the Geestkracht program of the Netherlands Organisation for Health Research and Development (ZonMw, grant number 10-000-1002) and financial contributions by participating universities and mental health care organizations (VU University Medical Center, GGZ inGeest, Leiden University Medical Center, Leiden University, GGZ Rivierduinen, University Medical Center Groningen, University of Groningen, Lentis, GGZ Friesland, GGZ Drenthe, and Rob Giel Onderzoekscentrum). Ege, M. A., Messias, E., Thapa, P. B., & Krain, L. P. (2015). Adverse childhood experiences and geriatric depression: Results from the 2010 BRFSS. The American Journal of Geriatric Psychiatry, 23(1), 110–114. https://doi.org/ 10.1016/J.JAGP.2014.08.014 Feaster, D., Brincks, A., Robbins, M., & Szapocznik, J. (2011). Multilevel mod- els to identify contextual effects on individual group member outcomes: A family example. 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J., Van Oppen, P., Spinhoven, P., Penninx, B. W. J. H., & Zitman, F. G. (2009). Conflict of interest. None. Glaser, D. (2002). Emotional abuse and neglect (psychological maltreatment): A conceptual framework. Child Abuse and Neglect, 26(6–7), 697–714. https://doi.org/10.1016/S0145-2134(02)00342-3 Strengths and limitations This is one of the few studies investigating multiple siblings per family, which contributes to the understanding of the family framework in which CM mostly occurs. Moreover, decomposing individual variables into a family level variable and relative scores helps us to differentiate between family-wide and individual- specific effects (Feaster et al., 2011; Jenkins et al., 2016). Another strength is the large clinically relevant sample, consisting of persons with a lifetime depressive or anxiety disorder and their affected and unaffected siblings. Considering the genetic back- ground of depression (Smoller, 2016), it should be acknowledged that the study sample includes families with at least one affected family member, symptom levels in the sample may therefore be higher than in the general population. It should be noted, how- ever, that PA and SA were less prevalent than EM which could have contributed to lower ICC for PA and SA. Nevertheless, des- pite the lower prevalence, PA and SA did contribute to depression in their unique way. Moreover, these numbers do seem to adequately reflect the sibling concordance for the three types of CM. Second, CM was measured using a retrospective instrument (the CTQ), which may be sensitive to recall bias. However, most studies indicate fairly good reliability of the CTQ (Hardt & Rutter, 2004) and are not critically affected by current mood disorders (Spinhoven et al., 2010). Previous studies have reported good test–retest reliability for the CTQ (Bernstein & Fink, 1998). 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https://openalex.org/W4294643580	https://www.researchsquare.com/article/rs-771346/latest.pdf	English	null	Escherichia coli from urine samples of pregnant women as an indicator for antimicrobial resistance in the community: a field study from rural Burkina Faso	Antimicrobial resistance and infection control	2,022	cc-by	7,487	Escherichia Coli From Urine Samples of Pregnant Women as an Indicator for Antimicrobial Resistance in the Community: a Field Study From Rural Burkina Faso. Escherichia Coli From Urine Samples of Pregnant Women as an Indicator for Antimicrobial Resistanc in the Community: a Field Study From Rural Burkina Faso. A.S. Post Radboudumc https://orcid.org/0000-0002-0471-8250 I. Guiraud CRUN M. Peeters Prince Leopold Institute of Tropical Medicine: Instituut voor Tropische Geneeskunde P. Lompo CRUN S. Ombelet Institute of Tropical Medicine: Instituut voor Tropische Geneeskunde I. Karama CRUN S. Yougbaré CRUN Z. Garba CRUN E. Rouamba CRUN H. Tinto CRUN J. Jacobs (  jjacobs@itg.be ) Institute of Tropical Medicine: Instituut voor Tropische Geneeskunde Faso. A.S. Post Radboudumc https://orcid.org/0000-0002-0471-8250 I. Guiraud CRUN M. Peeters Prince Leopold Institute of Tropical Medicine: Instituut voor Tropische Geneeskunde P. Lompo CRUN S. Ombelet Institute of Tropical Medicine: Instituut voor Tropische Geneeskunde I. Karama CRUN S. Yougbaré CRUN Z. Garba CRUN E. Rouamba CRUN H. Tinto CRUN J. Jacobs (  jjacobs@itg.be ) Institute of Tropical Medicine: Instituut voor Tropische Geneeskunde Research Keywords: Antimicrobial resistance, community, asymptomatic bacteriuria, ANC, pregnancy, Escherichia coli, rural Africa, Burkina Faso A.S. Post Radboudumc https://orcid.org/0000-0002-0471-8250 I. Guiraud CRUN M. Peeters Prince Leopold Institute of Tropical Medicine: Instituut voor Tropische Geneeskunde P. Lompo CRUN S. Ombelet Institute of Tropical Medicine: Instituut voor Tropische Geneeskunde I. Karama CRUN S. Yougbaré CRUN Z. Garba CRUN E. Rouamba CRUN H. Tinto CRUN J. Jacobs (  jjacobs@itg.be ) Institute of Tropical Medicine: Instituut voor Tropische Geneeskunde Research Posted Date: August 9th, 2021 Posted Date: August 9th, 2021 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. DOI: https://doi.org/10.21203/rs.3.rs-771346/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License DOI: https://doi.org/10.21203/rs.3.rs-771346/v1 DOI: https://doi.org/10.21203/rs.3.rs-771346/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 2/21 Abstract Introduction: In low- and middle-income countries, surveillance of antimicrobial resistance (AMR) is mostly hospital-based and, in view of poor access to clinical microbiology, biased to more resistant pathogens. We assessed AMR among Escherichia coli isolates obtained from urine cultures of pregnant women as an indicator for community AMR and compared the AMR results with those from E. coli isolates obtained from febrile patients in previously published clinical surveillance studies conducted within the same population in Nanoro, rural Burkina Faso. Results: Between October 2016 – September 2018, midstream urine samples collected as part of routine antenatal in Nanoro district were cultured by a dipslide method and screened for antibiotic residues. Among 6018 consenting women (median (IQR) age 25 (20 - 30)), 84 (1.4%) were excluded because of symptoms of urinary tract infection and 96 (1.6%) screened positive for antibiotic residues. Significant growth - defined as a monoculture of Enterobacterales at counts of ≥ 104 colony forming units/ml – was observed in 202 (3.4%) cultures; E. coli represented 155 (76.7%) of isolates. Among these E. coli isolates, resistance rates to ampicillin, cotrimoxazole and ciprofloxacin were respectively 65.8%, 64.4% 16.2%, compared to 89.5%, 89.5% and 62.5% among E. coli from historical clinical isolates (n = 48 of which 45 from blood cultures). Proportions of extended spectrum beta-lactamase producers and multidrug resistance were 3.2% and 5.2% among E. coli isolates from urine in pregnant women versus 35.4%, and 60.4% respectively among clinical isolates. Adding urine culture to the routine urine analysis (protein and glucose) of antenatal was feasible. The dipslide culture method was affordable and user-friendly and allowed on-site inoculation and easy transport; challenges were contamination (midstream urine sampling) and the semi-quantitative reading. Conclusions: The E. coli isolates obtained from healthy pregnant women had significantly lower AMR rates compared to clinical E. coli isolates, probably reflecting the lower antibiotic pressure in the pregnant women population. Provided confirmation of the present findings in other settings, E. coli from urine samples in pregnant women may be a potential indicator for benchmarking, comparing, and monitoring community AMR rates across populations over different countries and regions. Study design We conducted a cross-sectional study recruiting pregnant women attending routine antenatal care (ANC) in rural Burkina Faso. Urine samples routinely obtained for dipstick analysis (glucose and protein) were semi-quantitatively cultured by dipslide technique to assess for asymptomatic bacteriuria (ASB). For the purpose of this study, isolates growing in counts of ≥ 104 colony forming units (CFU/ml) belonging to the Enterobacterales species and Enterococcus faecalis were considered as "significant growth". Antibiotic susceptibility testing was done for E. coli isolates. AMR profiles were compared to E. coli isolates obtained from blood culture surveillance studies performed in the same district and the same laboratory. Women were asked about recent antibiotic use prior to sampling and urine samples were screened for antibiotic residues. Introduction Antimicrobial resistance (AMR) rises globally and is a threat to public health, particularly in low- and middle-income countries (LMIC) (1). Surveillance is one of the five domains of the World Health Organization's Global Action plan against AMR (2). The Global Antimicrobial Resistance Surveillance System (GLASS) set outs standards for collection, analysis and sharing of AMR data at a worldwide level. Aggregated surveillance data reported to GLASS rely on antibiotic susceptibility testing (AST) results of clinical samples (3). However, surveillance of clinical samples has shortcomings particularly when applied in LMIC. First, LMIC face problems of access to competent and quality-assured clinical bacteriology (4). As a result, samples Page 3/21 processed in LMIC settings may be biased to more advanced disease stages and collected under coverage of empiric antibiotic treatment. Second, surveillance by clinical samples may be influenced by the type of clinical specimen (e.g. blood versus respiratory tract secretions), previous antibiotic use as well as indications for sampling, factors that are often not standardized in LMIC (5). Finally, clinical bacteriology in LMIC is typically implemented at the second level of care, i.e. the district referral hospital (6). Although samples may be collected at the primary level of care (health post, health center), expertise and skills for sampling as well as reliable transport systems may be lacking (1). By consequence, the deducted surveillance data may tend towards an overestimation of AMR rates and may not reflect AMR rates at community level. With the present study we aimed to assess if Escherichia coli isolates recovered from urine samples of healthy pregnant women can serve as a proxy for AMR surveillance at the community level in rural West- Africa. We focused on E. coli as it is the most frequent isolate in asymptomatic bacteriuria in pregnancy (7). In addition, E. coli is a key pathogen in current programs that monitor AMR in human (3) and in One Health populations (AGISAR) (8). Furthermore, we assessed whether adding bacteriological culture of urine to the routine antenatal care is feasible in a rural West-African setting. Study site, period and participants, routine a The study was conducted from October 2016 to September 2018 at the Clinical Research Unit of Nanoro (CRUN) in the Center-West Region of Burkina Faso. Samples were obtained in 9 health centers within the Health and Demographic Surveillance System (HDSS) of CRUN, at 11 to 38 km from CRUN (Fig. 1). The HDSS monitors changes in a total population of 60,000 persons distributed over 24 villages (9). Routine ANC is provided at the health centers and is organized in morning hours between 7 and 12 a.m. A total of 4 ANC visits are recommended during each pregnancy (10) and comprise collection of demographic data (age, week of pregnancy and Gestation Parity Abortion score [GPA score]) and uranalysis for glucose and protein by dipstick test. Page 4/21 Page 4/21 Page 4/21 Study intervention: urine culture and collection of demographic and clinical data Informed consent was sought from all women attending routine ANC by the ANC nurse. After written consent was obtained, a study nurse provided a sterile cup and instructions on how to obtain a clean midstream urine sample. Apart from routinely collected ANC data, information on antibiotic use in the past two weeks, and signs and symptoms of urinary tract infection were collected. For semi-quantitative culture, dipslide devices (Uricult MC/CLED, International Medical Products, Brussels, Belgium and Servocults, Meus S.R.L, Padova, Italy) were used. The dipslide consisted of cysteine-, lactose, and electrolyte-deficient (CLED) agar on one side and MacConkey agar on the other side. Inoculation was done by the study nurse: dipslides were fully submerged in the urine sample, alternatively, a sterile pipet was used to inoculate both agar slides. The dipslide was then placed straight-up on a piece of absorbent paper to allow excess urine to leak off. Subsequently, the urine was tested for presence of glucose and protein using a dipstick analysis (UroColor strips [Standard Diagnostics, Gyeonggi-do, Republic of Korea] or Urine-10 strips [Cypress Diagnostics, Hulshout, Belgium]). Leukocyturia was quantified according to the manufacturers’ instruction as negative (-), + (25–74 cells/µL), ++ (75–499 cells/µL), or +++ (500 or more cells/µL). The urine dipslides and the left-over urine samples were stored in the fridge (2–8°C). Transport to the laboratory was done in a light protected box by motorcycle, within 24 hours after collection, at room temperature. Comparison with clinical isolates For comparison, AST results from clinical isolates of urine and blood cultures collected under different study protocols collected in the Nanoro district hospital and processed at CRUN during the same period were used. Two studies assessed the proportion and differentiation of malaria and bacteremia in the Nanoro district hospital (13, 14), a third study assessed the incidence and reservoir of non-typhoidal Salmonella bloodstream infection (15, 16) and finally, several isolates were obtained from a blood culture surveillance study for follow-up of antimicrobial resistance (unpublished data). p Semi-quantitative culture, bacterial identification and antibiotic susceptibility testing Upon reception at the laboratory of CRUN, dipslides were incubated for 16 to 24 hours at 35°C. Grown cultures were assessed for colony counts by comparing the number of colonies to the figure provided in the product's instructions for use. Bacterial isolates were identified using standardized biochemical techniques and API (bioMérieux, Marcy l’Etoile, France) in case of doubtful test reactions. The isolates were subsequently stored in Tryptic Soy Agar (CM0131, Oxoid Ltd). All bacteria growing in counts of ≥ 104 colony forming units/ml (CFU/ml) except for non-fermentative Gram-negative bacteria and bacteria considered as contaminants (Bacillus spp. and coagulase-negative staphylococci (CNS)) were shipped to the Institute of Tropical Medicine in Antwerp (Belgium) for confirmation of identification by matrix- assisted laser desorption/ionization time-of-flight (MALDI-TOF) technology (Bruker MALDI Biotyper, Bruker, Billerica, MA, US) at the University Hospital of Leuven (Belgium). Isolates identified as E. coli were processed for AST by disk diffusion (Neo-Sensitabs, Rosco Diagnostica A/S, Taastrup, Denmark) according to Clinical & Laboratory Standards Institute (CLSI) guidelines (11). Combination disk tests (Neo-Sensitabs, Rosco Diagnostica A/S, Taastrup, Denmark) were performed to assess the production of Extended Spectrum ß-lactamases (ESBL) (11). Antibiotic residue testing Page 5/21 To assess antibiotic use prior to urine collection, urine samples were tested for the presence of antibiotic residues as part of the work-up at the laboratory of CRUN. For each urine sample, a Mueller-Hinton plate was inoculated with 0.5 McFarland saline solutions of Bacillus spizizenii (ATCC 6633). An absorbent paper disk was saturated with urine and placed on the Mueller-Hinton agar. Plates were incubated at 35°C for 18–24 hours. The appearance of an inhibition zone around the urine disk was considered positive for the presence of residue antibiotics (12). Definitions For the purpose for this study, single-organism cultures with counts of ≥ 104 CFU/ml belonging to the Enterobacterales species or Enterococcus faecalis were considered as "significant growth" and the isolates were considered as “pathogens”. Isolates obtained from febrile patients are referred to as “clinical samples”. Asymptomatic bacteriuria was defined as the presence of actively multiplying bacteria in the urinary tract in patients that have no obvious symptoms of urinary tract infection (UTI) (17). Skin- or environmental bacteria (CNS, Bacillus spp.), non-fermentative Gram-negative bacteria and bacteria growing as mixed flora (≥ 2 different isolates) were considered as contaminants (12). In case a culture grew with mixed isolates including Enterobacterales, the latter were also subcultured for the purpose of antibiotic susceptibility testing (AST). Staphylococcus aureus isolates were not considered for antibiotic susceptibility testing. Multi-drug resistance (MDR) for Enterobacterales was defined as resistance to the three principal oral antibiotic categories for urinary tract infection (penicillins, cotrimoxazole and fluoroquinolones). The number of parities of each participant was categorized as in nullipara (never given birth), primipara (given birth once), multipara (≥ 2 births) or grand multipara (≥ 5 births) (18). Sample size, data registration and statistical analysis Ethics The study was approved by the national ethics committee of Burkina Faso (Comité d’Ethique pour la Recherche en Santé (Reference N˚2015-7-96 July 1st, 2015), the institutional review board of ITM, Antwerp (Reference 1008/15 from December 15th, 2015) and the ethics committee of the University Hospital of Antwerp (Reference 15/51/563, January 4th, 2016). Written informed consent was obtained before participation in the study. A screening log with reasons for refusal was completed at each health center included in the study. If ASB was diagnosed, laboratory staff of the study site communicated the recovery of clinically significant isolates and their AST results to the study investigator, who informed the ANC nurse or the clinician responsible of the ANC. Participants were treated according to national treatment guidelines. Sample size, data registration and statistical analysis In line with the CLSI M39 (19), a minimum number of 30 E. coli isolates was targeted for separate antibiotic susceptibility reporting. Assuming a prevalence of 5% − 10% asymptomatic bacteriuria with 10% contamination rate and E. coli being 40% of retrieved isolates, 6000 women were targeted. Data were recorded in a coded database (Microsoft Excel, Redmond, US). Differences in proportions were compared using as appropriate a Mann-Whitney-u test, a Kruskall Wallis test or a Chi-square test. For smaller sample sizes (value in one of the cells ≤ 5), the Fischer exact test was used. A p-value of 0.05 was Page 6/21 Page 6/21 considered as statistically significant. Reporting of the methods and results was done according to the STROBE guidelines for cross-sectional studies (20). considered as statistically significant. Reporting of the methods and results was done according to the STROBE guidelines for cross-sectional studies (20). Breakdown of samples, proportions of significant growth, species recovered. A breakdown of samples and significant growth is presented in Fig. 2. In total 2292 (38.6%) of dipslides did not have any growth, 2945 (49.6%) had growth of < 104 CFU/ml and 697 (11.6%) had growth of ≥ 104 CFU/ml. In total 202 samples (28.9% of grown cultures and 3.4% of all samples) showed significant growth, of which 122 (2.1%) reached ≥ 105 CFU/ml and therefore qualified as ASB. E. coli accounted for 76.7% of pathogens obtained (155/202) from cultures with ≥ 104 CFU/ml. Other species were Klebsiella spp. (n = 32 [15.8%]), Proteus mirabilis (n = 5 [2.5%]), Enterobacter spp. (n = 5 [2.5%]), Citrobacter spp. (n = 3 [1.5%]) and Pantoea (n = 1 [0.5%]). Species distribution between samples with ≥ 104 CFU/ml and ≥ 105 CFU/ml did not differ significantly (Mann-Whitney p < 0.001). Characteristics of study participants Over a time-period of two years (October 2016 to September 2018) a total of 6018 urine samples were collected. In 84 (1.4%) samples, participants reported signs suggestive of urinary tract infection: 77 (1.3%) participants declared symptoms of dysuria and 7 (0.1%) declared both hematuria and dysuria. Their samples were excluded for analysis, leaving 5934 samples representing 5907 unique participants; 27 participants were sampled on two separate ANC visits. Data on dipstick results were missing from 48 samples; of them, 36 were collected between June 11th and August 8th 2017, from the healthcare center of Seguedin. The age of participants ranged from 14 years to 50 years. Overall, the median (interquartile range [IQR]) age was 25 (20–30) years (Table 1). In total 24.4% of samples were obtained from nulli- or primipara. All other cases were multi (42.9%) or grand multipara (32.7%). Trimesters 1, 2 and 3 comprised respectively 3.9%, 32.7% and 63.5% of samples. The median (IQR) age was similar among the three trimester groups (25 [20–30], 24 [19–30] and 26 [21–30]) years respectively, although this difference was statistically significant (p < 0.0001). Page 7/21 Table 1 Overview of demographic data of unique study participants Participants as stratified by age* < 20 years 20–29 years > 30 years n = 1181 n = 2904 n = 1782 Age (median [IQR]) 18 (18–19) 24 (22–27) 33 (30–35) Trimester (median [IQR]) 3 (2–3) 3 (2–3) 3 (2–3) Gestation nullipara (% within agegroup) 5 (0.4%) 2 (0.1%) 0 (0) primipara (% within agegroup) 975 (82.6%) 399 (13.7%) 14 (0.8%) multipara (% within agegroup) 194 (16.4%) 1958 (67.4%) 388 (21.8%) grand multipara (% within agegroup) 7 (0.6%) 545 (18.8%) 1380 (77.4%) Reported antibiotics use prior to sampling (nr. [%]) 7 (0.6%) 14 (0.5%) 1 (0.1%) * unique participants = 5907; age missing for 40 participants \| data on trimester missing for 17 participants Table 1 Other growth In total 564 samples with bacterial growth were classified as non-significant growth because they (i) grew Enterobacterales in less than 104 CFU/ml (n = 52), (ii) grew with pathogens not usually associated with urinary tract infection (n = 37), (iii) grew skin contaminants (n = 325) or (iv) grew 2 or more pathogens (n = 150). A specification of these isolates can be found in Supplementary Table 1. E. coli from clinical samples E. coli from clinical samples Page 8/21 A total of 48 E. coli isolates were obtained from clinical samples in the Nanoro district hospital between 2012 and 2019. Forty-five isolates were retrieved from blood cultures and 3 isolates from urine cultures. None of the isolates from urine samples were obtained from pregnant women. Antibiotic resistance among E. coli from healthy pregnant women and febrile patients AMR profiles of both the clinical isolates and isolates from healthy pregnant women are presented in Table 2. Resistance to ampicillin, cotrimoxazole and ciprofloxacin were respectively 65.8%, 64.4% 16.2%. MDR was observed in 5.2% isolates; resistance to gentamicin occurred in 3.9% isolates. There were 5 (3.2%) ESBL producing E. coli isolates; 2 of which were co-resistant to cotrimoxazole and ciprofloxacin, of which one was co-resistant to gentamicin as well. There was no resistance to fosfomycin and only 3.9% resistance to nitrofurantoin among E. coli recovered from urine in healthy pregnant women. Page 9/21 Table 2 Supplementary Table 2 shows an overview of antibiotic susceptibility profiles of other major pathogens obtained from healthy pregnant women; other Enterobacterales growing in ≥ 104 CFU/ml, E. coli obtained from urine samples growing in mixed flora. This overview shows that antibiotic resistance among other potential pathogens (i.e. Klebsiella spp., Enterobacter spp., Proteus spp.) was also relatively low. One Klebsiella pneumoniae and one Enterobacter cloacae were ESBL-producers (2/44, 4.5% of non-E. coli Enterobacterales). T i d i Table 2 Proportions of antibiotic resistance and combined antibiotic resistance for Escherichia coli obtained from urine samples of healthy pregnant women and clinical samples from febrile patients Differences in proportions of clinical and urine isolates were assessed using chi-square test. For smaller sample sizes (value in one of the cells ≤ 5), the Fischer exact test was used. All differences between isolates obtained from healthy pregnant women and febrile patients were statistically significant (p < 0.001) urine samples of healthy pregnant women and clinical samples from febrile patients Differences in proportions of clinical and urine isolates were assessed using chi-square test. For smaller sample sizes (value in one of the cells ≤ 5), the Fischer exact test was used. All differences between isolates obtained from healthy pregnant women and febrile patients were statistically significant (p < 0.001) Healthy pregnant women Febrile patients Urine culture (n = 155) Urine culture (n = 3) blood culture (n = 45) Resistance per antibiotic n (%) n = 48 (%) nitrofurantoin 6 (3.9%) not done fosfomycin 0 (0%) not done ampicillin 102 (65.8%) 43 (89.6%) cotrimoxazole 97 (64.4%) 43 (89.6%) ciprofloxacin 25 (16.2%) 30 (62.5%) gentamicin 6 (3.9%) 11 (22.9%) ceftriaxone 5 (3.2%) 18 (37.5%) ESBL producers 5 (3.2%) 17 (35.4%) Combined resistance n (%) n (%) ampicillin + cotrimoxazole 80 (51.6%) 41 (85.4%) ampicillin + cotrimoxazole + ciprofloxacin 8 (5.2%) 29 (60.4%) ampicillin + cotrimoxazole + gentamicin 3 (1.9%) 11 (22.9%) ampicillin + cotrimoxazole + ciprofloxacin + gentamicin 2 (1.3%) 11 (22.9%) ESBL + cotrimoxazole 2 (1.3%) 17 (35.4%) ESBL + cotrimoxazole + ciprofloxacin 2 (1.3%) 16 (33.3%) ESBL + cotrimoxazole + gentamicin 1 (0.6%) 7 (14.5%) ESBL + cotrimoxazole + ciprofloxacin + gentamicin 1 (0.6%) 7 (14.5%) There was no statistical difference in resistance patterns between isolates growing in counts of 104 CFU/ml and 105 CFU/ml In contrast, resistance rates among isolates obtained from febrile patients were significantly higher for all individual antibiotics as well as combinations. Out of 48 isolates 43 (89.5%) were resistant to each ampicillin and cotrimoxazole, and 30 (62.5%) were resistant to ciprofloxacin; MDR was observed in 60.4% of isolates. Resistance to gentamicin was lowest at 22.9%. In total 18 isolates showed resistance to ceftriaxone; all but one (35.4% of total) were confirmed as ESBL producers. All ESBL producing isolates were co-resistant to cotrimoxazole and all but one were co-resistant to ciprofloxacin. Trimester and parity Data on trimester of pregnancy, gestation and parity was available for 5890 and 5934 participants respectively (Table 3). Most (120/202 [59.4%]) participants with significant growth were in their third trimester of pregnancy, differences with the first and second trimester were however not significant (Kruskall Wallis, p = 0.6). There was also no relation between proportion of significant growth and parity (Kruskall Wallis, p = 0.07). Page 11/21 Table 3 Total numbers of samples with numbers of significant growth for 5890 healthy pregnant women, matched by parity and trimester of pregnancy. Significant growth is defined as growth of Enterobacterales in counts of ≥ 104 CFU/ml. Parity was defined as follows: nullipara = never give birth, primipara = given birth once, multipara = parity ≥ 2, grand multipara = parity ≥ 5 (18). Patients with full data Total Trimester 1 Trimester 2 Trimester 3 n = 5890 n = 5890 n = 231 n = 1925 n = 3734 Gestation nr. Cases % growth nr. Cases % growth nr. Cases % growth nr. Cases % growth nullipara (percentage significant growth) 7 0% 1 0% 1 0% 5 0% primipara (percentage significant growth) 1396 4.4% 65 4.6% 545 4.0% 786 4.6% multipara (percentage significant growth) 2550 3.3% 101 3.9% 844 3.9% 1605 3.0% grand multipara (percentage significant growth) 1937 2.7% 64 0% 535 3.2% 1338 2.7% Age distribution (years) 25 (20–30%) 25 (20–30%) 24 (19–30%) 26 (21–30%) Dipstick results Nitrite* (n [%]) 74 (1.2%) 7 (3.0%) 31 (1.6%) 36 (1.0%) Leukocytes* (n [%]) 1083 (18.4%) 45 (19.5%) 321 (16.7%) 717 (19.2%) Dipslide results** < 104 CFU/ml 2920 (49.6%) 127 (55.0%) 918 (47.7%) 1875 (50.2%) ≥ 104 CFU/ml 687 (11.7%) 19 (8.2%) 194 (10.1%) 474 (12.7%) Clinically significant growth 199 (3.4%) 7 (3.0%) 72 (3.7%) 120 (3.2%) Asymptomatic bacteriuria 120 (2.0%) 4 (1.7%) 42 (2.2%) 74 (2.0%) Data differ slightly from those in Fig. 2 where data are presented for all samples Clinically significant growth is defined as growth with an Enterobacterales species in counts of ≥ 104 CFU/ml Asymptomatic bacteriuria is defined as clinically significant growth with counts of ≥ 105 CFU/ml * Dipstick was done for 5970 samples. The trimester was not recorded for 6 of them, of whom 1 with Total numbers of samples with numbers of significant growth for 5890 healthy pregnant women, matched by parity and trimester of pregnancy. Trimester and parity Significant growth is defined as growth of Enterobacterales in counts of ≥ 104 CFU/ml. Parity was defined as follows: nullipara = never give birth, primipara = given birth once, multipara = parity ≥ 2, grand multipara = parity ≥ 5 (18). Clinically significant growth is defined as growth with an Enterobacterales species in counts of ≥ 104 CFU/ml Clinically significant growth is defined as growth with an Enterobacterales species in counts of ≥ 104 CFU/ml * Dipstick was done for 5970 samples. The trimester was not recorded for 6 of them, of whom 1 with dysuria and 1 with leukocyturia. Leukocyturia as recorded here combines samples quantified as 25 cells/µl or more. Page 12/21 Page 12/21 Patients with full data Total Trimester 1 Trimester 2 Trimester 3 Trimester not recorded for 45 samples, of whom 10 had a urine culture of ≥ 104 CFU/ml and 2 has a urine culture of ≥ 105 CFU/ml Patients with full data T Trimester 1 Trimester 2 Trimester not recorded for 45 samples, of whom 10 had a urine culture of ≥ 104 CFU/ml and 2 has a urine culture of ≥ 105 CFU/ml ** Trimester not recorded for 45 samples, of whom 10 had a urine culture of ≥ 104 CFU/ml and 2 has a urine culture of ≥ 105 CFU/ml Antibiotics use prior to sampling Screening for antibiotics residue was available for 5800/5934 (97.7%) participants. In total 96 (1.7%) samples screened positive for antibiotic residue. Previous antibiotics use was reported by 29 patients, of whom 2 had antibiotic residues in their urine samples. One participant with significant growth reported antibiotics use prior to sampling and four participants screened positive for antibiotic residues. Summary of findings The present study assessed the AMR rates of E. coli present as significant growth (≥ 104 CFU/ml) in the urine of healthy pregnant women in rural Burkina Faso. Among 155 E. coli isolates obtained from 5934 healthy women, AMR rates were significantly lower compared to E. coli isolates obtained from clinical samples (mostly blood cultures) in the same district. Leukocyte esterase and nitrite Leukocyte esterase and nitrite of the dipstick were available for 5886/5934 (99.2%) samples (Supplementary Table 3). Leukocyturia was present in 1084 (18.4%) samples (quantification: +; n = 712 [12.1%], ++; n = 240 [4.1%] and +++; n = 132 [2.2%]) and nitrite in 74 (1.2%) samples. For detection of significant growth, the positive predictive and negative predictive values of leukocyturia were 6.7% and 97.3% respectively; for nitrite they were 37.8% and 97.0% and for leukocyturia and nitrite combined they were 48.7% and 96.9%. Comparison with other studies In the present study, ASB was defined as growth of one species of Enterobacterales in counts of 105 CFU/ml or more and was present in 2.1% of women. For antimicrobial susceptibility testing we used the quantitative cut-off of ≥ 104 CFU/ml to define ‘significant growth’, as previously done in an international survey of antimicrobial susceptibility in uncomplicated urinary tract infections (21). The ASB proportion in the present study was lower compared to some earlier studies from sub-Saharan Africa, citing proportions of 7% − 40% (22–25), but it was comparable to those found in other studies (26, 27). The presently lower proportions of ASB may be related to the stringent definition, i.e. including only Enterobacterales as significant organisms, whereas in most other studies with higher ASB proportions, Staphylococci represented a substantial number of cases (22–25). Page 13/21 AMR rates among the E. coli isolates obtained from urine of pregnant women were significantly lower compared to AMR rates of clinical isolates. Among the individual antibiotics, this difference was most apparent for ciprofloxacin, i.e. 16.2% for the urine isolates in pregnant women versus over 60% among clinical isolates. Likewise, proportions of ESBL producing and MDR isolates among E. coli from urine in healthy pregnant women were 3.2% and 5.2% versus 35.4%, and 60.4% respectively among clinical isolates. It is tempting to speculate that these differences reflect the use of antibiotics such as ciprofloxacin and third generation cephalosporins in the community setting. The proportion of ESBL producers among the clinical E. coli isolates of the comparator studies (35.4%) was slightly lower compared to the 45% reported for sub-Saharan Africa in recent meta-analyses (28–30). Carriage rates of ESBL producing E. coli from stool samples ranged from 38% in Chad to 58% in the Central African Republic (31, 32). For the urine isolates obtained in pregnant women, AMR rates were lower compared to those found in other cross-sectional studies assessing ASB among pregnant women in sub-Saharan Africa. A study from Ghana from 2018 reported high resistance rates among E. coli to nitrofurantoin (35.4%), ciprofloxacin (48.8%), gentamicin (41.5%) and cefuroxime (32.9%) (33). Two studies from Nigeria (2007 and 2010) reported resistance rates among E. coli of approximately 20% against second generation cephalosporins, 40% against gentamicin and 20–70% against ciprofloxacin (23, 34), which was similar to results from a study performed in Uganda in 2010 (35). Comparison with other studies A possible explanation for the observed difference to our results is the fact that we strictly excluded participants with symptoms and signs of urinary tract infection. E. coli from urine in healthy pregnant women as an indicator of AMR in the community In the present cohort of over 6000 pregnant women attending ANC, only 1.5% declared symptoms suggestive of an urinary tract infection and only 1.7% had evidence of antibiotic use as demonstrated by urine analysis. The latter proportion is very low compared to 30% − 40% antibiotic use (based on parents’ declaration) among children suspected of invasive bacterial infection in three of the comparator studies (13–15). As such, the presently observed low AMR rates among E. coli isolates from the urine of healthy pregnant women tends to confirm our pre-study assumption, i.e. there may be a risk of overestimation of AMR rates when performing surveillance on selected clinical samples (36). However, other factors must be taken into account when comparing resistance rates between both groups. First, the species E. coli has distinct pathotypes displaying different degrees in pathogenicity and AMR (37); further genetic studies are planned to assess the pathotypes of the isolates from pregnant women versus those of the clinical samples. Further, in view of low Infection Prevention & Control in healthcare facilities, it is not excluded that part of the clinical isolates were belonging to a particular hospital-associated cluster. Notwithstanding these considerations, it is tempting to forward E. coli in urine of pregnant women as a potential indicator for benchmarking, comparing and monitoring community AMR rates across communities over different countries and regions. Such community AMR data generate valuable Page 14/21 Page 14/21 information about the empiric choice of antibiotics in the local context (38, 39) but may also reflect the effect of AMR control measures. As shown at least in this study setting (and to be confirmed in other settings as well), pregnant women have limited illness and antibiotic use and are accessible through ANC clinics. As part of ANC clinics, urine is routinely sampled for dipstick analysis of glucose and protein and WHO recommends midstream urine culture for the diagnosis of ASB (40). The dipslide devices presently used were affordable (cost approximately 1 €/device) and user-friendly; they have a long shelf-life (6–9 months at room temperature) and allow for reliable inoculation on-site and subsequent transport to the laboratory. At the downside, there are the challenges of midstream-urine sampling (including contamination) and the reading of the colony counts on the dipstick devices as discussed above. Moreover, the proportion of significant growth is low. Limitations and strengths As noted above, reading of colony counts on dipslide devices tended to be subject to interpretation and this may have impacted the classification of non-significant growth, significant growth and ASB. However, our results showed that pathogen and AMR profiles were similar between E. coli from the latter two groups. Second, despite well-designed instructions and training, 8.3% of the samples were contaminated, probably related to the less stringent urine sampling in the context of a ANC compared to clinical care. Third, in retrospect, we realized that the GPA system of parity had not been fully understood by all study nurses, leading to possible too low reported numbers of nullipara compared to primipara. Additionally, there were missing data from 36 consecutive participants (0.6% of all included patients) at one of the healthcare centers. Strengths included the systematic methods used to perform this study, with high numbers of participants included and a consistent work-up by a small team of nurses and laboratory staff. The definitions for ASB and contamination were stringent, adding to the robustness of data. E. coli from urine in healthy pregnant women as an indicator of AMR in the community Leukocyte esterase and nitrite analysis (incorporated in most urine dipsticks) can be used as a screening tool to select samples for culture (40) but in the present study they were not very accurate to predict growth; further research for a reliable biomarker predicting growth is recommended. Conclusion In conclusion, in this cross-sectional study among healthy pregnant women attending ANC in rural Burkina Faso, we retrieved significant growth in 3.4% of urine samples, with E. coli representing over three-quarters of isolates. AMR rates were considerably lower among these urine samples compared to E. coli isolates obtained from clinical isolates in the same study area. Pending further research (geographic generalizability of proportions of growth and predominance of E. coli), E. coli obtained from urine culture during ANC visits has the potential of an indicator organisms for benchmarking and monitoring AMR rates across populations worldwide. Acknowledgments Acknowledgments Page 15/21 The authors are grateful to all participants and ANC nurses for their participation to the study. We would like to thank the laboratory staff of the CRUN unit in Nanoro and to the research nurses of the participating CSPS. Furthermore, a special thanks to Katrien Lagrou from UZ Leuven for assisting in the use of the MALDI-TOF system. Writing – review & editing – AP, IG, MP, LP, SO, IK, SY, ZG, ER, HT, JJ Writing – review & editing – AP, IG, MP, LP, SO, IK, SY, ZG, ER, HT, JJ Writing – original draft – AP, MP, SO, JJ Writing – original draft – AP, MP, SO, JJ Author contributions Conceptualization – AP, IG, JJ Data curation – IG, MP, LP, IK, SY, ZG Formal analysis – AP, MP, ER, JJ Methodology – AP, IG, JJ Supervision – HT, JJ Validation – MP, JJ Conceptualization – AP, IG, JJ Conceptualization – AP, IG, JJ Data curation – IG, MP, LP, IK, SY, ZG Formal analysis – AP, MP, ER, JJ Methodology – AP, IG, JJ Supervision – HT, JJ Validation – MP, JJ Conceptualization – AP, IG, JJ Data curation – IG, MP, LP, IK, SY, ZG References 1. Jacobs J, Hardy L, Semret M, Lunguya O, Phe T, Affolabi D, et al. Diagnostic Bacteriology in District Hospitals in Sub-Saharan Africa: At the Forefront of the Containment of Antimicrobial Resistance. Frontiers in medicine. 2019;6:205. 2. World Health Organisation. Global Action Plan on AMR. 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Meta-analysis of proportion estimates of Extended-Spectrum-Beta-Lactamase-producing Enterobacteriaceae in East Africa hospitals. Antimicrobial Resistance & Infection Control. 2016;5(1):18. 30. Mansouri F, Sheibani H, Javedani Masroor M, Afsharian M. Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae and urinary tract infections in pregnant/postpartum women: A systematic review and meta-analysis. International journal of clinical practice. 2019:e13422. 31. Farra A, Frank T, Tondeur L, Bata P, Gody JC, Onambele M, et al. High rate of faecal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae in healthy children in Bangui, Central African Republic. Clinical Microbiology and Infection. 2016;22(10):891.e1-.e4. 31. Farra A, Frank T, Tondeur L, Bata P, Gody JC, Onambele M, et al. High rate of faecal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae in healthy children in Bangui, Central African Republic. Clinical Microbiology and Infection. 2016;22(10):891.e1-.e4. 32. Ouchar Mahamat O, Tidjani A, Lounnas M, Hide M, Benavides J, Somasse C, et al. Fecal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae in hospital and community settings in Chad. Antimicrobial Resistance & Infection Control. 2019;8(1):169. 33. Forson AO, Tsidi WB, Nana-Adjei D, Quarchie MN, Obeng-Nkrumah N. Escherichia coli bacteriuria in pregnant women in Ghana: antibiotic resistance patterns and virulence factors. BMC research notes. 2018;11(1):901. 34. Imade PE, Izekor PE, Eghafona NO, Enabulele OI, Ophori E. Asymptomatic bacteriuria among pregnant women. North American journal of medical sciences. 2010;2(6):263-6. 34. Imade PE, Izekor PE, Eghafona NO, Enabulele OI, Ophori E. Asymptomatic bacteriuria among pregnant women. North American journal of medical sciences. 2010;2(6):263-6. 35. Andabati G, Byamugisha J. Microbial aetiology and sensitivity of asymptomatic bacteriuria among ante-natal mothers in Mulago hospital, Uganda. African health sciences. 2010;10(4):349-52. 36. Alós JI, Serrano MG, Gómez-Garcés JL, Perianes J. Antibiotic resistance of Escherichia coli from community-acquired urinary tract infections in relation to demographic and clinical data. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases. 2005;11(3):199-203. Page 18/21 Page 18/21 37. Allocati N, Masulli M, Alexeyev MF, Di Ilio C. Escherichia coli in Europe: an overview. References Int J Environ Res Public Health. 2013;10(12):6235-54. 38. Schechner V, Temkin E, Harbarth S, Carmeli Y, Schwaber MJ. Epidemiological interpretation of studies examining the effect of antibiotic usage on resistance. Clinical microbiology reviews. 2013;26(2):289-307. 39. Organisation WH. Surveillance standards for antimicrobial resistance. Geneva: World Health Organisation; 2001. 39. Organisation WH. Surveillance standards for antimicrobial resistance. Geneva: World Health Organisation; 2001. 40. World Health Organisation. WHO recommendation on the method for diagnosing anaemia in pregnancy. 2016. p. 1-5. 40. World Health Organisation. WHO recommendation on the method for diagnosing anaemia in pregnancy. 2016. p. 1-5. Figures Page 19/21 Figure 2 clinically significant growth is defined as isolates growing in counts of ≥ 104 CFU/ml belonging to the Enterobacterales species or Enterococcus faecalis. Clinically significant isolates growing in counts of ≥ 105 CFU/ml were defined as ASB. clinically significant growth is defined as isolates growing in counts of ≥ 104 CFU/ml belonging to the Enterobacterales species or Enterococcus faecalis. Clinically significant isolates growing in counts of ≥ 105 CFU/ml were defined as ASB. Figure 1 Study site, period and participants, routine antenatal care Page 20/21 SupplementaryTables.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 21/21 Page 21/21
https://openalex.org/W4206251090	https://dugi-doc.udg.edu/bitstream/10256/3583/1/Microtox-toxicity.pdf	English	null	Is Microtox® toxicity related to potentially harmful algae proliferation in Mediterranean salt marshes?	Limnética/Limnética	2,010	public-domain	6,730	Limnetica, 29 (2): 257-268 (2010) Limnetica, 29 (2): 257-268 (2010) , 29 (1): x-xx (2008) , 29 (1): x-xx (2008) Limnetica, 29 (2): 257-268 (2010) c⃝Asociaci´on Ib´erica de Limnolog´a, Madrid. Spain. ISSN: 0213-8409 , ( ) ( ) c⃝Asociaci´on Ib´erica de Limnolog´a, Madrid. Spain. ISSN: 0213-8409 otox R⃝toxicity related to potentially harmful algae proliferation in Mediterranean salt marshes? Mediterranean salt marshes are ecosystems that are highly inuenced by sea changes and freshwater inputs from runoff. In these ecosystems, toxic and non-toxic algae blooms often produce large and unpredictable biomasses of phytoplankton. The Microtox R⃝test has been described as a successful, quick method for detecting toxicity in various phytoplankton taxa. Our study sought to test the efciency of Microtox R⃝in detecting toxic HAB in Mediterranean salt marshes. The results showed that the Microtox R⃝test was able to detect toxic substances in the particulate matter of several lagoons in the Empord`a salt marshes. This Microtox R⃝toxicity coincided with periods when potentially harmful cyanobacteria, dinoagellates and haptophytes had a high biomass. The results suggest that potentially harmful phytoplankton cannot be ruled out as a source of Microtox R⃝toxicity or as a source of other organism kills and sub-acute effects in Mediterranean salt marshes. Key words: Toxicity assay, coastal lagoons, bloom, phytoplankton, connement. ∗Corresponding author: rocio.lopez@udg.edu 2 ∗Corresponding author: rocio.lopez@udg.edu 2 Accepted: 24/2/10 Received: 25/8/09 Received: 25/8/09 Received: 25/8/09 Is Microtox R ⃝toxicity related to potentially harmful algae proliferation in Mediterranean salt marshes? Roc´o L´opez-Flores1,∗, Dani Boix1, Anna Badosa2, Sandra Brucet3 and Xavie Flores1,∗, Dani Boix1, Anna Badosa2, Sandra Brucet3 and Xavier D. Quintan 1 Institute of Aquatic Ecology and Department of Environmental Sciences. University of Girona. Facultat de Ci`encies. Campus de Montilivi. 17071 Girona. Spain. 2 Department of Wetland Ecology, Do˜nana Biological Station-CSIC. c/Am´erico Vespucio s/n, Isla de la Cartuja. 41092 Sevilla, Spain. 3 European Commission, Joint Research Centre, Institute for Environment and Sustainability. Via E. Fermi, 2749, 21027 Ispra, Italy. 2 Palabras clave: Ensayo de toxicidad, lagunas costeras, proliferaci´on, toplancton, connamiento. INTRODUCTION the ecosystem has characteristics similar to the Empord`a salt marshes, where the variety of wa- terbodies (Trobajo et al., 2002; Gasc´on et al., 2005) and the high variability of the phytoplank- ton community in different environmental condi- tions (L´opez-Flores et al., 2006a; L´opez-Flores et al., 2006b; L´opez-Flores et al., 2009) necessitate an extensive method of toxicity-detection. Mediterranean salt marshes are ecosystems that are highly inuenced by sea changes and fresh- water inputs from runoff (Britton & Crivelli, 1993). The composition of the phytoplankton community in these areas is largely determined by hydrological variability (Quintana & Moreno- Amich, 2002; L´opez-Flores et al., 2006a). The hydrological pattern leading to phytoplankton production is characterised by sudden inputs dur- ing sea storms that cause the lagoons to be ooded, mainly by seawater. Later, these salt marsh basins become conned, leading to high concentrations of organic matter, nutrients and organisms (this process is most extreme during summer; see Quintana et al., (1998). In these ecosystems, toxic and non-toxic algae blooms of- ten and unpredictably produce large biomasses of dinoagellates, cryptophytes, haptophytes or cyanobacteria species (Quintana and Moreno- Amich, 2002; L´opez-Flores et al., 2006a). The proliferation of harmful phytoplankton in nearby Mediterranean beaches and harbours has been re- ported by several authors (Garc´es et al., 2000; Vila et al., 2001). Some authors have also re- ported HAB (Harmful Algal Blooms) events in other Mediterranean coastal lagoons (e.g., Com´n & Ferrer, 1978; Sarno et al., 1993). However, L´opez-Flores et al. (2006b) showed that, rather than reaching high concentrations of biomass and becoming potentially harmful, the taxa pro- liferating in the coastal lagoons were not the same as those taxa forming blooms in the nearby marine waters. Thus, the species composition and abundance variability in these ecosystems make it difcult to monitor toxicity because one needs a method that provides extensive and efcient measurements. The aim of this study was to perform an initial test of the efciency of Microtox R⃝in detecting toxic HAB in Mediterranean salt marshes. In or- der to reach this objective, we analysed the com- position of phytoplankton taxa during periods of high biomass development and its relationship to the results obtained through the Microtox R⃝test. ¿Est´a la toxicidad detectada por Microtox R⃝relacionada con las proliferaciones de algas potencialmente t´oxicas en las marismas mediterr´aneas? ¿Est´a la toxicidad detectada por Microtox R⃝relacionada con las proliferaciones de algas potencialmente t´oxicas en las marismas mediterr´aneas? Las marismas mediterr´aneas son ecosistemas altamente inuenciados por la entrada de agua marina y de agua dulce, proce- dente de la escorrent´a. En estos ecosistemas, las proliferaciones de algas t´oxicas y no t´oxicas sin patr´on temporal o es- pacial, alcanzan altas e impredecibles biomasas de toplancton. El ensayo Microtox R⃝ha sido descrito como un r´apido y eciente m´etodo para detectar la toxicidad de diferentes taxones de toplancton. Nosotros hemos hecho una primera aproxi- maci´on para determinar la eciencia del test Microtox R⃝en la detecci´on de proliferaciones algales t´oxicas en las marismas mediterr´aneas. Se ha podido concluir que hay substancias t´oxicas, en la materia particulada de diversas lagunas de las marismas del Empord`a, que el m´etodo Microtox R⃝es capaz de detectar. Esta toxicidad detectada por Microtox R⃝se da en periodos en que cianobacterias, dinoagelados y hapt´otos potencialmente t´oxicos presentan alta biomasa. Los resultados sugieren que el toplancton potencialmente t´oxico no puede ser descartado como causante de la mortalidad o de efectos sub-agudos a otros organismos de las marismas mediterr´aneas. Palabras clave: Ensayo de toxicidad, lagunas costeras, proliferaci´on, toplancton, connamiento. 258 L´opez-Flores et al. Study site Grid indicates UTM coordinates (1000 m).Zonas de muestreo en las marismas del Alt Empord`a (izquierda) y La Pletera (derecha). Las l´neas indican las coordenadas UTM (1000 m). uated between the mouths of the Muga and the Fluvi`a. Four lagoons were sampled in these salt marshes (Fig. 1): Litoral and Rogera (more eutrophic), and Turies and Connectada (less eutrophic). More data about the limnological characteristics of these lagoons can be found elsewhere (Gasc´on et al., 2005; Brucet et al., 2005; L´opez-Flores et al., 2009). were frozen for later laboratory analysis of NH+ 4, NO− 2, NO− 3 and soluble reactive phosphate (SRP). Filtered and unltered samples were refrigerated for total and dissolved organic carbon analysis (TOC and DOC, respectively). Inorganic samples and total nutrient samples were oxidised to ni- trate and orthophosphate and then analysed via colourimetry following Grasshoff et al., (1983). Total organic carbon was measured using a TOC analyser (TOC 5000 Shimadzu, Shimadzu Scien- tic Instruments, Columbia, USA). Study site The study was undertaken in the Empord`a wetlands. These wetlands include a group of Mediterranean coastal lagoons and salt marshes with variable depth (average depth of 0.60 m and maximum depth close to 2 m) located in the northeast NE Iberian Peninsula (Fig. 1). They show a typical Mediterranean hydrologic regime, which is greatly affected by the sea’s inuence (Quintana et al., 1998). The hydrology of this area depends primarily on sudden and irregu- lar intrusions during sea storms and/or intense rainfall. After sea storms, rainfall or freshwa- ter inputs from rivers, the marshes remain con- ned (lacking water supply) for a long period and tend towards desiccation. Lagoons from two salt marshes, La Pletera marshes and Alt Empord`a marshes, were included in this study. The La Pletera salt marshes are situated be- tween the urban centre of l’Estartit (Torroella de Mongr´, Girona) and the Ter River mouth. Five lagoons were sampled within these salt marshes (Fig. 1): two old (O1 and O2), more- eutrophic lagoons, and three new (N1, N2 and N3), less-eutrophic lagoons (Badosa et al., 2006; L´opez-Flores et al., 2006a). The Microtox R⃝test has been described as a successful, rapid method for detecting cyanobac- terial toxicity (Lawton et al., 1990; Volterra et al., 1992; Bruno et al., 1994; Campbell et al., 1994), dinoagellate toxicity (Bruno et al., 1990; Gia- cobbe & Yang, 1999; Derby et al., 2003), and diatom toxicity (Derby et al., 2005). Thus, the test could be a feasible method for taking an ini- tial measurement of an ecosystem’s toxicity if The Alt Empord`a salt marshes include a group of coastal lagoons and salt marshes sit- Microtox R⃝toxicity of HAB in Mediterranean salt marshes 259 Mediterranean Sea 508 509 510 4673 4674 4675 4676 Turies Litoral Rec Corredor Rogera Connectada Fluvià River Muga River Iberian Peninsula Ter River Fluvià River N Ter River O1 O2 N3 N2 N1 Mediterranean Sea 515.5 516.5 4653 4654 Mediterranean Sea Muga River Figure 1.Sampling sites in the Alt Empord`a (left) and La Pletera (right) salt marshes. Grid indicates UTM coordinates (1000 m).Zonas de muestreo en las marismas del Alt Empord`a (izquierda) y La Pletera (derecha). Las l´neas indican las coordenadas UTM (1000 m). Mediterranean Sea Mediterranean Sea Figure 1.Sampling sites in the Alt Empord`a (left) and La Pletera (right) salt marshes. Sampling Samples were taken simultaneously with obser- vations of water discoloration between August 2000 and December 2003 at a central point of each lagoon at a depth of 15-30 cm. Some ad- ditional samples were also taken during sh kill episodes (e.g., Rogera lagoon in August 2001). Electrical conductivity (CRISON 524, Crison In- struments, Barcelona, Spain), pH (CRISON 507, Crison Instruments, Barcelona, Spain), tempera- ture and oxygen concentration (OXI 320, WTW, Munich, Germany) and water level were mea- sured in situ. Filtered samples (Whatman GF/F) Biomass estimation Biovolume estimations were obtained following Hillebrand et al. (1999). The formula developed by Reynolds (1984) was then utilised to trans- form biovolume to cell biomass dry weight. τ = lost light remaining light = I0 I5 −1 Scanning electron microscopy (SEM) The identication of some athecated dinoag- ellates, cyanobacteria and other agellates was supported by SEM (Zeiss DSM 960A, G¨ottin- gen, Germany). Samples were centrifuged, dehy- drated, dried to the critical point and gold-coated. Concentration values (%) and luminescence inhibition (τ) were entered into a logarithmic equation, which allowed us to compute concen- tration values for a decrease in the luminescence of 50 % (Volterra et al., 1992). The model for the computation of light emissions, where toxic effects are expressed as the ratio of activity lost to activity remaining, was developed and named gamma (τ) by Johnson et al. (1974). τ is com- puted by the formula, Epiuorescence microscopy After staining the thecate dinoagellates with calcouor white, a specic stain for cellulose, ob- servations were conducted to identify the Alexan- drium species (Hallegraeff et al., 2003). A drop of calcouor (2 mg/l) was added to the sam- ple slide, which was then covered with a cover slip and examined under an epiuorescence lamp (HBO 50, G¨ottingen, Germany). Inverted microscopy Aliquots (150 ml) were xed with lugol (1 % nal concentration) for quantifying phytoplank- ton. The general procedure for identifying and quantifying phytoplankton cells involved sedi- mentation of a subsample in a settling chamber for 24 hours and then counting cells in an ap- propriate area (Uterm¨ohl, 1958) using an inverted 260 L´opez-Flores et al. For this study, we used a Microtox R⃝M500 Toxicity Analyser System from Microbics Cor- poration (Carlsbad, California). The system is equipped with a 30-well, temperature-controlled incubator block set at 15 ◦C and a storage cell kept at approximately 5 ◦C for the reconstituted bacteria before dilution. The Microtox R⃝test was carried out according to the trademark directives. The light emission of the bacterial suspensions was measured before and after exposure to ve different dilutions of the sample, including a con- trol sample (diluent alone). Because the bacteria came from a marine environment, and in order to avoid an osmotic shock, 0.25 ml of 20 % NaCl were added to the sample (1.5 ml) before the dilution. The sample concentrations tested were 50 %, 25 %, 12.5 % and 6.25 % of the initial concentration (original extract). Sample dilutions were always prepared by adding saline solution (2 % NaCl aqueous solution (Microtox Diluent)) to provide osmotic protection. microscope (Zeiss Axiovert 135, G¨ottingen, Ger- many). Several taxonomic works (Desikachary, 1959; Dodge, 1982; Sournia, 1986; Chr´etiennot- Dinet, 1990; Tomas, 1997; Faust & Gulledge, 2002; Hallegraeff et al., 2003) were used to de- termine the phytoplankton taxa. microscope (Zeiss Axiovert 135, G¨ottingen, Ger- many). Several taxonomic works (Desikachary, 1959; Dodge, 1982; Sournia, 1986; Chr´etiennot- Dinet, 1990; Tomas, 1997; Faust & Gulledge, 2002; Hallegraeff et al., 2003) were used to de- termine the phytoplankton taxa. Microtox R⃝toxicity of HAB in Mediterranean salt marshes 261 Table 1. List of phytoplankton taxa in the different lagoons studied. Potentially harmful taxons are in bold (Hallegraeff et al., 2003). The symbol + indicates mean values <105 cells l−1, and ++ indicates mean values between 105 and 106. Blooms are identied with b and B if the number of cells per litre is between 106-107 and >107, respectively. Lista de taxones de toplancton de las diferentes lagunas estudiadas. Los taxones potencialmente t´oxicos se muestran en negrita (Hallegraeff et al., 2003). El s´mbolo + corresponde a valores medios <10 5 celulas/l, y ++ a valores medios entre 10 5y10 6. Los blooms algales se identican con b y B, si el n´umero de celulas por litro es entre 10 6y10 7, y >10 7, respectivamente. Heavy metals analyses and pesticides data Toxicity was considered for values of EC505 lower than 100 % or TU5 > 1. For metals samples, 500 ml of water was l- tered through a Whatman GF/C lter and frozen for later analysis. Frozen lters from samples were digested for 6 hours by a mixture of 4 ml HNO3 (1:1) + 10 ml HCl (1:4) and ltered be- We also analysed the soluble fraction of the samples for which the Microtox R⃝results were positive for the particulate fraction. For the solu- ble fractions, none of the tested samples showed luminescent inhibition. p y y p Species O1 O2 N1 N2 N3 Litoral Turies Rogera Connectada Diatoms Amphora spp. + + b + + Chaetoceros sp. + B Cocconeis placentula Ehrenberg ++ + ++ b ++ + Cyclotella spp. + ++ + + + ++ Cylindrotheca closterium (Ehrenberg) Reimann & Lewin ++ ++ B ++ ++ b b Entonomeis spp. + + + ++ + Navicula spp. ++ ++ + ++ + + ++ ++ + Nitzchia sp. + + + + + + + Chlorophytes Nannochloris sp. B B Chrysophytes Ochromonas spp. ++ b b b Cryptophytes Chroomonas sp. b b ++ + + b Hemiselmis sp. ++ + Cyanobacteria Anabaena spp. + + + Aphanocapsa sp. B Dactylococcopsis cf. raphidioides Hansgirg B Merismopedia spp. + ++ ++ + Synechococcus sp. + + Dinoagellates Alexandrium pseudogonyaulax (Biecheler) Horiguchi ++ + ++ Amphidinium sp. B + b Glenodinium foliaceum Stein B + ++ b ++ b Gymnodinium spp. B B b b + ++ ++ Gyrodinium instriatum Freudenthal & Lee ++ Gyrodinium sp. ++ Heterocapsa spp. + b + Oxyrrhis marina Dujardin b B b b + B + Prorocentrum micans Ehrenberg + + + Prorocentrum minimum (Pavillard) Schiller + B B B Scrippsiella subsalsa (Ostenfeld) Steidinger & Balech b + + Scrippsiella spp. + ++ + b + ++ Euglenophytes Euglena cf. Pr´oxima Dangeard + + Prasinophytes Pyramimonas spp. b B b b B b Tetraselmis cf. gracilis (Kylin) Butcher b ++ ++ Haptophytes Chrysochromulina sp. + Pavlova cf. Lutheri (Droop) Green + ++ Prymnesium sp. B + + + Number of samples 12 16 7 3 7 7 3 2 1 Species O1 O2 N1 N2 N3 Litoral Turies Rogera Connectada Diatoms Amphora spp. + + b + + Chaetoceros sp. + B Cocconeis placentula Ehrenberg ++ + ++ b ++ + Cyclotella spp. Microtox R⃝toxicity assays where I0 and I5 were the luminescence at 0 and 5 minutes, respectively. The Microtox R⃝bioassay was used for study- ing the toxicity of potentially harmful algae. This method is based on the fading of light emitted by the luminescent bacteria Vibrio scheri when ex- posed to noxious substances. The concentration of the test chemical that causes τ to equal 1 was used to compute the EC50 value for the assay. The log transformation in the τ approach permits simple regression anal- yses to compute EC50 values (Johnson, 2005). Thus, high values of EC505 indicate low toxicity, whereas low values indicate high toxicity. Toxic- ity Units (TU5) have been calculated as, Frozen lters from natural samples were ex- tracted using a Pyrex tube with 5 ml of Milli Q water (Millipore). The tub was placed in an ul- trasonic bath with ice water and then stored for 24 hours in the refrigerator (4 ◦C) to prolong ex- traction time. Extracts were then centrifuged at 4000 rpm for 10 minutes, and the supernatant was immediately analysed. TU5 = 100 EC505 Microtox R⃝toxicity of HAB in Mediterranean salt marshes Heavy metals analyses and pesticides data + ++ + + + ++ Cylindrotheca closterium (Ehrenberg) Reimann & Lewin ++ ++ B ++ ++ b b Entonomeis spp. + + + ++ + Navicula spp. ++ ++ + ++ + + ++ ++ + Nitzchia sp. + + + + + + + Chlorophytes Nannochloris sp. B B Chrysophytes Ochromonas spp. ++ b b b Cryptophytes Chroomonas sp. b b ++ + + b Hemiselmis sp. ++ + Cyanobacteria Anabaena spp. + + + Aphanocapsa sp. B Dactylococcopsis cf. raphidioides Hansgirg B Merismopedia spp. + ++ ++ + Synechococcus sp. + + Dinoagellates Alexandrium pseudogonyaulax (Biecheler) Horiguchi ++ + ++ Amphidinium sp. B + b Glenodinium foliaceum Stein B + ++ b ++ b Gymnodinium spp. B B b b + ++ ++ Gyrodinium instriatum Freudenthal & Lee ++ Gyrodinium sp. ++ Heterocapsa spp. + b + Oxyrrhis marina Dujardin b B b b + B + Prorocentrum micans Ehrenberg + + + Prorocentrum minimum (Pavillard) Schiller + B B B Scrippsiella subsalsa (Ostenfeld) Steidinger & Balech b + + Scrippsiella spp. + ++ + b + ++ Euglenophytes Euglena cf. Pr´oxima Dangeard + + Prasinophytes Pyramimonas spp. b B b b B b Tetraselmis cf. gracilis (Kylin) Butcher b ++ ++ Haptophytes Chrysochromulina sp. + Pavlova cf. Lutheri (Droop) Green + ++ Prymnesium sp. B + + + Number of samples 12 16 7 3 7 7 3 2 1 262 L´opez-Flores et al. fore analysis (L´opez-Flores et al., 2003; Salvad´o et al., 2006). Flame atomic absorption spectrom- etry was employed to determine the metal con- centration. A SpectrAA-300 and a GTA-96 elec- trothermal atomisation unit with an automatic injector were used (all supplied by Varian). Sam- ples from La Pletera salt marshes were analysed in the present study (O1, O2, N1, N2 and N3), whereas the heavy metal concentration of the Empord`a wetlands (Litoral, Connectada, Rogera and Turies) was extracted from previous works (L´opez-Flores et al., 2003; Salvad´o et al., 2006). ed mainly to cyanobacteria and dinoagellate taxonomic groups and were found in both salt marshes; however, they were not detected in all of the lagoons. Diatom species from the genus Chaetoceros were also detected, forming a bloom in the N3 lagoon during spring 2003; however, no sh kills were related to this genus. Heavy metals analyses and pesticides data The harmful cyanobacteria genera identied were Anabaena, with low densities in different lagoons belonging to both salt marshes, and Aphanocapsa, whose blooms joined with Dactylococcopsis cf. raphid- iodes blooms during a sh kill in Rogera in Au- gust 2001. Haptophytes from the genus Prym- nesium were also found in high densities in O1 and with a lower abundance in the new lagoons (N1, N2, N3). Alexandrium pseudogonyaulax and Prorocentrum minimum were identied in the new lagoons of La Pletera salt marshes, the latter developing high biomass in all three la- goons. Scrippsiella subsalsa was found in O2 every summer between the years 2001 and 2003. At least two different species from the genus Gymno- dinium were identified in most of the lagoons. ⃝ The pesticide data used in the present study were part of a larger study commissioned by the local government (L´opez-Flores et al., 2003; Salvad´o et al., 2006), 15 years after the declaration of the Integral Reserve figures; thus, a subsequent increase in the concentration of pesticides is improbable. Statistical analysis Environmental and biological variables were cor- related with TU5 using lineal correlation (Pear- son coefcients; p < 0.05). Calculations and sta- tistical analyses were performed with SPSS 15.0. The Microtox R⃝test obtained positive values for the particulate fraction in samples from the two salt marshes (Table 2); however, not all of the lagoons produced positive results. The results for the soluble fraction were always negative, in- dicating that positive toxicity results are only re- lated to the particulate fraction. Table 2.List of samples with TU5 >1(EC505 <100 %). Samples are sorted by TU5. Lista de muestras con TU5 >1 (EC505 <100 %). Las muestras est´an ordenadas por TU5. Microtox R⃝toxicity of HAB in Mediterranean salt marshes Microtox R⃝toxicity of HAB in Mediterranean salt marshes Table 3. Heavy metal concentrations in the studied lagoons. Microtox R⃝test EC505 values extracted from (1) Villaescusa et al. (1996); (2) Villaescusa et al. (1997); (3) Ronco (1992); (4) Villaescusa et al. (1998). Concentraci´on de metales pesados en las lagunas estudiadas. Valores EC505 del test Microtox R⃝extraidos de: (1) Villaescusa et al., (1996); (2) Villaescusa et al. (1997); (3) Ronco (1992); (4) Villaescusa et al. (1998). Table 3. Heavy metal concentrations in the studied lagoons. Microtox R⃝test EC505 values extracted from (1) Villaescusa et al. (1996); (2) Villaescusa et al. (1997); (3) Ronco (1992); (4) Villaescusa et al. (1998). Concentraci´on de metales pesados en las lagunas estudiadas. Valores EC505 del test Microtox R⃝extraidos de: (1) Villaescusa et al., (1996); (2) Villaescusa et al. (1997); (3) Ronco (1992); (4) Villaescusa et al. (1998). Lagoon Cadmium (mg/l) Chrome (mg/l) Copper (mg/l) Nickel (mg/l) Lead (mg/l) O1 < 0.02 < 0.03 0.03 < 0.02 < 0.07 O2 < 0.02 < 0.03 < 0.02 < 0.02 < 0.07 N1 < 0.02 < 0.03 < 0.02 < 0.02 < 0.07 N2 < 0.02 < 0.03 < 0.02 < 0.02 < 0.07 N3 < 0.02 0.034 < 0.02 < 0.02 < 0.07 Litoral < 0.02 0.149 < 0.02 < 0.02 < 0.07 Connectada < 0.02 0.130 < 0.02 < 0.02 < 0.07 Rogera < 0.02 0.089 < 0.02 < 0.02 < 0.07 Turies < 0.02 0.148 < 0.02 < 0.02 < 0.07 EC505 (mg/l) 7.82(1) 4.30(2) 8.00(3) 10.00(4) 0.36(4) ues of TU5 > 1 (EC505 <100 %). Only in one sample of N1 in later spring (May 2003) was TU5 > 1. Note that high TU5 generally coin- cided with mid to late summer. Often, high TU5 coincided with a high biomass of cyanobacteria, dinoagellates or haptophytes. However, the proliferation of some potentially harmful species was also identied in lagoons where TU5 was close to zero (Table 4). In these samples, P. minimum was the main species found, but A. pseudogonyaulax and Gymno- dinium spp. were also found. Heavy metal concentrations in the lagoons were compared to the corresponding EC505 found in the literature (Ronco, 1992; Villaescusa et al., 1996; Villaescusa et al., 1997; Villa- escusa et al., 1998) in order to measure their contribution to sample toxicity (Table 3). Gen- erally, concentrations did not reach the method’s detection limit. RESULTS The phytoplankton taxa identied are shown in Table 1. The potentially harmful species belong- Samples from the less-eutrophic lagoons (N1, N2, N3, Turies and Connectada) never had val- Table 2.List of samples with TU5 >1(EC505 <100 %). Samples are sorted by TU5. Lista de muestras con TU5 >1 (EC505 <100 %). Las muestras est´an ordenadas por TU5. Biomass (mg/l) Lagoon Season TU5 Gymnodinium spp. P. minimum S. subsalsa Prymnesium sp. Aphanocapsa sp. O2 Summer (17.07.03) 36.54 — — 00.01 — — O1 Summer (17.07.03) 14.91 — — — 0.16 — O2 Summer (18.09.02) 12.75 172.65 — — — — O1 Summer (18.09.02) 09.54 004.05 — — — — O1 Summer (20.08.03) 06.42 — — — 0.15 — O2 Spring (18.06.03) 05.67 — — 00.18 — — O2 Summer (18.08.01) 04.10 — — 09.35 — — N1 Spring (14.05.03) 02.75 000.79 0.12 — — — O2 Autumn (15.10.02) 02.48 045.56 — — — — Litoral Summer (01.07.02) 01.99 024.24 — — — — O2 Summer (15.07.02) 01.90 — — 17.38 — — O2 Summer (13.08.02) 01.92 005.69 — — — — Rogera Summer (18.08.01) 01.90 — — — — 33.45 Table 2.List of samples with TU5 >1(EC505 <100 %). Samples are sorted by TU5. Lista de muestras con TU5 >1 (EC505 <100 %). Las muestras est´an ordenadas por TU5. 263 Table 4. List of samples with a high biomass of potentially harmful species and with TU5 <1 (EC505 <100 %). Lista de muestras con biomasa alta de especies potencialmente t´oxicas y con TU5 <1 (EC505 <100 %). Microtox R⃝toxicity of HAB in Mediterranean salt marshes Only chrome concentrations were detectable in some lagoons; however, they never reached the EC505 values established in studies with Microtox R⃝. TU5 was positively correlated with the biomass of bacterioplankton (r2 = 0.521; p < 0.0001) and phytoplankton (r2 = 0.392; p = 0.007), chlorophyll a (r2 = 0.454, p = 0.005), TOC concentration (r2 = 0.441, p = 0.002), con- ductivity (r2 = 0.460, p = 0.001) and tempera- ture (r2 = 0.310, p = 0.038), whereas the wa- Table 4. List of samples with a high biomass of potentially harmful species and with TU5 <1 (EC505 <100 %). Lista de muestras con biomasa alta de especies potencialmente t´oxicas y con TU5 <1 (EC505 <100 %). Table 4. List of samples with a high biomass of potentially harmful species and with TU5 <1 (EC505 < con biomasa alta de especies potencialmente t´oxicas y con TU5 <1 (EC505 <100 %). p g p y p 5 ( 5 ) con biomasa alta de especies potencialmente t´oxicas y con TU5 <1 (EC505 <100 %). Biomass (mg/l) Lagoon Season Gymnodinium spp. P. minimum A. pseudogonyaulax N1 Autumn (15.10.02) — — — Autumn (12.11.02) — 10.05 10.42 Winter (19.02.03) 10.76 10.10 — Winter (17.03.03) — 13.76 — Spring (15.04.03) — 14.48 — Spring (18.06.03) 27.98 — — N2 Winter (19.02.03) 10.83 10.42 — Winter (17.03.03) — 12.45 — Spring (15.04.03) — 18.96 — Spring (18.06.03) 18.46 — — N3 Summer (18.09.02) — 10.02 0.12 Winter (19.02.03) — 10.52 — Winter (17.03.03) — 24.23 — Spring (15.04.03) — 14.48 — 264 L´opez-Flores et al. study, high biomass densities developed by some harmful species could not be related to Microtox R⃝toxicity (e.g. P. minimum prolifera- tion). These negative Microtox R⃝results could be due to the lack of toxic substances in the wa- ter, but also could be accounted for by their low toxicity for Vibrio scheri or to the chemistry of toxins (because only hydrosoluble toxins were tested). The species P. minimum has often been associated with sh mortality and the produc- tion of venerupin, a hepatotoxin (Tangen, 1983; Faust & Gulledge, 2002). This species has also been shown to contain a water-soluble, neuro- toxic component that can kill mice (Heil et al., 2005). However, its toxicity is known to be con- ditioned by the species clone, and the toxic ef- fects may be elicited under certain growth con- ditions (Glibert & Sellner, 2005). Microtox R⃝toxicity of HAB in Mediterranean salt marshes These contra- dictions bring into doubt the efciency of the Microtox R⃝method in detecting harmful algae toxins. Moreover, some authors criticise immer- sion exposure methods like Microtox R⃝because they assume an indiscriminate, whole-organism exposure, whereas in natural conditions, expo- sure to toxins is mainly through the grazer’s digestive system (Caldwell et al., 2004). In ad- dition, Microtox R⃝toxicity detection is limited because not all phytoplankton toxins affect the viability of the tested bacteria in the same way. Other methods, such as high-performance liquid chromatography (HPLC), lipid emulsions and liposome or microparticulate zooplankter diets (Caldwell et al., 2004), are more rigorous but are only recommended if taxonomic knowledge of the community composition is available and the chemical structure of the toxin is well known be- cause the analysis is highly focussed on a partic- ular toxin or organism. Even with perfect knowl- edge of the taxonomic composition, variations of the species strain (Saker et al., 2005) or the nu- trient availability (Gran´eli et al., 1999; Johansson et al., 2006) could lead to changes in the toxicity level. Moreover, these methods have the disad- vantage of being more expensive and slower and are generally not available for non-qualiedtech- nicians. As an example, the harmful species A. tay- ter level was negatively correlated with TU5 (r2 = −0.401, p = 0.019). No signicant cor- relations were found between TU5 and the biomass of each harmful species or with the total harmful species biomass. ACKNOWLEDGEMENTS CHR´ETIENNOT-DINET, M. J. 1990. Chlorarachnio- phyc´ees, chlorophyc´ees, chrysophyc´ees, crypto- phyc´ees, eugl´enophyc´ees, eustigmatophyc´ees, pra- sinophyc´ees, prymn´esiophyc´ees, rhodophyc´ees et tribophyc´ees. Editions du CNRS, Paris (France). 261 pp. The authors wish to thank E. Fulladosa and I. Villaescusa for Microtox R⃝assistance. This work was supported by a grant from the Comisi´on de Investigaci´on Cient´ca y T´ecnica (CICYT), Pro- grama de Investigaci´on Fundamental (ref. 518 CGL2008-05778/BOS) and by a BR grant of the University of Girona. COM´IN, F. A. & X. FERRER. 1978. Desarrollo ma- sivo del toagelado Prymnesium parvum Carter (Haptophyceae) en una laguna costera del delta del Ebro. Oecol. Aquat., 3: 207-210. DERBY, M. L., M. GALLIANO, J. J. KRZANOW- SKI & D. F. MARTIN. 2003. Studies of the ef- fect of [Psi]-APONIN from Nannochloris sp. on the Florida red tide organism Karenia brevis. Toxi- con, 41: 245-249. Microtox R⃝toxicity of HAB in Mediterranean salt marshes Microtox R⃝toxicity of HAB in Mediterranean salt marshes 265 planctonica from blooms found in Lake Mulargia (Italy). Toxicon, 32: 369-373. (Paralytic Shellfish Poison) toxins were detected using HPLC (Giacobbe and Yang, 1999), although PSP is the syndrome most related to this species. (Paralytic Shellfish Poison) toxins were detected using HPLC (Giacobbe and Yang, 1999), although PSP is the syndrome most related to this species. The pulse rhythm of phytoplankton prolifer- ation and the spatial variability of species com- position did not allow us to correlate harmful species biomass to Microtox R⃝toxicity. Despite this, we discovered a contemporaneity between HABs and Microtox R⃝toxicity. The results sug- gest that potentially harmful phytoplankton can- not be ruled out either as a source of Microtox R⃝ toxicity or as a source of other organism kills or sub-acute effects in Mediterranean salt marshes. BRUNO, M., P. M. B. GUCCI, E. PIERDOMINICI, A. IOPPOLO & L. VOLTERRA. 1990. Presence of saxitoxin in toxic extracts from Gonyaulax polyedra. Toxicon, 28: 1113-1116. The pulse rhythm of phytoplankton prolifer- ation and the spatial variability of species com- position did not allow us to correlate harmful species biomass to Microtox R⃝toxicity. Despite this, we discovered a contemporaneity between HABs and Microtox R⃝toxicity. The results sug- gest that potentially harmful phytoplankton can- not be ruled out either as a source of Microtox R⃝ toxicity or as a source of other organism kills or sub-acute effects in Mediterranean salt marshes. CALDWELL, G. S., S. B. WATSON & M. G. BENT- LEY. 2004. How to assess toxin ingestion and post- ingestion partitioning in zooplankton? J. Plankton Res., 26: 1369-1377. CAMPBELL, D. L., K. A. LAWTON, K. A. BEAT- TIE & G. A. CODD. 1994. Comparative asses- ment of the specicity of the brine shrimp and Microtox assays to hepatotoxic (microcystin-LR- containing) cyanobacteria. Environ. Toxicol. Water Qual., 9: 71-77. DISCUSSION There are substances in the particulate matter of several lagoons in the Empord`a salt marshes that produce toxicity detected by the Microtox R⃝ test. This Microtox R⃝toxicity coincides with periods when potentially harmful cyanobacte- ria, dinoagellates and haptophytes have a high biomass. In addition, in one case, HABs and Microtox R⃝toxicity co-occurred with sh kills. In the Empord`a salt marshes, Microtox R⃝toxi- city occurs during summer, coinciding with high temperatures, high bacterial biomass and high or- ganic matter concentration as well as high densi- ties of chlorophyll a and high pico- and nanophy- toplankton biomasses. Although the correlations found are low, they suggest that toxicity events are related to connement situations when wa- ter level decreases, increasing salinity, organic matter and phytoplankton biomass (L´opez-Flores et al., 2006a). The toxicity measured was not caused by substances dissolved in the water be- cause the Microtox R⃝test was negative for this fraction and was not caused by heavy metals in particulate matter because their concentrations were under the EC505 for the metals analysed. Furthermore, previous studies have ruled out the presence of harmful concentrations of pesticides in the Empord`a salt marshes (L´opez-Flores et al., 2003; Salvad´o et al., 2006). The lack of oxy- gen is one of the most likely causes of sh and zooplankton mortality in this ecosystem because, during summer, high phytoplankton biomasses occur and anoxia situations are frequent. How- ever, the lack of oxygen in the water has no ef- fect on Microtox R⃝toxicity; thus, positive values cannot be attributed to anoxia. Pre io s st dies ha e fo nd a relationship be Previous studies have found a relationship be- tweenMicrotox R⃝toxicity and harmful algae pro- liferation (Bruno et al., 1990; Bruno et al., 1994; Derby et al., 2003). However, in the present REFERENCES BADOSA, A., D. BOIX, S. BRUCET, R. L ´OPEZ- FLORES & X. D. QUINTANA. 2006. Nutrients and zooplankton composition and dynamics in re- lation to the hydrological pattern in a conned Mediterranean salt marsh (NE Iberian Peninsula). 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Comparison of nutrient and Microtox R⃝toxicity of HAB in Mediterranean salt marshes 267 VILA, M., J. CAMP, E. GARC´ES, M. MAS ´O & M. DELGADO. 2001. High resolution spatio- temporal detection of potentially harmful dinoag- ellates in conned waters of the NW Mediter- ranean. J. Plankton Res., 23: 497-514. SARNO, D., A. ZINGONE, V. SAGGIOMO & G. C. CARRADA. 1993. Phytoplankton biomass and species composition in a Mediterranean coastal la- goon. Hydrobiologia, 271: 27-40. SOURNIA, A. 1986. Cyanophyc´ees, dictyochophy- c´ees, dinophyc´ees et raphidophyc´ees. Editions du CNRS, Paris (France). 219 pp. VILLAESCUSA, I., S. MART´I, C. MATAS, M. MART´INEZ & J. M. RIB ´OS. 1997. Chromium (VI) toxicity to luminescent bacteria. Environ. Tox- icol. Chem., 16: 871-874. TANGEN, K. 1983. Shellsh poisoning and the oc- currence of potentially toxic dinoagellates in Norwegian waters. Sarsia, 68: 1-7. VILLAESCUSA, I., M, MARTINEZ, M. PILAR & C. MURAT. 1996. Toxicity of cadmium species on luminescent bacteria. Fresenius J. Anal. Chem., 354: 566-570. TOMAS, C. R. 1997. Identifying marine phytoplank- ton. Academic Press, St. Petersburg (Florida). 858 pp. VILLAESCUSA, I., C. MATAS, C. HOSTA, M. MARTINEZ & J. C. MURAT. 1998. Evaluation of lead (II) and nickel (II) toxicity in NaCl and Na- ClO4 solutions by using Microtox bioassay. Frese- nius J. Anal. Chem., 361: 355-358. TROBAJO, R., X. D. QUINTANA & R. MORENO- AMICH. 2002. Model of alternative predomi- nance of phytoplankton-periphyton-macrophytes in lentic waters of Mediterranean coastal wetlands. Arch. Hydrobiol., 154: 19-40. VOLTERRA, L., M. BRUNO & P. M. B. GUCCI. 1992. Fast method for detecting toxic cyanophyte blooms. Environ. Toxicol. Water Qual., 7: 215-222. UTERM ¨OHL, H. 1958. Zur vervollkommnung der quantitativen Phytoplankton-Metodik. Mitt. Int. Ver. Limnol., 9: 1-38.
https://openalex.org/W4308008350	https://www.qeios.com/read/5GHH6L/pdf	English	null	Review of: "The Universal Accessibility Provisions in Hospitals of New Delhi, India"	null	2,022	cc-by	353	Qeios, CC-BY 4.0 · Review, November 1, 2022 Qeios ID: 5GHH6L · https://doi.org/10.32388/5GHH6L Review of: "The Universal Accessibility Provisions in Hospitals of New Delhi, India" Sunita Kathuria Potential competing interests: No potential competing interests to declare. I appreciate and congratulate the authors for researching in the area which is much needed to be explored and reported at the public forum. I am satisfied with the research work of the authors as the research questions are well addressed by the findings at the end. The research methodology and presentation of the findings seems good to me. However, I have a few suggestions that if possible may please be incorporated, or otherwise may be ignored. The inclusion criteria of selecting these 32 Government hospitals may please be added to the research methodology section. The readers would have a clear picture of why only these govt. hospitals were approached in the study and why not others (rationalize pls). The inclusion criteria of selecting these 32 Government hospitals may please be added to the research methodology section. The readers would have a clear picture of why only these govt. hospitals were approached in the study and why not others (rationalize pls). The Discussion section if may please be enriched further with few of the Indian studies that reflects on the status of accessibility to medical support or other dimensions like education, employment etc. to the persons with disabilities in other parts of India, or also in global context is possible. The Discussion section if may please be enriched further with few of the Indian studies that reflects on the status of accessibility to medical support or other dimensions like education, employment etc. to the persons with disabilities in other parts of India, or also in global context is possible. The paper is nicely developed however at the end I felt as if some specific suggestions and recommendations to different stakeholders could have been made. This would make the research work significant to the concerned people and other departments associated with persons with disabilities. All the best for your future research work. Qeios ID: 5GHH6L · https://doi.org/10.32388/5GHH6L 1/1
https://openalex.org/W4385827554	https://humanfactors.jmir.org/2023/1/e42506/PDF	English	null	A Beta-Prototype Chatbot for Increasing Health Literacy of Patients With Decompensated Cirrhosis: Usability Study	JMIR human factors	2,023	cc-by	7,926	Abstract Background: Health literacy is low among patients with chronic liver disease (CLD) and associated with poor health outcomes and increased health care use. Lucy LiverBot, an artificial intelligence chatbot was created by a multidisciplinary team at Monash Health, Australia, to improve health literacy and self-efficacy in patients with decompensated CLD. Objective: The aim of this study was to explore users’ experience with Lucy LiverBot using an unmoderated, in-person, qualitative test. Methods: Lucy LiverBot is a simple, low cost, and scalable digital intervention, which was at the beta prototype development phase at the time of usability testing. The concept and prototype development was realized in 2 phases: concept development and usability testing. We conducted a mixed methods study to assess usability of Lucy LiverBot as a tool for health literacy education among ambulatory and hospitalized patients with decompensated CLD at Monash Health. Patients were provided with free reign to interact with Lucy LiverBot on an iPad device under moderator observation. A 3-part survey (preuser, user, and postuser) was developed using the Unified Acceptance Theory Framework to capture the user experience. Results: There were 20 participants with a median age of 55.5 (IQR 46.0-60.5) years, 55% (n=11) of them were female, and 85% (n=17) of them were White. In total, 35% (n=7) of them reported having difficulty reading and understanding written medical information. Alcohol was the predominant etiology in 70% (n=14) of users. Participants actively engaged with Lucy LiverBot and identified it as a potential educational tool and device that could act as a social companion to improve well-being. In total, 25% (n=5) of them reported finding it difficult to learn about their health problems and 20% (n=4) of them found it difficult to find medical information they could trust. Qualitative interviews revealed the conversational nature of Lucy LiverBot was considered highly appealing with improvement in mental health and well-being reported as an unintended benefit of Lucy LiverBot. Patients who had been managing their liver cirrhosis for several years identified that they would be less likely to use Lucy LiverBot, but that it would have been more useful at the time of their diagnosis. Overall, Lucy LiverBot was perceived as a reliable and trustworthy source of information. Conclusions: Lucy LiverBot was well received and may be used to improve health literacy and address barriers to health care provision in patients with decompensated CLD. A Beta-Prototype Chatbot for Increasing Health Literacy of Patients With Decompensated Cirrhosis: Usability Study A Beta-Prototype Chatbot for Increasing Health Literacy of Patients With Decompensated Cirrhosis: Usability Study Jessica Au1, BMedSci, MD; Caitlin Falloon1, BMedSci, MD; Ayngaran Ravi1, MBBS; Phil Ha2, MBBS; Suong Le2,3, MBBS 1School of Clinical Sciences, Monash University, Clayton, Australia 2Department of Gastroenterology and Hepatology, Monash Health, Clayton, Australia 3Monash Digital Therapeutics and Innovation Laboratory, Monash University, Clayton, Australia these authors contributed equally Corresponding Author: Jessica Au, BMedSci, MD School of Clinical Sciences Monash University 30 Wellington Road Clayton, 3800 Australia Phone: 61 431201056 Email: jessicaau7@gmail.com Corresponding Author: Jessica Au, BMedSci, MD School of Clinical Sciences Monash University 30 Wellington Road Clayton, 3800 Australia Phone: 61 431201056 Email: jessicaau7@gmail.com Abstract The study revealed important feedback that has been used to further optimize Lucy LiverBot. Further acceptability and validation studies are being undertaken to investigate whether Lucy LiverBot can improve clinical outcomes and health related quality of life in patients with decompensated CLD. (JMIR Hum Factors 2023;10:e42506) doi: 10.2196/42506 Original Paper A Beta-Prototype Chatbot for Increasing Health Literacy of Patients With Decompensated Cirrhosis: Usability Study Jessica Au1, BMedSci, MD; Caitlin Falloon1, BMedSci, MD; Ayngaran Ravi1, MBBS; Phil Ha2, MBBS; Suong Le2,3, MBBS 1School of Clinical Sciences, Monash University, Clayton, Australia 2Department of Gastroenterology and Hepatology, Monash Health, Clayton, Australia 3Monash Digital Therapeutics and Innovation Laboratory, Monash University, Clayton, Australia these authors contributed equally Au et al JMIR HUMAN FACTORS JMIR HUMAN FACTORS Au et al Original Paper (JMIR Hum Factors 2023;10:e42506) doi: 10.2196/42506 https://humanfactors.jmir.org/2023/1/e42506 Introduction Chronic liver disease (CLD) is a major global public health burden and results in 2 million deaths annually [1,2]. Decompensated CLD is a significant contributor to patient morbidity and mortality and is defined as an acute deterioration in hepatic function resulting in jaundice, hepatic encephalopathy, ascites, hepatorenal syndrome, or spontaneous bacterial peritonitis [3,4]. In 2012, the direct health care costs associated with the treatment of liver disease was estimated at US $448 million in Australia [5] and US $32.5 billion in the United States [6]. Lost productivity costs in Australia were estimated at US $4.3 billion in 2012, mainly from lost lifetime earnings due to reduced life expectancies and lower employment participation [3]. The World Health Organization [7] defines health literacy as “the achievement of a level of knowledge, personal skills and confidence to take action to improve personal and community health by changing personal lifestyles and living conditions. Thus, health literacy means more than being able to read pamphlets and make appointments. By improving people’s access to health information, and their capacity to use it effectively, health literacy is critical to empowerment.” A novel strategy to improve chronic disease patient engagement and self-management are artificial intelligence (AI) “Chatbots.” Chatbots are an emerging health care technology used for basic diagnostic or monitoring purposes in ambulatory settings [18,19]. An AI chatbot has the ability to use natural language processing (NLP) to decipher human language in order to retrieve relevant data using conversational algorithms [20]. This interactive user interface, which is intended to simulate a bidirectional conversation with a clinician aims to increase patient engagement and reduce information overload [21,22]. AI chatbots can be deployed through an omnichannel strategy: web-based, Facebook messenger, and mobile apps [23]. Recent studies have shown high levels of acceptance of health specific chatbots by users and physicians [21,24]. In psychiatry, a discipline where chatbots are more prevalent, they are used to screen for mental health disorders and are also capable of delivering cognitive behavioral therapy [25,26]. A key limitation of existing health care chatbots is their lack of human emotion [27,28] and limited focus on education [29] when they could be leveraged as a tool to improve health literacy among patients with complex chronic conditions. Providing targeted information to improve health literacy digitally could also help bridge the communication gap between patient and clinicians, while increasing patient autonomy [30]. KEYWORDS chronic liver disease; chatbot; artificial intelligence; health literacy; acceptability self-titration, fluid and salt restriction, and nutrition optimization in the community. It has not yet been demonstrated whether improved health literacy is associated with increased patient self-sufficiency in these domains of cirrhosis self-management. Patients with CLD also have significant carer requirements, which negatively impacts the mental, physical, and social well-being of patients and caregivers [16]. This burden is further amplified in patients with hepatic encephalopathy and cognitive dysfunction [17]. (JMIR Hum Factors 2023;10:e42506) doi: 10.2196/42506 JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 1 (page number not for citation purposes) https://humanfactors.jmir.org/2023/1/e42506 XSL•FO RenderX XSL•FO RenderX Au et al JMIR HUMAN FACTORS Introduction Poor health literacy has been demonstrated in patients with CLD, which may contribute to the high morbidity, mortality, and economic burden experienced by this specific chronic disease cohort [8,9]. Adherence to chronic disease treatment regimes has also been associated with health literacy, with adherence rates being 14% higher in patients with higher levels of health literacy [10]. In patients with liver cirrhosis, simple educational interventions increased patient’s disease knowledge by 26% [11]. Furthermore, the low levels of health literacy combined with high unemployment rates act as significant barriers for such patients to navigate complex health care systems and communicate with clinicians [12,13]. Studies have demonstrated an association between education level and CLD mortality, and this association was magnified for those with alcohol-related etiology [14,15]. The epidemiology of CLD is shifting away from chronic viral hepatitis toward lifestyle related etiologies including alcohol abuse and metabolic syndrome. This highlights the need for targeted interventions which address health literacy to improve self-management by reducing alcohol consumption, and addressing obesity, malnutrition, and sarcopenia [3]. Hepatic encephalopathy also impacts patient’s ability to understand health information, as it impairs executive function, problem-solving, and attention [9]. A liver specific “Chatbot” that promotes CLD health literacy through an interactive conversational interface has not been reported in the literature. Our study aims to investigate whether a novel AI chatbot is an acceptable tool to provide health information to patients with decompensated CLD. Study Design We conducted a prospective mixed method study to determine patient usability of “Lucy LiverBot,” an AI chatbot designed and built by a clinical multidisciplinary team (MDT) at Monash Health, Australia using a no code platform provided by software developers Andi Chatterton and Mark Chatterton from inGeniousAI, an industry partner. There are limited studies reporting the true prevalence of poor health literacy, its etiology, and the identification and management of potentially modifiable or preventable risk factors for poorer health literacy in decompensated CLD. Liver cirrhosis is a multisystem disorder, which is difficult for both clinicians and patients to optimize according to guideline-based management. There are high expectations placed on patients and carers to manage complicated medication regimes, lactulose https://humanfactors.jmir.org/2023/1/e42506 JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 2 (page number not for citation purposes) frequently asked questions library. Questions that cannot be answered by Lucy LiverBot are notified by email to the Liver Pharmacist who consults the relevant subject matter expert and then builds the response to this new question in the backend. Although all patients used written cues, the device also allows for voice-to-text recognition. Emojis were also used to reduce the amount of written text. Concept Development Lucy LiverBot was developed by a MDT from Monash and Austin Health in 2019 comprising a Hepatologist, a Liver Nurse Consultant, a Liver Pharmacist, and a Liver Transplant Dietitian. Each member of the MDT team was responsible for identifying a list of 10 questions commonly posed by patients with CLD in their area of subject matter expertise. Each MDT member was then responsible for creating the answers to these questions and for the veracity of the content. In-depth interviews and small focus groups were also conducted with 10 patients attending the Complex Liver Care Clinic, Monash Health—an ambulatory care program for adults with decompensated cirrhosis to validate the questions to be answered by Lucy LiverBot, identify any missing questions, and confirm a patient need for the product. https://humanfactors.jmir.org/2023/1/e42506 Overview Lucy LiverBot is an AI chatbot developed by a MDT to deliver disease, medication, and nutrition-specific health information to patients with decompensated CLD (Figure 1). A key function JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 2 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX JMIR HUMAN FACTORS Au et al of Lucy LiverBot is the emphasis on health literacy and education [8]. Information is presented to patients through conve conversational scripts, visuals, and videos in English. of Lucy LiverBot is the emphasis on health literacy and education [8]. Information is presented to patients through conversational scripts, visuals, and videos in English. Figure 1. An example of a simple flow conversation with Lucy LiverBot which allows patients to type any questions they may have about chronic liver disease. Usability Testing We conducted the study from when standardized tools to assess user’s satisfaction with the experience of using chatbots were unavailable. We did not use other usability tools such as the System Usability Scale or the Usability Metric for User Experience, as these tools were not developed to consider the conversational aspects which relate to a user’s interaction with a chatbot. Instead, we used a mixed methods approach to gather preliminary insights into a patient’s experience with Lucy LiverBot. Each participant engaged in a 1-time only testing session consisting of (1) a preuser testing survey to determine patient demographics, their baseline confidence levels managing their own health, their understanding of CLD nutrition and possible barriers to optimal health; (2) a user-testing survey to determine patient satisfaction and to analyze whether the user interface is patient friendly; (3) a postuser testing survey to determine overall satisfaction with the app and its likely use in the management of CLD. All 3 surveys were developed using the Unified Acceptance Theory Framework [31]. The Liver Pharmacist was trained as a superuser, built the Lucy LiverBot, tested the NLP, and was the primary data custodian of the frequently asked questions bank. Technical support was provided by inGeniousAI to the Liver Pharmacist who built Lucy LiverBot on the inGeniousAI no code proprietary platform. This cross functional collaboration used an agile development approach, which involved iterative cycles of design, build, and testing with clinicians and patients. To use the chatbot, patients type in words or phrases into the text section. The chatbot then uses NLP to understand the intent of what the patient has typed and responds by extracting a scripted answer in our proprietary JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 3 (page number not for citation purposes) JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 3 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX JMIR HUMAN FACTORS Au et al unable to read, understand, or answer questions fluently in English. The presurvey was developed by JA and PH and reviewed by SL, a consultant gastroenterologist and hepatologist. The presurvey was a validated questionnaire based on patient reported measures of treatment burden—the “Patient Experience with Treatment and Self-Management (PETS)” [32]. The user testing phase was made up of 2 sections: participant use of the chatbot and a subsequent survey. Usability Testing Lucy LiverBot was preloaded onto iPads which were provided to participants who could ask any nutrition-related questions for approximately 15 minutes. Within this time frame, Lucy LiverBot would guide participants through specific conversation flows depending on the key words used in their initiating question. The user testing survey questions were developed with input from inGeniousAI as their experience in chatbot design and deployment provided a valuable insight into the strategies required for successful user testing. The post user testing survey occurred immediately after the user testing survey and was developed to assess overall patient acceptance and usability of the app. The surveys were self-administered but with the moderator present, documenting additional feedback verbally provided by the patients throughout the testing session. Statistical Analysis Baseline clinical and disease demographics including current state of liver cirrhosis, decompensation complications, and total burden of hospital admissions in the past 12 months were extracted from the patient medical record. Summary data are presented as means (SD), proportions, or median (IQR) depending on the data distribution. All verbal patient feedback was documented and captured by the moderator verbatim. Anonymized transcripts were uploaded onto NVivo (Lumivero) for Windows (version 1.3; Microsoft Corp) for data management and coding. Qualitative data were reviewed by 2 independent assessors and 2 sets of key themes were identified. A third independent assessor then synthesized these results to produce a final set of key themes. Illustrative quotes were reported to support themes. Ethics Approval Ethics approval (RES-19-461A) was granted by the Human Research Ethics Committee Monash Health and was carried out according to the National Statement on Ethical Conduct in Human Research (2018). Recruitment Adult patients were recruited from liver clinics and inpatient wards at Monash Health, the second largest tertiary health care network in Australia. Monash Health provides 4.1 million episodes of care per year to a population of 1.5 million people. Given the intent to conduct a study, only 20 participants were recruited into this study. Study inclusion criteria were adults with decompensated CLD and capacity to provide informed consent. Clinical, laboratory, and imaging data were used to confirm patient CLD decompensation status. Decompensation was defined as per English as a second language criteria [33]. Participants were excluded from the study if they had greater than grade 1 hepatic encephalopathy at the time of consent, did not complete all components of the survey, or if they were JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 4 (page number not for citation purposes) https://humanfactors.jmir.org/2023/1/e42506 JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 5 (page number not for citation purposes) Overview The median age was 55.5 (IQR 46.0-60.5) years, with 55% (n=11) of them being female and 85% (n=17) of them being White (Table 1). The median BMI was 31.2 (IQR 22.6-36.55). Active alcohol consumption was cross referenced from both self-reporting and clinical documentation of alcohol being the confirmed etiology for CLD. No current alcohol intake was reported among 25% (n=5) of participants, 50% (n=10) of them were still current drinkers and 25% (n=5) of them had quit drinking alcohol. https://humanfactors.jmir.org/2023/1/e42506 JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 4 (page number not for citation purposes) XSL•FO RenderX Table 1. Baseline demographics (n=20). Value Characteristics 55.5 (46.0-60.5) Age (years), median (IQR) Sex, n (%) 9 (45) Male 31.2 (22.6-36.55) BMI, median (IQR) Ethnicity, n (%) 17 (85) White Smoking status, n (%) 11 (55) Smoker or ex-smoker Alcohol intake, n (%) 15 (75) Previous or current alcohol intake English fluency, n (%) 18 (90) Fluent Highest level of education, n (%) 12 (60) Less than high school 8 (30) High school graduate 2 (10) Tertiary Do you have difficulty reading and understanding medical information? n (%) 13 (65) Never 5 (25) Sometimes 2 (10) Usually Employment status, n (%) 19 (95) Unemployed Carer, n (%) 14 (70) Yes Owns a device, n (%) 18 (90) Yes Cause of cirrhosis, n (%) 14 (70) Alcohol 3 (15) Viral 3 (15) Other Time since diagnosis of cirrhosis, n (%) 7 (35) Unsure 2 (10) <6 months 4 (20) 6 months to 2 years 4 (15) 2-4 years 3 (15) >4 years In total, 35% (n=7) of participants reported having difficulty reading and understanding written medical information, despite 90% (n=18) of participants being fluent in English. A large proportion had not completed high school (n=12, 60%) and was alcohol (n=14, 70%), followed by viral (n=3, 15%) and other (n=3, 15%). Of the 20 participants, 25% (n=5) of participants found it Au et al JMIR HUMAN FACTORS JMIR HUMAN FACTORS Au et al was alcohol (n=14, 70%), followed by viral (n=3, 15%) and other (n=3, 15%). was alcohol (n=14, 70%), followed by viral (n=3, 15%) and other (n=3, 15%). was alcohol (n=14, 70%), followed by viral (n=3, 15%) and other (n=3, 15%). In total, 35% (n=7) of participants reported having difficulty reading and understanding written medical information, despite 90% (n=18) of participants being fluent in English. JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 6 (page number not for citation purposes) https://humanfactors.jmir.org/2023/1/e42506 Overview A large proportion had not completed high school (n=12, 60%) and were unemployed (n=19, 95%); a majority (n=14, 70%) required a carer. The primary cause of liver cirrhosis in this patient group In total, 35% (n=7) of participants reported having difficulty reading and understanding written medical information, despite 90% (n=18) of participants being fluent in English. A large proportion had not completed high school (n=12, 60%) and were unemployed (n=19, 95%); a majority (n=14, 70%) required a carer. The primary cause of liver cirrhosis in this patient group Of the 20 participants, 25% (n=5) of participants found it difficult to learn about their health problems and 20% (n=4) of them found it difficult to find medical information they could trust (Table 2). Although 65% (n=13) of them found it easy to understand advice provided directly by their health care https://humanfactors.jmir.org/2023/1/e42506 JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 5 (page number not for citation purposes) JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 5 (page number not for citation purposes) XSL•FO RenderX JMIR HUMAN FACTORS Au et al reminded them to do things for their health. Regarding emotional well-being, 50% (n=10) of them felt preoccupied by their self-care, with 55% (n=11) of them depressed about their CLD. A large proportion felt worn out by self-care (n=14, 70%) and were frustrated (n=15, 75%) with their health situation (Table 2). providers, and 25% (n=5) of them found it difficult to understand. In addition, 20% (n=4) of them found it difficult to find information on what foods they should eat to stay healthy and 45% (n=9) of them reported issues monitoring their eating and drinking habits. In addition, 70% (n=14) of them were bothered by feeling dependent on others for health care needs, with 35% (n=7) of them bothered when family or friends https://humanfactors.jmir.org/2023/1/e42506 https://humanfactors.jmir.org/2023/1/e42506 XSL•FO RenderX XSL•FO RenderX JMIR HUMAN FACTORS Au et al Understanding medical information How easy or difficult has it been to learn about your health problems? 12 (60) Easy 3 (15) Neither easy or difficult 5 (25) Difficult How easy or difficult has it been to learn what foods you should eat to stay healthy? 14 (70) Easy 2 (10) Neither easy or difficult 4 (20) Difficult How easy or difficult has it been to find sources of medical information that you trust? JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 7 (page number not for citation purposes) https://humanfactors.jmir.org/2023/1/e42506 JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 7 https://humanfactors.jmir.org/2023/1/e42506 (page number not for citation purposes) L•FO Something for anxiety and depression could help, particularly being able to write a journal. Lucy LiverBot was overtly identified as a potential “companion” by patients with CLD by providing a sense of social connection for patients who are socially isolated. Principal Findings You can talk like you are talking to somebody else like a friend. There is a clinical urgency for cost-effective and scalable interventions that address the poor health literacy of patients with CLD [34] in order to improve patient engagement and self-management of this complex condition [35,36]. Lucy LiverBot was well received by participants and the results suggest that it could provide targeted CLD information via an engaging channel. Participants were actively engaged while using Lucy LiverBot throughout the in-depth user testing process, which took approximately 1 hour. We were also able Overview 4 (20) Agree 4 (20) Neither agree or disagree 10 (50) Disagree 2 (10) Not applicable LiverBot at later stages of CLD, but that it would have been useful at the time of their diagnosis. Keep checking up on you. Having information that is not conflicting. Very informative. Would explain everything if I didn't know anything about cirrhosis. It's quick and very simple to use. I like how I can ask questions as soon as they arise rather than wait for an appointment or google world wide. Having information that is not conflicting. Very informative. Would explain everything if I didn't know anything about cirrhosis. It's quick and very simple to use. I like how I can ask questions as soon as they arise rather than wait for an appointment or google world wide. It was 1 participant who suggested additions that could be incorporated into the chatbot to specifically focus on the mental health of users. Mental Health and Well-Being Telling stuff I already know so not that useful. Brilliant, can help many people, for young people. Several participants identified improvement in mental health and well-being as an unintended benefit of Lucy LiverBot. Beyond providing disease specific information, the conversational nature of the chatbot appealed to many as it provided a well-received reminder to maintain habits conducive to their well-being and health habits. https://humanfactors.jmir.org/2023/1/e42506 Overview 15 (75) Easy 1 (5) Neither easy or difficult 4 (20) Difficult How easy or difficult has it been to understand advice from different health care providers? 13 (65) Easy 2 (10) Neither easy or difficult 5 (25) Difficult Monitoring health behaviors How much of a problem has it been for you to monitor your health behaviors, for example, exercise, diet and medication adherence? 12 (60) A little 3 (15) Somewhat 4 (20) Quite a bit 1 (5) Not applicable How bothered have you been by feeling dependent on others for your health care needs? 5 (25) A little 7 (35) Somewhat 6 (30) Quite a bit 2 (10) Not applicable How bothered have you been by others reminding you to do things for your health, for example, take medications, eat healthy, schedule appointments? 14 (70) A little 1 (5) Somewhat 4 (20) Quite a bit 1 (5) Not applicable Managing emotional well-being How often did your self-care make you feel preoccupied? 8 (40) Rarely 8 (40) Sometimes 2 (10) Often 2 (10) Not applicable How often did your self-care make you feel depressed? 8 (40) Rarely appointments? 14 (70) A little 1 (5) Somewhat 4 (20) Quite a bit 1 (5) Not applicable Managing emotional well-being How often did your self-care make you feel preoccupied? 8 (40) Rarely 8 (40) Sometimes 2 (10) Often 2 (10) Not applicable How often did your self-care make you feel depressed? 8 (40) Rarely https://humanfactors.jmir.org/2023/1/e42506 https://humanfactors.jmir.org/2023/1/e42506 XSL•FO RenderX JMIR HUMAN FACTORS Participants, n (%) Survey questions 9 (45) Sometimes 2 (10) Often 1 (5) Not applicable How often did your self-care make you feel worn out? 6 (30) Rarely 6 (30) Sometimes 8 (40) Often 0 (0) Not applicable How often did your self-care make you feel frustrated? 5 (25) Rarely 8 (40) Sometimes 7 (35) Often 0 (0) Not applicable Problems with multidisciplinary communication I have problems with different health care providers not communicating with each other about my medical care? Au et al JMIR HUMAN FACTORS Survey questions I have problems with different health care providers not communicating with each other about my medical care? I have problems with different health care providers not communicating with each other about my medical care? Reliable Source of Medical Information Lucy LiverBot was generally perceived as a reliable and trustworthy source of information as it was produced by medical professionals in the field of CLD. Participants recognized their potential to provide a trusted reference for nutritional information, rather than resorting to the internet. Maintaining wellbeing. Keep checking up on you. JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 8 (page number not for citation purposes) Timing of Chatbot Implementation It is also hoped that a centralized digital device designed by the MDT will help bridge the communication gap between patients and clinicians. Many participants identified that the conversational tone and companion-like nature of the chatbot was one of its key strengths. Lucy LiverBot’s ability to engage with users provided a social platform for them to ask concerns and may have the potential to extend its disease specific content to directly address mental well-being and provide a sense of social connection. By addressing these identified barriers, Lucy LiverBot has the potential to fill a gap in the provision of health care to this group of complex chronic disease patients. Further validation studies are required to determine whether Lucy LiverBot as an intervention would prevent clinical outcomes such as readmission related to decompensated CLD. The efficacy of novel digital health interventions such as Lucy LiverBot, which lack a formal evaluation framework akin to pharmacotherapy and device trials, would benefit from a multidisciplinary evaluation strategy tailored to the specific study end point. We conducted the study from when standardized tools to assess user’s satisfaction with the experience of using chatbots were unavailable. If we were to repeat the study again we would leverage new tools such as the Chatbot Usability Scale. An assessment of human computer interactions will also be required to determine the real-world patient usage patterns of Lucy LiverBot. A randomized controlled trial would be the ideal format in determining whether Lucy LiverBot is effective in improving health literacy and reducing hospital readmission. However, as ambulatory medical care models become increasingly multidisciplinary, it may become difficult to delineate which arms of the multimodal health care model are responsible for changes in clinical outcome. For example, if an improvement in admissions for hepatic encephalopathy were to be observed, this could be attributed to the increased communication with clinical staff through chatbot alerts, the health education provided by the chatbot, or perhaps an improvement in other indices such as nutrition and adherence. Further prospective studies based on the principles of implementation science are warranted to assess the benefits that Lucy LiverBot may provide to clinical end points such as decompensation rate, morbidity, quality of life, and clinic attendance. Timing of Chatbot Implementation A common theme that emerged from participants was that the use of Lucy LiverBot may depend on the timing of its implementation in the patient’s disease progress. Patients who had been managing their liver cirrhosis for longer periods of time identified that they would be less likely to use Lucy JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 8 (page number not for citation purposes) XSL•FO RenderX JMIR HUMAN FACTORS Au et al what features are most beneficial within Lucy LiverBot and whether patients will be committed to using it for an extended period of their own volition beyond a study context. There was a potential selection bias as participants who agreed to join the study may be more motivated to improve their health and more likely to engage with Lucy LiverBot. In addition, response bias may have played a role in the study as participants completed the survey while investigators were in the room for technical support. This may have influenced patients to select answers that they believed were more acceptable. To remove any potential responder bias due to a perceived impact on their care, participants were assured that the results from the study would not be viewed by their treating team. We also trained final year medical students to conduct the testing rather than physicians to reduce the perceived power imbalance between participants and interviewers. The study also did not assess the stage of hepatic encephalopathy in participants. In future studies, we plan to assess this both at baseline and longitudinally to further delineate the effect of hepatic encephalopathy on a patient’s ability to remember health specific information. to capture users from a variety of age groups and at different stages of their disease process which allowed us to determine at what stage of CLD Lucy LiverBot would be most useful. In addition, our extensive testing process ensured that all available chat flows were tested and NLP continued to improve with each consecutive patient. Our results highlighted key barriers faced by patients with CLD which have the potential to impact their health outcomes—understanding health information, monitoring health behaviors, managing emotional well-being, and multidisciplinary communication. Lucy LiverBot has been specifically designed to assist patients with the understanding of health information and the monitoring of their health behaviors. Timing of Chatbot Implementation A longitudinal component of the study should be established whereby participants are tracked throughout their disease progression and compared to those who did not use a health chatbot to determine whether Lucy LiverBot prevented hospital readmissions and led to improved patient outcomes. Such studies will need to perform costings analyses and assess long-term patient participation, and adherence to digital health care models. It would be important to continue monitoring the performance of Lucy LiverBot after its launch to identify any errors in NLP so that necessary adjustments can be made. The NLP feature in Lucy LiverBot is basic and further advancements in this technology will be required to improve future iterations capable of providing an even more engaging user experience. The user testing allowed us to gauge how patients were most likely to phrase questions which allowed us to alter recognized terms. This was evident as Lucy LiverBot was unable to recognize some patient questions during the user testing stage if they were not worded in a similar manner to the initial input options. Unfortunately, the chatbot is currently only available in English, which limits its generalizability and scalability for participants from Culturally and Linguistically Diverse communities. This precluded some patients from participating in this study, however this is a technical limitation of NLP in general, rather than of Lucy LiverBot specifically. Ideally, future versions of Lucy LiverBot will be available in multiple languages. The study was limited by a paucity of research on health chatbots, which made it difficult to determine the sample size required to adequately power the study and the ideal study design to assess patient usability. Our small study population allowed preliminary information to be obtained regarding the usability of Lucy LiverBot and its potential to act as an educational tool for patients with CLD. However, future studies with larger cohorts of patients will be required to definitively demonstrate Lucy LiverBot’s ability to improve health literacy and health outcomes. It is likely that solutions such as Lucy LiverBot will require frequent cycles of iteration and user testing to become maximally effective. This will lend more insight into https://humanfactors.jmir.org/2023/1/e42506 JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 9 (page number not for citation purposes) Acknowledgments We are incredibly grateful to our patients for taking the time to contribute to research into the use of chatbots in chronic liver disease. Conclusions Our study identified barriers to health care provision and found that Lucy LiverBot was well received by patients with decompensated chronic liver disease. Lucy LiverBot can JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 9 (page number not for citation purposes) XSL•FO RenderX JMIR HUMAN FACTORS Au et al the potential for Lucy LiverBot to improve patient engagement and self-management and its use as an engagement tool with multidisciplinary teams. the potential for Lucy LiverBot to improve patient engagement and self-management and its use as an engagement tool with multidisciplinary teams. specifically address these barriers and be introduced as a potential educational intervention to address the impact of poor health literacy on disease outcomes and a health related quality of life. Further validation studies are required to demonstrate Authors' Contributions JA, CF, AR, PH, and SL were all involved in data curation, conceptualization, formal analysis, investigation, methodology, project administration, writing-original draft, and writing-reviewing and editing. SL was also involved in funding acquisition, validation, and supervision. 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JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 11 (page number not for citation purposes) References JAMA Netw Open 2020;3(4):e201997 [FREE Full text] [doi: 10.1001/jamanetworkopen.2020.1997] [Medline: 32239220] JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 11 (page number not for citation purposes) https://humanfactors.jmir.org/2023/1/e42506 XSL•FO RenderX Au et al JMIR HUMAN FACTORS JMIR Hum Factors 2023 \| vol. 10 \| e42506 \| p. 12 (page number not for citation purposes) https://humanfactors.jmir.org/2023/1/e42506 Abbreviations AI: artificial intelligence CLD: chronic liver disease MDT: multidisciplinary team NLP: natural language processing PETS: Patient Experience with Treatment and Self-Management Edited by A Kushniruk; submitted 06.09.22; peer-reviewed by J Lander, M Serper, D Chao; comments to author 01.01.23; revised version received 25.02.23; accepted 14.05.23; published 15.08.23 Please cite as: Au J, Falloon C, Ravi A, Ha P, Le S A Beta-Prototype Chatbot for Increasing Health Literacy of Patients With Decompensated Cirrhosis: Usability Study JMIR Hum Factors 2023;10:e42506 URL: https://humanfactors.jmir.org/2023/1/e42506 doi: 10.2196/42506 PMID: 37581920 ©Jessica Au, Caitlin Falloon, Ayngaran Ravi, Phil Ha, Suong Le. Originally published in JMIR Human Factors (https://humanfactors.jmir.org), 15.08.2023. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Human Factors, is properly cited. The complete bibliographic information, a link to the original publication on https://humanfactors.jmir.org, as well as this copyright and license information must be included. https://humanfactors.jmir.org/2023/1/e42506 https://humanfactors.jmir.org/2023/1/e42506 XSL•FO RenderX XSL•FO RenderX
https://openalex.org/W2018520635	https://www.scielo.br/j/ni/a/47Fg4MZfCqCVqDKr8NVmLSS/?lang=en&format=pdf	English	null	New species of Moenkhausia Eigenmann, 1903 (Characiformes: Characidae) with comments on the Moenkhausia oligolepis species complex	Neotropical ichthyology/Neotropical Ichthyology	2,009	cc-by	5,407	Neotropical Ichthyology, 7(2):161-168, 2009 Copyright © 2009 Sociedade Brasileira de Ictiologia Neotropical Ichthyology, 7(2):161-168, 2009 Copyright © 2009 Sociedade Brasileira de Ictiologia Neotropical Ichthyology, 7(2):161-168, 2009 p y gy, ( ) , Copyright © 2009 Sociedade Brasileira de Ictiologia Ricardo C. Benine, Tatiane C. Mariguela and Claudio Oliveira A new species of Moenkhausia is described from tributaries of the rio Paraguay, Brazil. The new species is diagnosed from congeners by characters related to body coloration, the number of lateral line scales, the degree of poring of the lateral line, and number of scales rows above and below the lateral line. Molecular analyses using partial sequences of the mitochondrial gene Cytochrome Oxidase I from specimens of the new species and specimens belonging to morphologically similar species demonstrated that the new species is easily differentiated by their high genetic distance and by their position in the phylogenetic hypothesis obtained through the Maximum Parsimony methodology. The analyses of three samples of M. oligolepis also revealed that they have high genetic distances and belong to different monophyletic groups suggesting that this species corresponds to a species complex rather than a single species. Uma nova espécie de Moenkhausia é descrita de tributários do rio Paraguai, Brasil. Essa nova espécie é diagnosticada de seus congêneres por caracteres relacionados ao padrão de colorido do corpo, número de escamas da linha lateral, grau de desenvolvimento dos poros sensoriais na linha lateral e número de séries de escamas acima e abaixo da linha lateral. Um análise molecular usando sequências parciais do gene mitocondrial Citocromo Oxidase I de espécimes representativos da nova espécie e espécimes pertencentes a espécies morfologicamente similares demonstrou que a nova espécie é facilmente diferenciada por sua elevada distância genética e por sua posição na hipótese filogenética obtida pelo método de máxima parcimônia. A análise de três amostras de M. oligolepis também revelou que estas apresentam distâncias genéticas elevadas e pertencem a grupos monofiléticos distintos, sugerindo que esta espécie corresponda a um complexo de espécies e não uma única espécie. Key words: Taxonomy, Systematic, Molecular Phylogeny, COI gene, Moenkhausia sanctaefilomenae. Laboratório de Biologia e Genética de Peixes, Departamento de Morfologia, IBB-UNESP, Campus de Botucatu, Botucatu, SP 18618-000, Brazil. rbenine@ibb.unesp.br (RCB); tatimariguela@gmail.com (TCM); claudio@ibb.unesp.br (CO) New species of Moenkhausia Eigenmann, 1903 (Characiformes: Characidae) with comments on the Moenkhausia oligolepis species complex Ricardo C. Benine, Tatiane C. Mariguela and Claudio Oliveira Introduction Cis-Andean river basins, except for those in Patagonia, with its greatest diversity occurring in the basins of the Amazon and Guianas (Lima et al., 2003). The distributed characid genus Moenkhausia was proposed by Eigenmann (1903) based mainly on the combination of the presence of premaxillary teeth in two rows, an inner premaxillary tooth row with five or more teeth, a completely pored lateral line without a downward curve anteriorly, and a sheath of scales covering the base of caudal- fin lobes, characters still used to substantiate the generic allocation of new species to Moenkhausia (see Benine et al., 2007; Lima et al., 2007; Lucinda et al., 2007), considering the nonexistence of a phylogenetic definition. Currently allocated as incertae sedis in Characidae, Moenkhausia comprises, at present, 65 valid species widely distributed in the Neotropical Albeit the genus Moenkhausia cannot be defined as monophyletic at this time, a group of species within Moenkhausia shares an identical or, at least, very similar color pattern consisting of an overall reticulate pattern formed by dark borders on the scales, a vertically-elongate humeral blotch, a conspicuous dark blotch on the caudal peduncle preceded by a lighter area, and frequently a deep red eye in life. This group consists of M. oligolepis Günther, M. sanctaefilomenae Steindachner, M. cotinho Eigenmann, and M. pyrophthalma Costa, as suggested by Costa (1994). Lima & Toledo-Piza (2001) discussed a putatively close relationship 161 New species of Moenkhausia and comments on the Moenkhausia oligolepis species complex 162 of M. dyktiota Lima & Toledo-Piza with these cited species also on the basis of its overall similar colour pattern, albeit without the characteristic round, dark, blotch on the caudal peduncle. (forward and reverse). Individual sequences of each species were initially analyzed using BioEdit 5.0.9 (Hall, 1999) and a consensus sequence was obtained for each DNA segment for each species. All sequences were then aligned with DAMBE (Xia & Xie, 2001). Genetic distances (Kimura, 1980) were examined using MEGA 4.0 (Kumar et al., 2004). Despite the similar color pattern, these species are easily differentiated from each other on the basis of number of lateral line scales and number of series of scales above and below lateral line. Nonetheless, misidentifications between Moenkhausia oligolepis and M. sanctaefilomenae are common. According to Lima et al. (2003), M. oligolepis occurs in the Guianas and in the Amazon River basin, whereas M. Material and Methods Holotype. MZUSP 97827, 34.0 mm SL, undetermined sex, Brazil, Mato Grosso State, Cáceres, rio Sepotuba, rio Paraguay basin,15º46’07”S 57º38’54”W, 3-4 Mar 2002, H. A. Britski, O. Fröehlich, A. Catella & F. Marques. Holotype. MZUSP 97827, 34.0 mm SL, undetermined sex, Brazil, Mato Grosso State, Cáceres, rio Sepotuba, rio Paraguay basin,15º46’07”S 57º38’54”W, 3-4 Mar 2002, H. A. Britski, O. Fröehlich, A. Catella & F. Marques. Material examined in this study is deposited in the California Academy of Sciences (CAS), San Francisco; Laboratório de Biologia e Genética de Peixes do Departamento de Morfologia da Universidade Estadual Paulista (LBP), Botucatu, SP; Museu de Ciências e Tecnologia, Pontifícia Universidade Católica do Rio Grande do Sul (MCP), Porto Alegre, RS; and Museu de Zoologia da Universidade de São Paulo (MZUSP), São Paulo, SP. Vouchers of the material used in the molecular analysis are deposited at LBP. Paratypes. MZUSP 90270, 14, 24.0-36.4 mm SL, (2 c&s, 25.6- 33.1 mm SL), same data as the holotype. MZUSP 19111, 10, 30.7- 35.0 mm SL, Brazil, Mato Grosso State, Descalvados, rio Paraguay, 16º46’S 57º44’W, 9 Aug 1980, R. M. C. Castro & H. Ortega. LBP 3793, 10, 28.3-33.4 mm SL, Brazil, Mato Grosso do Sul State, Aquidauana, rio Negro, rio Paraguay basin, 19º34’02.3”S 56º14’09.1”W, 2 Aug 2006, C. Oliveira & L. H. G. Pereira. Paratypes. MZUSP 90270, 14, 24.0-36.4 mm SL, (2 c&s, 25.6- 33.1 mm SL), same data as the holotype. MZUSP 19111, 10, 30.7- 35.0 mm SL, Brazil, Mato Grosso State, Descalvados, rio Paraguay, 16º46’S 57º44’W, 9 Aug 1980, R. M. C. Castro & H. Ortega. LBP 3793, 10, 28.3-33.4 mm SL, Brazil, Mato Grosso do Sul State, Aquidauana, rio Negro, rio Paraguay basin, 19º34’02.3”S 56º14’09.1”W, 2 Aug 2006, C. Oliveira & L. H. G. Pereira. Morphometric and meristic data were taken following Fink & Weitzman (1974), except for head depth (HD), which was measured in the vertical through the middle of the orbit. Counts are followed by their frequency in parentheses. Asterisks indicate values for the holotype. Vertebrae of the Weberian apparatus were counted as four elements and the fused PU1+U1 as a single element. Vertebra and supraneural counts were taken from two cleared and stained (c&s) specimens prepared following the method of Taylor & Van Dyke (1985). Non-types. LBP 5074, 10, 25.7-32.1 mm SL, Brazil, Mato Grosso State, Cáceres, Baía do Caiçara, 16º03’11.3”S 57º48’32.0”W. Introduction sanctaefilomenae is distributed in the Parnaíba, São Francisco, upper Paraná, Paraguay and Uruguay River systems. Detailed examination revealed that not does only Moenkhausia oligolepis occur in the rio Paraguay basin but also that new similar species occur in sympatry revealing a species complex. In the present paper, we describe a new species of the M. oligolepis species complex and provide an analysis of its phylogenetic position based on molecular data. Phylogenetic analyses based on the maximum-parsimony method (Swofford & Berlocher, 1987) were performed using PAUP* beta version 4.0b10 (Swofford, 2002) with heuristic searches using random addition of sequences and the tree bisection and reconnection (TBR) algorithm. In all analyses the character-state optimization method employed was the accelerated transformation (ACCTRAN). Parsimony trees were generated using Ti/Tv ratio of 1:1 and considering terminal gaps as missing data. Bootstrap resampling (Felsenstein, 1985) was applied to assess support for individual nodes using 1000 replicates with random additions and TBR branch swapping. Material and Methods LBP 3739, 10, 22.4 - 32.5 mm SL, Brazil, Mato Grosso do Sul State, Aquidauana, rio Negro, rio Paraguay basin, 19º34’54.6”S 56º15’16.5”W. LBP 4655, 65, 21.9-32.9 mm SL, Brazil, Mato Grosso do Sul State, Batayporã, rio Baía, rio Paraná basin, 22º43’46.2”S 53º19’04.2”W. LBP 2630, 18, 23.0-37.9 mm SL, Brazil, Paraná State, Porto Rico, rio Paraná, 22º43’03.2”S 53º17’27.6”W. LBP 5225, 10, 25.0-32.8 mm SL, Brazil, Paraná State, Porto Rico, rio Paraná, 22º47’29”S 53º20’58”W. Total DNA was extracted from ethanol-preserved muscle tissues with the Wizard Genomic DNA Purification Kit (Promega). Partial sequences of COI gene were amplified with the primers Fish F1 and Fish R1 (Ward et al., 2005). Primer final concentrations were 5 mM. Amplifications were performed by the polymerase chain reaction (PCR) in a total volume of 25 ml for 35 cycles (30 s at 95 oC, 60 s at 50-60 oC, and 120 s at 72 oC). The PCR products were identified in a 1% agarose gel. The amplified segments were extracted from the gel with the kit GFXTM PCR DNA and Gel Purification (Amersham Pharmacy Biotech Inc). The sequencing reactions were done with the kit DYEnamic ET Terminator Cycle Sequencing (Amersham Pharmacy Biotech Inc.) and analyzed in an automated sequencer ABI PrismTM 377 DNA Sequencer (Perking-Elmer). All sequences were read at least twice Diagnosis. Moenkhausia forestii is readly distinguished from all congeners, except M. oligolepis, M. sanctaefilomenae, M. pyrophthalma, and M. diktyota, by the presence of a reticulated body pigmentation pattern, and a conspicuous dark blotch on the caudal peduncle extending to the base of caudal-fin rays preceded by a lighter area (vs. absence of a reticulated body pigmentation pattern, and a conspicuous dark blotch on the caudal peduncle extending to the base of caudal-fin rays preceded by a lighter area). Moenkhausia forestii is readily distinguished from M. oligolepis in the degree of poring of the lateral line (incomplete, vs. complete, 163 R. C. Benine, T. C. Mariguela & C. Oliveira Fig. 1. Moenkhausia forestii, holotype, MZUSP 97827, undetermined sex, 34.0 mm SL, Cáceres, rio Sepotuba, Mato Grosso State, Brazil. Fig. 1. Moenkhausia forestii, holotype, MZUSP 97827, undetermined sex, 34.0 mm SL, Cáceres, rio Sepotuba, Mato Grosso State, Brazil. Fig. 1. Moenkhausia forestii, holotype, MZUSP 97827, undetermined sex, 34.0 mm SL, Cáceres, rio Sepotuba, Mato Grosso St t B il Fig. 2. Material and Methods Moenkhausia forestii, new species, paratype, MZUSP 90270, 33.1 mm SL, premaxillary, maxillary and dentary; right side, lateral view (maxillary and dentary) or frontal view (premaxillary). Scale bar = 1 mm. respectively), and the total number of lateral line scales (23- 26, vs. 28-31 scales, respectively). Moenkhausia forestii can be distinguished from M. sanctaefilomenae in the number of transverse series of scales above the lateral line (5, vs. 4 scales, respectively), by the number of transverse series of scales below the lateral line (4, vs. 3 scales, respectively). Moenkhausia forestii is distinguished from M. pyrophthalma by its higher body depth (38.3-45.2% in SL vs. 31.2-33.0% in SL, respectively), by its shorter lateral line (23-26 vs. 27-31 scales, respectively), and by the form of the humeral spot (an inconspicuous dark inverse triangular humeral blotch located on second and third lateral line scales vs. a conspicuous black inverse drop-shaped humeral blotch located on second to fourth lateral line scales, respectively). Moenkhausia forestii is distinguished from M. diktyota by the color pattern of the caudal peduncle (caudal peduncle with very widely scattered dark pigment resulting in conspicuous lighter area preceding a conspicuous caudal-fin blotch which extends between the dorsal to ventral margins of the proximal one-third of caudal- fin base vs. caudal peduncle with elongate black stripe extending from the vertical through the last anal-fin ray to the tips of middle caudal fin rays, respectively). Description. Morphometric data summarized in Table 1. Overall size small (maximum of 36.4 mm SL). Greatest body depth at origin of dorsal fin. Dorsal profile of head straight or slightly convex. Dorsal profile of body sligthly convex from posterior tip of supraocciptal to end of dorsal-fin base; slightly convex from rear of dorsal-fin base to end of adipose-fin base. Caudal peduncle profile slightly concave both dorsally and ventrally. Ventral profile of body convex from tip of lower jaw to caudal Fig. 2. Moenkhausia forestii, new species, paratype, MZUSP 90270, 33.1 mm SL, premaxillary, maxillary and dentary; right side, lateral view (maxillary and dentary) or frontal view (premaxillary). Scale bar = 1 mm. New species of Moenkhausia and comments on the Moenkhausia oligolepis species complex 164 Table 1. Morphometric data of Moenkhausia forestii, holotype and paratypes. Material and Methods Holotype N Range Mean SD Standard length (mm) 34.0 35 24.0-36.4 30.6 3.13 Percents of standard length Greatest depth 43.1 35 38.3-45.2 42.6 1.75 Snout to dorsal-fin origin 56.8 35 53.5-58.6 56.3 1.28 Snout to pectoral-fin origin 31.0 35 28.5-33.1 30.7 1.11 Snout to pelvic-fin origin 51.2 35 46.9-52.4 49.6 1.34 Snout to anal-fin origin 71.1 35 64.4-72.3 68.5 1.91 Caudal peduncle depth 12.6 35 11.5-13.6 12.5 0.52 Caudal peduncle length 9.8 35 7.2-10.6 9.1 0.84 Pectoral-fin length 26.2 35 24.4-28.9 26.2 1.04 Pelvic-fin length 22.1 34 20.5-25.6 21.9 0.99 Dorsal-fin length 28.1 35 26.6-33.4 29.7 1.39 Dorsal-fin base 14.2 35 13.0-15.4 14.4 0.51 Anal-fin length 20.8 35 16.1-24.6 20.8 1.77 Anal-fin base 28.4 35 25.7-31.9 28.8 1.41 Eye to dorsal-fin origin 44.4 34 40.6-45.8 43.2 1.40 Dorsal-fin origin to caudal-fin origin 48.9 35 46.0-50.9 49.0 1.22 Head length 28.5 35 27.2-30.8 28.9 1.06 Head depth 21.1 35 19.6-22.7 21.0 0.79 Percents of head length Snout length 28.7 34 25.3-33.5 28.4 1.75 Upper jaw length 42.0 35 39.9-44.0 42.4 1.31 Horizontal orbital diameter 37.9 35 34.6-43.9 39.0 1.83 Least interorbital width 39.7 35 36.8-44.3 40.6 1.74 Table 1. Morphometric data of Moenkhausia forestii, holotype and paratypes. scales. Lateral line slightly ventrally curved anteriorly; with 5 (n = 35) series of scales above and 4 (n = 35) series of scales below. Scales around caudal peduncle 7-9* (mode = 9, n = 35). Single row of scales overlaying basal portion of anterior rays of anal fin. Sheet of scales covering proximal one-third of upper caudal-fin lobe and proximal one-half of lower caudal- fin lobe. First gill arch with 6-7* (mode = 7, n = 35) gill-rakers on upper limb and 10-11* (mode = 11, n = 28) gill-rakers on lower limb. Precaudal vertebrae 13, caudal vertebrae 17 (n = 2). Supraneurals 4 (n = 2). Color in alcohol. Overall ground coloration dark silver or yellowish tan (depending on degree of retention of guanine). Dark chromatophores concentrated on distal margin of scales resulting in conspicuous reticulated pattern. Mid-dorsal region darker than flanks. Humeral region with dark inverse triangle-shaped blotch located on second to third lateral line scales, extending 3-4 scales vertically, including lateral line. Few dark chromatophores scattered on infraorbitals and opercle. Dark thin stripe extending along horizontal septum on posterior half of body. Caudal fin with conspicuous dark blotch extending from its anterior portion onto proximal one- third of caudal-fin rays. Material and Methods Caudal peduncle with very widely scattered dark pigment resulting in conspicuous lighter area preceding caudal-fin blotch. Dorsal fin with scattered dark pigmentation, more concentrated on anterior half. Anal fin with scattered dark pigmentation. Paired fins hyaline with scattered dark pigmentation, more so on unbranched rays. peduncle origin. Prepelvic region transversely flattened, flattening more pronounced proximate to pelvic-fin insertion. Postpelvic median keel extending from pelvic-fin insertion to anal-fin origin. Mouth terminal, with lower jaw as long as, to somewhat longer than, upper jaw. Premaxillary teeth in two rows; outer row teeth 3-5* (mode = 5, n = 35), with 3-5 cusps, central cusp longer than other cusps; inner row teeth 5, with 4-5 cusps and rarely with lateral-most tooth with 3 cusps. Maxillary teeth 1-2* (mode = 2, n = 35), each with 5 cusps. Dentary with 4 larger teeth anteriorly, each with 4-5 cusps with central cusp longest (n =35). Larger anterior teeth followed by 5-7 small teeth with 1-3 cusps in two c&s paratypes (Fig. 2). Sexual dimorphism. One paratype (30.9 mm SL, MZUSP 90270) has small hooks on the segments of each anal-fin ray (up to four hooks per segment in each side of the anal fin). Additional non-type material from the upper rio Paraná system (LBP 5225) collected during the reproductive season included males with the same pattern of hooks. No further dimorphic characters were observed. Dorsal-fin rays ii,9. Dorsal-fin origin at middle body length, slightly posterior to vertical through pelvic-fin origin. Posterior margin of dorsal fin slightly concave. Anal-fin rays iv,17-23, iv,22* (mode = iv,22, n = 35). Distal margin of anal fin straight to somewhat concave with 4th unbranched and anterior 1st-3rd branched fin ray longer. Remaining anal-fin rays decreasing gradually in length rearward. Pectoral-fin rays i,11-12* (mode = i,12, n = 35). Pectoral fin pointed, 1st-2nd branched rays longer, lateral and medial margins straight, posterior margin oblique and straight. Tip of adpressed pectoral fin extends posterior of mid-length of adpressed pelvic fin. Pelvic-fin rays i,7. Pelvic fin pointed, lateral and medial margins straight, posterior margin oblique and straight.Tip of pelvic fin reaches anal-fin origin. Adipose fin origin at vertical through insertion point of antepenultimate branched anal fin. Principal caudal-fin rays i,9,8,i. Caudal-fin lobes equal. Distribution. Material and Methods Moenkhausia forestii is known from tributaries from rio Paraguay drainage (Mato Grosso and Mato Grosso do Sul States), from rio Baía (upper rio Paraná system at Batayporã, Mato Grosso do Sul State) and from rio Paraná (at Porto Rico, Paraná State) (Fig. 3). Etymology. The specific epithet, forestii, is in honour of Fausto Foresti for his contributions to our knowledge of fish genetics. Molecular Analyses. DNA sequences were obtained from: Moenkhausia forestii LBP 3739, LBP 4655 and LBP 5074; Moenkhausia oligolepis LBP 4098; LBP 1498 and LBP 5073; Moenkhausia sanctaefilomenae LBP 4695. The sequences obtained in this study have been deposited in GenBank (Table 2). Genetic distances (Kimura, 1980) range from zero among specimens of Moenkhausia forestii from Paraná basin to 0.200 ± 0.021 between M. oligolepis from the rio Araguaia Basin and M. Molecular Analyses. DNA sequences were obtained from: Moenkhausia forestii LBP 3739, LBP 4655 and LBP 5074; Moenkhausia oligolepis LBP 4098; LBP 1498 and LBP 5073; Moenkhausia sanctaefilomenae LBP 4695. Molecular Analyses. DNA sequences were obtained from: Moenkhausia forestii LBP 3739, LBP 4655 and LBP 5074; Moenkhausia oligolepis LBP 4098; LBP 1498 and LBP 5073; Moenkhausia sanctaefilomenae LBP 4695. The sequences obtained in this study have been deposited in GenBank (Table 2). Genetic distances (Kimura, 1980) range from zero among specimens of Moenkhausia forestii from Paraná basin to 0.200 ± 0.021 between M. oligolepis from the rio Araguaia Basin and M. Scales cycloids. Lateral line with 7-11 (10; mode = 9, n = 34) pored scales and 23-26 (25; mode = 25, n = 34) total 165 R. C. Benine, T. C. Mariguela & C. Oliveira Fig. 3. Map of the rio La Plata basin showing the localities of collection of Moenkhausia forestii, new species. Star represents the type locality. Fig. 3. Map of the rio La Plata basin showing the localities of collection of Moenkhausia forestii, new species. Star represents the type locality. Table 2. Genetic distance (mean ± standard error) among fishes of each locality (diagonal) and among samples from different localities (below diagonal). Numbers after species names are Genbank accession numbers. Material and Methods 1 2 3 4 5 6 7 1 Moenkhausia sanctaefilomenae Paraná Basin EU177008 to EU177012 0.003±0.002 2 Moenkhausia oligolepis Amazon Basin EU177038 to EU177042 0.190±0.019 0.010±0.003 3 Moenkhausia oligolepis Araguaia Basin EU177013 to EU177017 0.200±0.021 0.128±0.015 0.010±0.003 4 Moenkhausia oligolepis Paraguay Basin EU177033 to EU177037 0.184±0.018 0.109±0.013 0.139±0.016 0.014±0.003 5 Moenkhausia forestii Paraguay Basin -Aquidauana EU177023 to EU177027 0.183±0.019 0.174±0.019 0.180±0.019 0.197±0.019 0.005±0.002 6 Moenkhausia forestii Paraná Basin EU177018 to EU177022 0.182±0.019 0.172±0.019 0.180±0.019 0.197±0.019 0.006±0.002 0.000±0.000 7 Moenkhausia forestii Paraguay Basin - Cáceres EU177028 to EU177032 0.183±0.019 0.173±0.019 0.178±0.019 0.198±0.020 0.007±0.003 0.001±0.000 0.001±0.001 Table 2. Genetic distance (mean ± standard error) among fishes of each locality (diagonal) and among samples from different ocalities (below diagonal). Numbers after species names are Genbank accession numbers. Table 2. Genetic distance (mean ± standard error) among fishes of each locality (diagonal) and among samples from different localities (below diagonal). Numbers after species names are Genbank accession numbers. 1 2 3 4 5 6 7 1 Moenkhausia sanctaefilomenae Paraná Basin EU177008 to EU177012 0.003±0.002 2 Moenkhausia oligolepis Amazon Basin EU177038 to EU177042 0.190±0.019 0.010±0.003 3 Moenkhausia oligolepis Araguaia Basin EU177013 to EU177017 0.200±0.021 0.128±0.015 0.010±0.003 4 Moenkhausia oligolepis Paraguay Basin EU177033 to EU177037 0.184±0.018 0.109±0.013 0.139±0.016 0.014±0.003 5 Moenkhausia forestii Paraguay Basin -Aquidauana EU177023 to EU177027 0.183±0.019 0.174±0.019 0.180±0.019 0.197±0.019 0.005±0.002 6 Moenkhausia forestii Paraná Basin EU177018 to EU177022 0.182±0.019 0.172±0.019 0.180±0.019 0.197±0.019 0.006±0.002 0.000±0.000 7 Moenkhausia forestii Paraguay Basin - Cáceres EU177028 to EU177032 0.183±0.019 0.173±0.019 0.178±0.019 0.198±0.020 0.007±0.003 0.001±0.000 0.001±0.001 Table 2. Genetic distance (mean ± standard error) among fishes of each locality (diagonal) and among samples from different ocalities (below diagonal). Numbers after species names are Genbank accession numbers. The MP analyses showed that there are six monophyletic groups supported by values of equal or higher than 99% (Fig. 4). Three monophyletic groups corresponding to the species Moenkhausia sanctaefilomenae, M. oligolepis and M. forestii and three monophyletic groups correspond to the different samples of M. oligolepis. The relationship between sanctaefilomenae from the upper rio Paraná system (Table 2). The combined sequence data of the 35 specimens resulted in a matrix with 611 base pairs (bp), from which 419 were conserved sites and 181 were phylogenetically informative. The MP consensus tree obtained from the analysis of 1000 bootstrap replicates is presented in Fig. 4. New species of Moenkhausia and comments on the Moenkhausia oligolepis species complex 166 Fig. 4. Material and Methods Maximum Parsimony consensus tree for the mitochondrial gene Cytochrome Oxidase I. Numbers represent values of 1000 bootstrap replicates. Fig. 4. Maximum Parsimony consensus tree for the mitochondrial gene Cytochrome Oxidase I. Numbers represent values of 1000 bootstrap replicates. M. oligolepis from the Amazon and Paraguay river basins was weakly supported but they are apparently different from the sample from the rio Araguaia basin. and, thus failed to conform to the traditional definition of the genus. They based their generic assignments on other evidence, specifically overall similarity with other species of Moenkhausia that they felt justified this generic assignment. Such procedure was equally adopted herein. Discussion Eigenmann (1917: 84) included specimens with incomplete, interrupted, and complete lateral line in a single species, Moenkhausia sanctaefilomenae. In fact, this author raised the potential of the existence of more than one species in those samples, noting that the forms with incomplete lateral line would correspond to his M. australe, and those with both interrupted and complete lateral line would correspond to M. sanctaefilomenae. The basis for Eigenmann’s actions was unclear, but he presumably included samples with different degrees of development of the lateral line in a single species in the absence of broad samples at his disposal and consequently placed M. australe in the synonymy of M. According to Eigenmann (1907), the degree of development of the lateral-line (number and position of perforated scales) distinguishes Moenkhausia (complete lateral line) from Hemigrammus (incomplete lateral line) and Psellogrammus (interrupted lateral line). This purported difference was recently discussed by Costa (1994), Lima & Toledo-Piza (2001), who highlighted the weakness of the phylogenetic information in this feature. These authors described their new species in Moenkhausia, even though those forms demonstrated incompletely pored lateral-lines R. C. Benine, T. C. Mariguela & C. Oliveira 167 7, paratypes, 19.8-29.8 mm SL, rio das Mortes, between Água Boa and Cocalinho, Mato Grosso State, Brazil; LBP 4922, 5, 27.6-34.0 mm SL, rio das Mortes, Barra do Garças, Mato Grosso State, Brazil. Moenkhausia sanctaefilomenae: LBP 4695, 8, 25.1-61.3 mm SL, rio Paraná Basin, Marapoama, São Paulo State, Brazil; LBP 5541, 44, 31.6 - 47.1 mm SL, MCP 42016, 5, 32.6-45.6 mm SL, rio Parnaíba, Santa Filomena, Piauí State, Brazil; MZUSP 94090, 5, 25.9-43.2 mm, SL, rio Parnaíba, Santa Filomena, Piauí State, Brazil. 7, paratypes, 19.8-29.8 mm SL, rio das Mortes, between Água Boa and Cocalinho, Mato Grosso State, Brazil; LBP 4922, 5, 27.6-34.0 mm SL, rio das Mortes, Barra do Garças, Mato Grosso State, Brazil. Moenkhausia sanctaefilomenae: LBP 4695, 8, 25.1-61.3 mm SL, rio Paraná Basin, Marapoama, São Paulo State, Brazil; LBP 5541, 44, 31.6 - 47.1 mm SL, MCP 42016, 5, 32.6-45.6 mm SL, rio Parnaíba, Santa Filomena, Piauí State, Brazil; MZUSP 94090, 5, 25.9-43.2 mm, SL, rio Parnaíba, Santa Filomena, Piauí State, Brazil. sanctaefilomenae. Moenkhausia australe was, however, described by Eigenmann (1908) from two specimens collected at two different localities in Paraguay, Arroyo Trementina and Arroyo Chagalalina. Literature Cited Benine, R. C., R. M. C. Castro & A. C. A. Santos. 2007. A new Moenkhausia Eigenmann, 1903 (Characiformes: Characidae) from Chapada Diamantina, rio Paraguaçu basin, Bahia, Northeastern Brazil. Neotropical Ichthyology, 5(3): 259-262. g p p y Searches through the collections at MZUSP revealed that Moenkhausia forestii has been only recently collected in the upper portions of the rio Paraná system, most probably as a consequence of movement of the species through the “Piracema Channel” (a fish ladder constructed around Itaipu Hydroelectric). This fish ladder linked the middle and upper portions of rio Paraná, that were originally separated by the natural barrier formed by the Sete Quedas falls. Several other examples of similar establishment of species in the upper Paraná as a consequence of movement via this fish ladder are cited by Graça & Pavanelli (2007). Due to this secondary establishment, the specimens from rio Paraná drainage were included as non-type material. Non-types also includes dissected, alcohol-fixed, and poorly preserved specimens. Costa, W. J. E. M. 1994. Description of two new species of the genus Moenkhausia (Characiformes: Characidae) from the cen- tral Brazil. Zoologischer Anzeiger, 232: 21-29. Eigenmann, C. H. 1903. New genera of South America fresh-water fishes, and new names for some old genera. Smithsonian Miscellaneous Collection, 45: 144-148. Eigenmann, C. H. 1907. On further collections of fishes from Paraguay. Annals of the Carnegie Museum, 4: 108-157. Eigenmann, C. H. 1908. Preliminary descriptions of new genera and species of tetragonopterid characins. (Zoölogical Results of the Thayer Brazilian Expedition). Bulletin of the Museum of Comparative Zoology, 52: 91-106. Eigenmann, C. H. 1917. The American Characidae. Part I. Memoirs of the Museum of Comparative Zoology, 43: 1-102. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39: 783-791. Comparative material. Moenkhausia australe: CAS 70818, syntype, 38.9 mm SL, arroyo Trementina, rio Paraguay, Paraguay; CAS 70819, syntype, 29.7 mm SL, arroyo Chagalalina, rio Paraguay, Paraguay; MCP 25198, 5, 44.0-54.8 mm SL, rio Uruguay, Rio Grande do Sul State; MCP 27464, 5, 34.1-49.0 mm SL, rio Uruguay, Rio Grande do Sul State, Brazil. Moenkhausia diktyota: MZUSP 64107, 2,06 paratypes, 35.4-44.8 mm SL, rio Tiquié, Amazonas State, Brazil. Acknowledgements The authors are indebted to Osvaldo T. Oyakawa (MZUSP) for loan of material and curatorial assistance, to Dave Catania (CAS) and Zilda M. Lucena (MCP) for loan of material. Richard P. Vari (USNM) provided valuable suggestions and comments on various drafts of the manuscript. Waldo P. Troy provided specimens for molecular analysis. Guilherme A. M. Lopes assisted with Fig. 1. RCB and TCM are financially supported by FAPESP (proc. 06/00545-3; 06/04551-8, respectively) and CO by FAPESP and CNPq (proc. 306054/2006-0). This study is part of the FAPESP Thematic Project “Phylogenetic relationships in the Characidae (Ostariophysi: Characiformes) (proc. 04/09219-6). The observed values of genetic distances (Kimura, 1980) among species were 0.182 ± 0.019 between M. sanctaefilomenae and M. forestii, 0.183 ± 0.017 between M. oligolepis and M. forestii, and 0.191 ± 0.017 between M. sanctaefilomenae and M. oligolepis. These values are higher than those obtained in the similar analyses of within-genus COI sequences (0.0993) of marine fishes from Australia (Ward et al., 2005) and of freshwater fishes from Canada (Hubert et al., 2008) and from Mexico and Guatemala (Valdez-Moreno et al., 2009). The observed values of genetic distances within- species were 0.003 ± 0.001 for M. sanctaefilomenae, 0.004 ± 0.001 for M. forestii, and 0.092 ± 0.009 for M. oligolepis. This high value observed for M. oligolepis leads us to hypothesize that M. oligolepis still represents a species complex rather than a single species which will be explored in a further study. Discussion Those specimens were examined as part of this study and, contrary to Eigenmann (1917), these specimens do not have an incompletely pored lateral line and present a higher upper jaw length in comparison with Moenkhausia forestii (48.9-52.6% vs. 39.9-43.7% in HL, respectively), thus, M. australe is not equivalent to the new species described herein. Additional material from rio Uruguay (MCP 25198 and MCP 27464) with a complete lateral line has a comparable upper jaw length (47.2-52.4% in HL) with the syntypes of M. australe. Literature Cited Moenkhausia oligolepis: LBP 5365, 1, 37.3 mm SL, rio Jari, Laranjal do Jari, Amapá State, Brazil; LBP 4098, 9, 34.8- 47.7 mm SL, rio Japiim, rio Amazonas basin, Mâncio Lima, Acre State, Brazil; LBP 1498, 10, 46.1-65.1 mm SL, córrego Fundo, rio Araguaia basin, Barra do Garças, Mato Grosso State, Brazil; LBP 5073, 29, 39.0-52.5 mm SL, rio Paraguay Basin, Cáceres, Mato Grosso State, Brazil. Moenkhausia pyrophthalma: MZUSP 45290, Fink, W. L. & S. H. Weitzman. 1974. The so-called Cheirodontin fishes of Central America with descriptions of two new species (Pisces: Characidae). Smithsonian Contributions to Zoology, 172: 1-46. gy Graça, W. J. & C. S. Pavanelli. 2007. Peixes da planície de inundação do alto rio Paraná e áreas adjacentes. Maringá, EDUEM, 241p. Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41: 95-98. Hubert, N., R. Hanner, E. Holm, N. E. Mandrak, E. Taylor, M. Burridge, D. Watkinson, P. Dumont, A. Curry, P. Bentzen, J. Zhang, J. April & L. Bernatchez. 2008. Identifying canadian freshwater fishes through DNA barcodes. PLoS ONE, 3: e2490. Kimura, M. 1980. A simple method of estimating evolutionary rate of base substitutions through comparative studies of nucleotide New species of Moenkhausia and comments on the Moenkhausia oligolepis species complex New species of Moenkhausia and comments on the Moenkhausia oligolepis species complex 168 sequences. Journal of Molecular Evolution, 16: 111-120. sequences. Journal of Molecular Evolution, 16: 111-120. Kumar, S., K. Tamura & M. Nei. 2004. MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Briefings in Bioinformatics, 5: 150-163. Lima, F. C. T., H. A. Britski & F. A. Machado. 2007. A new Moenkhausia (Characiformes: Characidae) from central Brazil, with comments on the area relationship between the upper rio Tapajós and upper rio Paraguai systems. Aqua International Journal of Ichthyology, 13(2): 45-54. Lima, F. C. T., L. R. Malabarba, P. A. Buckup, J. F. P, Silva, R. P. Vari, A. Harold, R. Benine, O. T. Oyakawa, C. S. Pavanelli, N. A. Menezes, C. A. S. Lucena, R. E. Reis, F. Langeani, L. Casatti, V. A. Bertaco, C. R. Moreira & P. H. F. Lucinda. 2003. Genera Incertae Sedis in Characidae. Pp. 106-169. In: Reis, R. E., S. O. Kullander & C. J. Ferraris Jr. (Eds.). Check List of the Freshwater fishes of South and Central America. Porto Alegre, Edipucrs, 729p. Lima, F. C. Literature Cited T. & M. Toledo-Piza. 2001. New species of Moenkhausia (Characiformes: Characidae) from the rio Negro of Brazil. Copeia, 2004(4): 1058-1063. Lucinda, P. H. F., L. R. Malabarba & R. C. Benine. 2007. On a new species of the genus Moenkhausia Eigenmann (Ostariophysi: Characidae). Zootaxa, 1525: 61-68. Swofford, D. L. 2002. PAUP - Phylogenetic analysis using parsimony (*and other methods). Version 4b10. Sinauer, Sunderland. Swofford, D. L. & S. H. Berlocher. 1987. Inferring evolutionary trees from gene frequency data under the principle of maximum parsimony. Systematic Zoology, 36(3): 293-325. Taylor, W. R. & G. C. Van Dyke. 1985. Revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage. Cybium, 9(2): 107-119. Valdez-Moreno, M., N. V. Ivanova, M. Elias-Gutierrez, S. Contreras-Balderas & P. D. N. Hebert. 2009. Probing diversity in freshwater fishes from Mexico and Guatemala with DNA barcodes. Journal of Fish Biology, 74(2): 377-402. Ward, R. D., T. S. Zemlak, B. H. Innes, P. R. Last & P. D. N. Hebert. 2005. DNA barcoding Australia’s fish species. Philosophical Transactions of Royal Society, 359: 1847-1857. Xia, X. & Z. Xie. 2001. DAMBE: Data analysis in molecular biology and evolution. Journal of Heredity, 92: 371-373. Accepted April 2009 Published June 17, 2009 Accepted April 2009 Published June 17, 2009
https://openalex.org/W4307541367	https://zenodo.org/record/7263659/files/30-29-2022.pdf	English	null	Development of Interactive Multimedia Assisted by Virtual Laboratory for Critical and Creative Thinking Skills in Respiration and Excretion System Materials for Class XI High School Students	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	8,256	KEYWORDS: Critical and Creative, Interactive Multimedia, Virtual Laboratory KEYWORDS: Critical and Creative, Interactive Multimedia, Virtual Laboratory. www.ijcsrr.org www.ijcsrr.org Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 Development of Interactive Multimedia Assisted by Virtual Laboratory for Critical and Creative Thinking Skills in Respiration and Excretion System Materials for Class XI High School Students Tika Apriani Risdiyanto1, Syamsurizal2, Abdul Razak3, Violita4 1 Postgraduate Student of Biology Education FMIPA Padang State University 2 Lecturer of the Postgraduate Biology Education Program, Padang State University 3 Master of Biology Education, Padang State University, Padang, Indonesia 4 Jl. Prof. Dr. West Freshwater Hamka, Padang, Indonesia Tika Apriani Risdiyanto1, Syamsurizal2, Abdul Razak3, Violita4 1 Postgraduate Student of Biology Education FMIPA Padang State University 2 Lecturer of the Postgraduate Biology Education Program, Padang State University 3 Master of Biology Education, Padang State University, Padang, Indonesia 4 Jl. Prof. Dr. West Freshwater Hamka, Padang, Indonesia ABSTRACT: The 21st century learning summarizes students' thinking skills in four competencies, namely critical thinking skills, creative thinking, collaboration, and good communication. Teaching materials in schools should be able to help students in practicing thinking skills. This study aims to produce interactive multimedia assisted by a virtual laboratory for respiratory and excretory system materials in humans that can improve the critical and creative thinking skills of class XI students that are valid, practical, and effective. This type of research is development research with the Plomp modeldevelopment research which consists of three phases, namely the initial preliminary research phase, the development or prototyping phase, and the assessment phase. The research subjects are students of class XI SMAN 1 Sitiung Academic Year 2021-2022. The instrument in this study is a questionnaire of validity and practicality as well as a matter of effectiveness. The data analysis technique was validity, practicality using a Likert scale scoring and effectiveness using ANOVA using SPSS 21. The results showed interactive multimedia assisted by a virtual laboratory obtained very valid criteria according to expert review with a value of 81.26%. The practicality of the teacher is very practical with a value of 96.66% and the student response criteria are very practical with a value of 95.71%. The effectiveness test of interactive multimedia assisted by a virtual laboratory is effective because the experimental class shows an increase in critical thinking skills. Based on these results, it can be concluded that the interactive multimedia assisted by a virtual laboratory on the respiratory and excretory system materials developed in humans is valid, practical and effective. International Journal of Current Science Research and Review DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 Critical and creative thinking skills are the key to learning and should be applied to the 2013 curriculum, especially in learning biology. Biology learning today requires thinking skills to analyze and understand phenomena or problems related to biology concepts in everyday life (Sofnidar, 2012). Critical and creative thinking skills are interrelated skills. Critical and creative thinking skills are significantly related because critical thinking skills contribute to creative thinking skills (Mayarni, 2020). Based on the problem analysis conducted to the Biology teacher, students have difficulty understanding the learning material because the available teaching materials have not been able to maximize students' understanding of concepts and materials. Teaching materials in schools have not been able to visualize concepts and materials optimally. This problem has an impact on schools so that they do not maximize students' thinking skills which affect students' mindsets to be low (Sutama, 2014). The limitations of existing learning tools in schools and those owned by teachers are one of the factors that can determine the quality of learning. The required teaching materials must contain activity based so that students can be active in the learning process and interesting teaching materials so that they can visualize the material densely and clearly so that they can encourage the improvement of their critical and creative thinking skills. Based on the needs analysis at SMAN 1 Sitiung, students need media that can visualize the material briefly, densely and clearly so that it can help students to be active and assist in understanding learning. A total of 77.1% of students want the development of media, namely interactive multimedia assisted by the Virtual Laboratory (Appendix 6). The developed media is Interactive Multimedia assisted by Virtual Laboratory. Interactive Multimedia assisted by Virtual Laboratory which was developed contains respiration and excretion material and is assisted by practicum on each material. International Journal of Current Science Research and Review Based on the results of observations on 70 students, 75% of students (Appendix 6) still consider the respiration and excretion material difficult to understand and if there is no practice in the laboratory, it will be more difficult to understand the material, as many as 80.1% of students stated that learning resources only from textbooks and powerpoints made independently by the teacher (Appendix 6), so that students cannot get visual examples of the respiratory system and excretory system in humans without the help of pictures and are less able to visualize the material clearly. Based on the needs analysis, the media created by the teacher were sourced from textbooks and the internet. The developed media contains respiration and excretion material. The researcher chose the material for the respiratory system and the excretory system because this material is material that requires visual examples of the shape of the respiratory and excretory organs that must be shown to students so that they are easy to understand. There are many media made by teachers for KD 3.8 and KD 3.9 only. The concepts presented in the media made by the teacher have not facilitated and have not trained students' critical and creative thinking skills. Based on the questionnaire, it was found that 100% of the teachers stated that they had never measured or tested the thinking skills of students (Appendix 3), this was evidenced from the analysis test of students' thinking abilities with an average score of only 41, In this case the researchers chose SMAN 1 Sitiung as a place for observation, research and data collection because at SMAN 1 Sitiung there was no media developed using interactive multimedia assisted by a virtual laboratory, andthe results of observations at SMAN 1 Sitiung, it was proven that previously there had been no development of interactive multimedia learning media assisted by a virtual laboratory. This has encouraged researchers to conduct research on the development of interactive learning media on respiration and excretion material for critical and creative thinking of students in class XI SMAN 1 Sitiung on biology subjects supported by virtual laboratories as teaching materials in order to improve students' understanding in learning and be able to improve critical and creative thinking skills. 2. RESEARCH METHOD This type of research is design and development research. The development model used in the development of interactive multimedia assisted by a virtual laboratory is the Plomp development model. The Plomp development model consists of three stages, namely the preliminary research stage, the development or prototyping stage, and the assessment stage. The type of formative evaluation that will be used is described in the following details of the development and prototyping activities. 1. BACKGROUND OF THE PROBLEM 21st century learning summarizes students' thinking skills in four competencies, namely critical thinking skills, creative thinking, collaboration, and good communication. Higher order thinking ability is an ability that includes three aspects, namely analysis, evaluation, and creation in accordance with the implementation of the 2013 curriculum (Anazalia, et al). Thinking skills that play a role are critical and creative thinking skills. Students who are able to face global competition in the 21st century world of work are creative and critical students (Agnafia, 2019). Critical thinking skills should be owned by every student. Critical thinking skills are skills to train students in analyzing and identifying problems in depth to get brilliant new innovations. Critical thinking is important to develop because advances in information and global competition demand in analyzing phenomena or solving problems (Hayati, 2016). Creative thinking skills are skills that train students to develop ideas, imagination and increase sensitivity to phenomena. Creative thinking skills of students are very important in the learning process, thinking can affect the ability, speed and effectiveness of students' learning (Widiawati: 2019) Creative thinking skills are one of the skills that important for students who are applied in learning to enrich their knowledge and skills. Students who think creatively tend to prefer to export new ideas to solve problems (Fuad, 2015). 4043 Corresponding Author: Syamsurizal Volume 05 Issue 10 October 2022 olume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 Available at: ijcsrr.org Page No.-4043-4053 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.o 3. Prototype III The revised product will be assessed by the validator, and will be continued at the one to one evaluation stage. The one to one test was carried out by three students with different abilities, namely low, medium and high. Students provide suggestions in the form of input from aspects of content feasibility, language and graphics. Based on the comments given, students gave positive responses to the development of interactive multimedia assisted by a virtual laboratory. 2. Prototype II At this stage, consultation with experts or experts (expert review) is carried out to get interactive multimedia assisted by a valid virtual laboratory. Aspects assessed in the validity questionnaire are didactic, construct and technical. 1. Prototype I yp The design of Prototype I development starts from designing interactive multimedia storyboards. After that, it was continued by designing a systematic presentation of material and learning objectives to be achieved which were divided into 4044 Corresponding Author: Syamsurizal Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 Available at: ijcsrr.org Page No.-4043-4053 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org several learning activities and guided by KD 3.9 and KD 3.10 in the 2013 Curriculum. Creating interactive multimedia products using the Adobe Animate CC program. Entering the Prototype I stage, it conducts self-evaluation activities and this stage is developed based on several components, such as the feasibility of content, language and graphics. International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org several learning activities and guided by KD 3.9 and KD 3.10 in the 2013 Curriculum. Creating interactive multimedia products using the Adobe Animate CC program. Entering the Prototype I stage, it conducts self-evaluation activities and this stage is developed based on several components, such as the feasibility of content, language and graphics. several learning activities and guided by KD 3.9 and KD 3.10 in the 2013 Curriculum. Creating interactive multimedia products using the Adobe Animate CC program. Entering the Prototype I stage, it conducts self-evaluation activities and this stage is developed based on several components, such as the feasibility of content, language and graphics. 4. Prototype IV The development of prototype IV is the stage of conducting interactive multimedia practicality tests which were developed through small group evaluation activities. The small group evaluation was carried out by six students. Taking students based on the category of the ability level of students, namely low, medium and high, is known from interviews with teachers and students' scores. Each category consists of two students. 4045 Corresponding Author: Syamsurizal a. Prototype I . Development Results a. Prototype I . Development Results The design for the development of Prototype I begins with designing an interactive multimedia storyboard (Appendix 13). After that, it was continued by designing a systematic presentation of material and learning objectives to be achieved which were divided into several learning activities and guided by KD 3.9 and KD 3.10 in the 2013 Curriculum. Creating interactive multimedia products using the Adobe Animate CC program. The development of interactive multimedia products can be seen at the following link:https://bit.ly/3PxEaAx 3.1 Research Results The results obtained from the preliminary research stage are used as guidelines for developing interactive multimedia assisted by a virtual laboratory on the respiratory system and excretory system material. This interactive multimedia assisted virtual laboratory was created using the Adobe Animate CC program. The activities carried out at this stage of development are as follows. 1) Development and Prototyping Stage b. Prototype II Development Results yp p The development of interactive multimedia in prototype II is carried out by an expert review of the validity of which aims to see the feasibility of interactive multimedia assisted by a virtual laboratory which was developed based on didactic aspects, construct aspects, and technical aspects. The interactive multimedia validation was carried out by three experts consisting of media, material and language experts. Aspects assessed in the validity questionnaire are didactic, construct and technical. The didactic aspect includes the suitability of interactive multimedia developed with the 2013 revised 2017 curriculum and based on the learning needs of students. Aspects of the construct assessed for the suitability of interactive multimedia developed with criteria that must be met in the development of developed interactive multimedia, and the rules for using the Indonesian language. The technical aspects assessed were the suitability of writing, pictures, and graphics in the developed textbooks. The results of the validity of interactive multimedia can be seen in Table 11. Table1. Results of Data Analysis Validation of Three Validators No Aspect Score (%) Criteria 1 didactic 80.56 Valid 2 Construct 82.29 Very Valid 3 Technical 80.95 Valid Average Vlidity Value 81.26 Very Valid The results of the average validity value of 81.26% indicate that interactive multimedia assisted by a virtual laboratory on the respiratory system and excretory system materials developed in humans have very valid criteria based on aspects of the feasibility of content, language, presentation, and graphics. The results of the average validity value of 81.26% indicate that interactive multimedia assisted by a virtual laboratory on the respiratory system and excretory system materials developed in humans have very valid criteria based on aspects of the feasibility of content, language, presentation, and graphics. 4046 Corresponding Author: Syamsurizal link:https://bit.ly/3PxEaAx Entering the Prototype I stage, it conducts self-evaluation activities and this stage is developed based on several components, such as the feasibility of content, language and graphics. The design of prototype I includes the following things. Figure 1. Interactive Multimedia Cover Display assisted by virtual laboratory 4045 Corresponding Author: Syamsurizal Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 Figure 1. Interactive Multimedia Cover Display assisted by virtual laboratory Figure 1. Interactive Multimedia Cover Display assisted by virtual laboratory 4045 Corresponding Author: Syamsurizal Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 Volume 05 Issue 10 October 2022 4045 Corresponding Author: Syamsurizal Available at: ijcsrr.org Page No.-4043-4053 Volume 05 Issue 10 October 2022 d. Results of Development of Prototype IV The development of prototype IV is the stage of conducting interactive multimedia practicality tests which were developed through small group evaluation activities. The small group evaluation was carried out by six students. Taking students based on the category of the ability level of students, namely low, medium and high, is known from interviews with teachers and students' scores. Each category consists of two students. The results of the small group evaluation obtained can be seen in Table 12. Table2. Small Group Evaluation Results. No. Aspect Score (%) Criteria 1 Ease of Use 94.64 Very practical 2 Efficiency 92.86 Very practical 3 Attractiveness 100 Very practical 4 Easy to interpret 94.05 Very practical 5 Have Equivalence 100 Very practical Average Practicality Score 95.71 Very practical Table2. Small Group Evaluation Results. Based on the practicality test questionnaire, the results obtained were 95.71, it can be explained that interactive multimedia assisted by the virtual laboratory material for the respiratory system and excretory system in humans that was developed was considered very practical and could be used for class experiments and further tests. Students are also asked to provide suggestions for improving the developed multimedia. 4047 Corresponding Author: Syamsurizal International Journal of Current Science Research and Review DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 The average value of the One To One Evaluation questionnaire that has been filled out by students is 96.66% (Appendix 18). Based on these results, it can be described that the assessment of the aspects of component completeness, Content Feasibility, and Language is appropriate. While in the graphic aspect, there are several suggestions from students for revisions such as the presence of several buttons that do not work and images that are not in accordance with the material. c. Prototype III Development Results The revised product will be assessed by the validator, and will be continued at the one to one evaluation stage. The one to one test was carried out by three students with different abilities, namely low, medium and high. Students provide suggestions in the form of input from aspects of content feasibility, language and graphics. Based on the comments given, students gave positive responses to the development of interactive multimedia assisted by a virtual laboratory. p p y y The results of the accumulated One to One Evaluation Questionnaire on interactive multimedia that have been implemented can be seen in the image below: * Picture1. Questionnaire RatingOne To One 0 20 40 60 80 100 120 Kelengkapan komponen Kelayakan Isi Kebahasaan Kegrafikaan Peserta didik dengan kemampuan tinggi Peserta didik dengan kemampuan sedang Peserta didik dengan kemampuan rendah 4046 Corresponding Author: Syamsurizal 4046 Corresponding Author: Syamsurizal Volume 05 Issue 10 October 2022 Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 2) Assessment Stage The activities carried out at this stage of the assessment are multimedia assessments developed in actual conditions. Tests were carried out on two test classes, namely the experimental class and the control class. The purpose of the large group assessment is to determine the level of practicality and effectiveness of the developed multimedia. 1) The results of the Practicality Assessment by the Teacher 1) The results of the Practicality Assessment by the Teacher The practicality of computer-assisted interactive multimedia was also assessed by the teacher based on aspects of the use of multimedia in the classroom. The teacher who assesses is a Biology teacher who teaches in the experimental class for the 2021/2022 academic year. The teacher assesses the practicality of multimedia by filling out the questionnaire that has been provided. ResultsThe questionnaire filled out by the teacher is as follows. 4047 Corresponding Author: Syamsurizal Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 Table3. Practical Assessment by Teacher No. Aspect Score (%) Criteria 1 Ease of Use 91.67 Very practical 2 Efficiency 100.00 Very practical 3 Attractiveness 100.00 Very practical 4 Easy to interpret 91.67 Very practical 5 Have Equivalence 100 Very practical Average Practicality Score 96.66 Very practical Table3. Practical Assessment by Teacher No. Aspect Score (%) Criteria 1 Ease of Use 91.67 Very practical 2 Efficiency 100.00 Very practical 3 Attractiveness 100.00 Very practical 4 Easy to interpret 91.67 Very practical 5 Have Equivalence 100 Very practical Average Practicality Score 96.66 Very practical 4048 Corresponding Author: Syamsurizal Table4. Results of Field Test Evaluation. Based on the table above, it is known that the average practicality value of interactive multimedia assisted by virtual laboratories filled by experimental class students is 94.57% categorized as very practical to use (Appendix 24). Based on the table above, it is known that the average practicality value of interactive multimedia assisted by virtual laboratories filled by experimental class students is 94.57% categorized as very practical to use (Appendix 24). Table3. Practical Assessment by Teacher International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org International Journal of Current Science Research and Review Based on the table above, it is known that the average practicality value of interactive multimedia assisted by virtual laboratories filled by teachers is 96.66% (Appendix 20) with very practical criteria. 1) Results of Practicality Assessment by Students 1) Results of Practicality Assessment by Students Interactive multimedia assisted by a virtual laboratory that has been assessed as practical by small groups is revised and tested in the experimental class. Furthermore, at the end of the meeting, students provide an assessment of the interactive multimedia developed. The questionnaire given is a practical field test evaluation questionnaire. This test is called the large group test. The results of the field test evaluation can be seen in the table below. The results of the field test evaluation can be seen in the table below. Table4. Results of Field Test Evaluation. Table4. Results of Field Test Evaluation. No. Aspect Score (%) Criteria 1 Ease of Use 96.10 Very practical 2 Efficiency 92.49 Very practical 3 Attractiveness 92.49 Very practical 4 Easy to interpret 94.16 Very practical 5 Have Equivalence 89.99 Very practical Average Practicality Score 94.57 Very practical International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org thinking ability data had a significance of > 0.05, which means that all data were homogeneous. Data that have been normal and homogeneous, then tested the hypothesis using ANOVA. The results of the hypothesis test of critical and creative thinking skills in the experimental and control classes can be seen in the table. Table6. Results of ANOVA Calculation of Critical and Creative Thinking Skills. Skills Think Class Significance Information Critical Experiment 0.000 Hypothesis Received Control Creative Experiment 0.000 Hypothesis Accepted Control Table6. Results of ANOVA Calculation of Critical and Creative Thinking Skills. Based on the table above, it is known that the significance value of students' critical thinking skills is 0.000 and creative thinking skills is 0.000. This indicates that the value of sig. < 0.05, which means that the hypothesis is accepted. Furthermore, further tests were carried out, namely the bonferoni test. Bonferoni further test results can be seen in Based on the table above, it is known that the significance value of students' critical thinking skills is 0.000 and creative thinking skills is 0.000. This indicates that the value of sig. < 0.05, which means that the hypothesis is accepted. Furthermore, further tests were carried out, namely the bonferoni test. Bonferoni further test results can be seen in Table7. Bonferoni further test results Group Group Average Test Score ( X ) level Sig. Experimental Critical Critical Control 71.09* .000 Creative Experiment 83.23* .000 Creative Control 69.25* .000 Critical Control Experimental Critical 71.09* .000 Creative Experiment 71.63* .000 Creative Control 57.65 .884 Creative Experiment Experimental Critical 83.23* .000 Critical Control 71.63* .000 Creative Control 69.79* .000 Creative Control Experimental Critical 69.25* .000 Critical Control 57.65 .884 Creative Experiment 69.79* .000 Note: * = significant Based on the Bonferroni further test, the experimental critical group was significantly different from the critical control group, the creative experimental group, and the creative control group. While the critical control group was not significantly different from the creative control. Thus, it is known that the use of interactive multimedia assisted by a virtual laboratory has an effect on improving the critical and creative thinking skills of class XI students of SMAN 1 Sitiung. 2) Effectiveness Test The results of the homogeneity test of the critical and creative values of the control and experimental classes showed that the critical and creative 4048 Corresponding Author: Syamsurizal Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org thinking ability data had a significance of > 0.05, which means that all data were homogeneous. Data that have been normal and homogeneous, then tested the hypothesis using ANOVA. The results of the hypothesis test of critical and creative thinking skills in the experimental and control classes can be seen in the table. International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org thinking ability data had a significance of > 0.05, which means that all data were homogeneous. Data that have been normal and homogeneous, then tested the hypothesis using ANOVA. The results of the hypothesis test of critical and creative thinking skills in International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org 2) Effectiveness Test ) The results of the effectiveness test obtained in the form of learning outcomes data. The learning outcomes include students' critical and creative thinking skills. Data on critical and creative thinking skills were obtained from the learning outcomes test at the end of the lesson. The learning process in the experimental class uses interactive multimedia assisted by a virtual laboratory. While the learning process in the control class uses teaching materials commonly used by teachers in schools. Learning outcomes data were obtained through learning outcomes tests conducted at the last meeting. The test is given in the form of essay questions. Data on the assessment of students' critical and creative thinking skills are presented in the table below. Table5. Assessment of the Results of Students' Critical and Creative Thinking Skills Thinking Skills Class N Test Average Critical Experiment 35 82.69 Control 32 59.5 Creative Experiment 35 83.77 Control 32 55.81 le5. Assessment of the Results of Students' Critical and Creative Thinking Skills Based on the table above, it can be seen that the average critical and creative thinking skills of the experimental class students are higher than the control class average. The experimental class is a class that is given treatment in the form of interactive multimedia assisted by a virtual laboratory on the respiratory system material in humans, while the control class is a class without treatment. The average value of critical and creative thinking skills in the experimental class is 82.69 and 83.77 (Appendix 35). Meanwhile, the average value of critical and creative thinking skills in the control class is 59.5 and 55.81 (Appendix 36). The next stage is hypothesis testing, but first the normality test and homogeneity test are carried out as prerequisites for data analysis. Prerequisite tests carried out are normality test and homogeneity test. Researchers conducted a normality test using the Kolmogorov-Smirnov test. The results of the normality test of the critical and creative thinking ability of the control and experimental classes resulted that all critical and creative values of the control class, experimental significance > 0.05, meaning that the data was normally distributed. Furthermore, the homogeneity of the data was tested using Levene's test. 3.3 Discussion The prototype development stage directs researchers to create valid and practical interactive multimedia. The steps taken are interactive multimedia must be valid in terms of content, construct and technical. Validation is the accuracy, meaning, and usefulness of something made by researchers (Lufri, 2015). This stage consists of self-evaluation, expert review, one-to-one evaluation, small group evaluation, and field test. This evaluation is carried out to find out whether the problems that occur in the school can be resolved properly. The first stage is self-evaluation, which is self-assessment of the instrument prototype by checking itself about the construct, language and content, whether it is appropriate and correct, after it is deemed sufficient to proceed to the next evaluation stage (Agustine, 2014). Researchers evaluate themselves to see whether the components contained in virtual-assisted interactive Multimedialaboratorydeveloped has been fulfilled or not through filling out a self-evaluation questionnaire. The results of the self- evaluation carried out by researchers showed that the interactive multimedia developed had met the completeness of interactive multimedia with the intro page, login, main menu, user manual, to profile, then the material presented was in accordance with the 2013 curriculum, the presentation of the material was in accordance with KD 3.9 and KD 3.10 as well as the use of good and correct Indonesian and pictures that have been equipped with descriptions. The next stage is validation which is done by expert review. Validation Interactive multimedia assisted by a virtual laboratory is validated by three experts or experts. The validity of the interactive multimedia developed is assessed by experts or experts based on 3 aspects, namely didactic aspects, construct aspects and technical aspects. The validity of interactive multimedia assisted by a virtual laboratory conducted by an expert review showed that it was very valid overall with an average validity value of 81.27%. The description of the average value of validity is from the didactic aspect of 80.56% with very valid criteria, the construct aspect of 82.29% with very valid criteria and 80.95% technical aspects with valid criteria. The validator provides suggestions, criticisms, Based on the criteria in the didactic aspect, it is known that the multimedia developed is in accordance with the 2013 curriculum where there is a suitability of the material with indicators of competency achievement, the suitability of the material with learning objectives and the material is arranged systematically. 3.2. Relationship between Critical and Creative Thinking Skills This study uses data regression analysis of variables X and Y, where variable X is critical thinking and variable Y is creative thinking. This was done in order to see whether there was a relationship between critical thinking skills and creative students in the experimental class. The results of the research conducted were based on the test scores of students' critical and creative thinking skills. The results of the analysis of critical and creative thinking skills in the experimental class can be seen in the table below: 4049 Corresponding Author: Syamsurizal Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 4049 Corresponding Author: Syamsurizal Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 Volume 05 Issue 10 October 2022 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org Table8. The Result of the Analysis of the Relationship between Critical and Creative Thinking Skills in the Experimental Class Variable R R-square F Sig. Critical (X) independent 0.876 76.7% 108,487 0.000 Creative (Y) dependent International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org Table8. The Result of the Analysis of the Relationship between Critical and Creative Thinking Skills in the Experimental Class International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org Table8. The Result of the Analysis of the Relationship between Critical and Creative Thinking Skills in the Experimental Class Variable R R-square F Sig. Critical (X) independent 0.876 76.7% 108,487 0.000 Creative (Y) dependent Based on the table above, it can be seen that the critical variable is the independent variable and the creative variable is the dependent variable. Significance 0.000 <0.05 this means that there is a relationship between variance which indicates that the relationship between the dependent and independent variables is strong. The correlation coefficient value is 0.876. While the coefficient of determination (R-square) is 76.7%. 4050 Corresponding Author: Syamsurizal International Journal of Current Science Research and Review DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 The next aspect is the technical aspect which has very valid criteria by the validator with a value of 80.95%. According to (Trianto, 2011), the terms or criteria for technical aspects are related to writing, images and appearance in the manufacture of a product. Based on the criteria on the technical aspect, it can be stated that the interactive multimedia developed uses the type and size of letters that can be read clearly and uses appropriate punctuation marks. The presentation of images in interactive multimedia has a size and explanation that can be read clearly. According to (Sudjana, 2011), picture illustrations and animations can help students understand and remember the accompanying material. Overall interactive multimedia developed has very valid criteria, so it can be tested for the next test stage. (Arikunto, 2009) which also explains the data generated from a product that is already valid, it can be said that the product has provided an overview of the development objectives correctly and in accordance with the actual reality and circumstances. Based on the results of the one- to-one evaluation, it was found that the interactive multimedia of the respiratory system and excretory system in humans that was developed received a positive response by students. Students in the one to one evaluation stated that the interactive multimedia developed had good writing, grammar, pictures and displays to help students understand the material. The stage carried out after the one to one evaluation is the multimedia practicality stage. Practicality testing is carried out with two techniques, namely field trials and teacher assessments (Setiawan H, 2017). The first practical assessment was carried out in a small group. Students are selected based on low, medium and high ability levels. Each level of ability consists of two students. The next assessment is an assessment in a large group or field test consisting of 35 students and an assessment by one biology teacher. Practicality of interactive multimedia assisted by virtual laboratory is seen from the aspect of ease of use, efficiency of interactive multimedia, attractiveness, ease of interpretation and equivalence with existing learning resources. Small group evaluation which has very practical criteria with a value of 95.71%. The results of the large group practicality (field group evaluation) also have very practical criteria with a value of 94.57%. International Journal of Current Science Research and Review Furthermore, the results of the assessment by the teacher have very practical criteria with a value of 96.66%. The interactive multimedia that has been developed provides convenience in terms of use, both in the presentation of material using language that is easy to understand and also in clear font sizes. This interactive multimedia is also supported by images and animations, where from the images and animations students can focus and be interested in understanding the material so that it affects the level of understanding of the students' material. On the aspect of ease of use of interactive multimedia in accordance with the available time so that it does not interfere with other learning. Practicality can be seen from the implementation time which should be short, fast and precise. Then, the attractiveness aspect has very practical criteria (Sukardi, 2012). 4051 Corresponding Author: Syamsurizal 3.3 Discussion The teaching materials developed must be in accordance with the applicable curriculum (Wiyanto, 208). The problems given in interactive multimedia assisted by a virtual laboratory require students to provide problem solving solutions by working together. According to (Bengi, 2015) states that at this stage students participate actively to create solutions to everyday problems that students often encounter, the solutions expressed are very innovative and this is a learning experience for students. At this stage to train students' creative thinking skills. The assessment of the construct aspect has very valid criteria by the validator with a value of 82.29%. interactive multimedia that has been developed has a clear identity and is systematically arranged, interactive multimedia is equipped with learning outcomes, the description of material in interactive multimedia is clear. Based on the use of language, the interactive multimedia developed has been in accordance with the rules of good and correct Indonesian and the use of appropriate terms. According to (Hamdani, 2011), language is one aspect that needs to be considered in the preparation of teaching materials and the language used should be simple and easy to understand. The opinion (Zulyusri S, 2019) states that the language used in interactive multimedia must be good, clear and does not cause confusion, 4050 Corresponding Author: Syamsurizal Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 4050 Corresponding Author: Syamsurizal Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org Relationship between Students' Critical and Creative Thinking Skills Critical and creative thinking are abilities that must be possessed by someone where these thinking abilities are two sides that cannot be separated and become the goals of national education. Both are said to be a necessary skill in life either alone or in groups. Based on research data, critical thinking skills have a relationship with students' creative thinking skills. The correlation/relationship between variables X and Y is 0.876 which indicates that the relationship between critical and creative variables is strong. Critical thinking skills affect 76.7% of the creative thinking skills of experimental class students. The relationship between critical and creative thinking skills is in line with (Treffinger DJ, 2007) that critical thinking skills and creative thinking skills are interconnected in producing an effective thought in solving a problem. The results of the study (Gunawan, 2014) also concluded that critical and creative thinking skills are simultaneously and significantly related to student learning achievement. Critical and creative thinking is needed to support the development of the 21st century. Critical and creative thinking plays an important role in the development of education and has a main goal in learning where there are four important components to build these abilities, namely (a) explaining and clarifying; (b) ask appropriate questions to clarify or challenge; (c) consider the credibility of the source; (d) problem solving and drawing conclusions (Iakovos, 2011). The ability to think critically and creatively is very necessary considering that today's science and technology is developing very rapidly and allows anyone to obtain information quickly and easily in abundance from various sources and any place. This has resulted in rapid changes in the order of life and global changes in life. If they are not equipped with critical and creative thinking skills, they will not be able to process, assess and retrieve the information needed to face these challenges. The ability to think critically and creatively is an important ability in life. Critical and creative thinking skills can be trained through learning that requires students to explore, inquiry, discover and solve problems. Learning that can lead students to develop and or overcome critical and creative thinking can be assumed by using a problem-based learning model which requires students to be active in the learning process and have the opportunity to find and or apply their own ideas. 4052 Corresponding Author: Syamsurizal Relationship between Students' Critical and Creative Thinking Skills Problem-based learning is an innovation in learning because in this learning students' thinking abilities are truly optimized through a systematic group or team work process, so that students can empower, hone, test, and develop their thinking skills on an ongoing basis. International Journal of Current Science Research and Review DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 Based on the results of the hypothesis test presented in CHAPTER IV, the results obtained are that there are significant differences in the critical and creative thinking skills of students in the experimental class and the control class. The results for critical thinking in the experimental and control classes obtained p-value = 0.000 < (α = 0.05). The results for creative thinking in the experimental and control classes obtained p-value = 0.001 < (α = 0.05). The hypothesis is accepted if the significant value is <0.05 on the ANOVA test. The treatment in the experimental class used interactive multimedia assisted by a virtual laboratory, while the control class used teaching materials commonly used by teachers. Increasing the results of students' critical thinking skills is closely related to the learning process carried out. This is in accordance with Hanafi and Samsudin (2012) who stated that interactive skills, easy access, and fun are some of the advantages provided by electronic learning materials. In addition, creative thinking is also integrated where students are directed to be able to express ideas or ideas in more detail, clearly, and differently than in general. Students think in groups to relate problems to interactive multimedia by conducting literature studies so that problems can be solved optimally. and different than usual. Students think in groups to relate problems to interactive multimedia by conducting literature studies so that problems can be solved optimally. and different than usual. Students think in groups to relate problems to interactive multimedia by conducting literature studies so that problems can be solved optimally. Based on the average value of the p test obtained by students and based on the results of research, interactive multimedia assisted by virtual laboratories is able to improve students' critical and creative thinking skills. In line with the research of Arda et al. (2015) which states that learning science with interactive learning materials is better than using conventional learning strategies, and being able to improve understanding of concepts also makes the learning process more interesting. Therefore, it can be concluded that interactive multimedia assisted by a virtual laboratory based on research results can improve students' critical and creative thinking skills. 1. Interactive multimedia has very valid, very practical and effective criteria. 1. Assessment Stage The assessment phase aims to determine the quality of interactive multimedia assisted by a virtual laboratory that was developed. The effectiveness of interactive multimedia assisted by a virtual laboratory that was developed can be seen from the students' critical and creative thinking skills obtained after students use interactive multimedia in the learning process. Product effectiveness is a product quality criterion developed based on the presence or absence of influence on users (Nieveen, 2007). The results of the effectiveness assessment show that the developed interactive multimedia is effectively used as teaching material in the learning process. Students' critical and creative thinking skills are assessed through daily tests of the human respiratory system. The instrument used in the form of essay questions as many as 6 items. Testing the effectiveness of interactive multimedia was carried out using the ANOVA test, because the data were normally distributed and homogeneous. Based on the ANOVA test, it was found that the hypothesis was accepted. Furthermore, the Bonferroni test was carried out to see the difference in the value of each item. Based on the Bonferroni test, it is known that the critical and creative thinking ability test scores in the experimental class are higher than the control class. Therefore, it indicates that the use of interactive multimedia assisted by a virtual laboratory on the respiratory system and excretory system materials in humans is effective for learning. Researchers continue to strive for an effective learning process using interactive multimedia assisted by a virtual laboratory. In line with the opinion (Zuriah, Based on the prerequisite test, namely the normality test, all values in the experimental and control classes were normally distributed and all data were homogeneous. This means that the test value has met the prerequisites and is continued with hypothesis testing. Achievements in training students' critical and creative thinking skills can be proven by the average score of each aspect of students' critical and creative thinking skills in both classes. 4051 Corresponding Author: Syamsurizal Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 International Journal of Current Science Research and R ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 International Journal of Current Science Research and Review w www.ijcsrr.org 2. The use of interactive multimedia can empower students' critical and creative thinking skills. 3. Critical thinking skills are related to creative thinking skills after students use interactive multimedia 3. Critical thinking skills are related to creative thinking skills after students use interactive multimedia REFERENCES 9. Widiawati., Sa'diatul F., Syamsurizal,. Ardi. (2019). Analysis of the Creative Thinking Ability of Class VII Students in Biology Learning at SMPN 25 Padang. Biology Education Atria. 4(4). 4. CONCLUSION 4052 Corresponding Author: Syamsurizal Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 4. CONCLUSION Based on the development of interactive multimedia assisted by a virtual laboratory for class XI students of SMAN 1 Sitiung, the following conclusions were obtained. 1. Interactive multimedia has very valid, very practical and effective criteria. Based on the development of interactive multimedia assisted by a virtual laboratory for class XI students of SMAN 1 Sitiung, the following conclusions were obtained. 1. Interactive multimedia has very valid, very practical and effective criteria. 1. Interactive multimedia has very valid, very practical and effective criteria. Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 4052 Corresponding Author: Syamsurizal International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 10 October 2022 DOI: 10.47191/ijcsrr/V5-i10-30, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org 6. Mayarni. (2020). The Relationship between Critical Thinking Ability and Students' Creative Thi Ecological Materials. Pendipa Journal of Science Education. 4(3) , 39-45. Cite this Article: Tika Apriani Risdiyanto, Syamsurizal, Abdul Razak, Violita (2022). Development of Interactive Multimedia Assisted by Virtual Laboratory for Critical and Creative Thinking Skills in Respiration and Excretion System Materials for Class XI High School Students. International Journal of Current Science Research and Review, 5(10), 4043-4053 REFERENCES 1. Agnafia, DN (2019). Analysis of Students' Critical Thinking Ability in Biology Learning. Florea.6(1), 45-5 1. Agnafia, DN (2019). Analysis of Students' Critical Thinking Ability in Biology Learning. Florea.6(1), 45-53. 3. Anazalia, M. Darussyamsu. R., Lufri., Syamsurizal and Yusni, A. (2020). The validity of the Higher Order Thinking Test Instrument (HOTS) on Digestive System Materials for Students of Class Xi SMA/MA. Biotic Journal. Vol.8 : 232- 244 3. Anazalia, M. Darussyamsu. R., Lufri., Syamsurizal and Yusni, A. (2020). The validity of the Higher Order Thinking Test Instrument (HOTS) on Digestive System Materials for Students of Class Xi SMA/MA. Biotic Journal. Vol.8 : 232- 244 4. Farah. A., Syamsurizal., Lufro., Fitri. A. (2022). Content Validation of Critical Thinking Ability Test Questions for Class XII High School Students on Biotechnology Materials. Journal of On Teacher Education. 4(1): 348-355 5. Husein S., Herayanti L., & Gunawan (2015) The Effect of Using Interactive Multimedia on Mastery of Concepts and Critical Thinking Skills of Students on Temperature and Heat Material. Physics and Technology Education.1(3). 5. Husein S., Herayanti L., & Gunawan (2015) The Effect of Using Interactive Multimedia on Mastery of Concepts and Critical Thinking Skills of Students on Temperature and Heat Material. Physics and Technology Education.1(3). 6. Mayarni. (2020). The Relationship between Critical Thinking Ability and Students' Creative Thinking Ability on Ecological Materials. Pendipa Journal of Science Education. 4(3) , 39-45. 6. Mayarni. (2020). The Relationship between Critical Thinking Ability and Students' Creative Thinking Ability on Ecological Materials. Pendipa Journal of Science Education. 4(3) , 39-45. 7. Sofnidar. (2012). Development of Mathematics Education Teaching Materials I with Contextual Approach. Edumatica. 2(2), 57-67. 7. Sofnidar. (2012). Development of Mathematics Education Teaching Materials I with Contextual Approach. Edumatica. 2(2), 57-67. 8. Wati NN., Leny, & Saadi P (2017) improve creative thinking skills and learning outcomes through the creative problem solving learning model assisted by interactive multimedia on buffer solution material at SMAN 4 Banjarmasin. Journal of Chemistry and Education. 1(1). 9. Widiawati., Sa'diatul F., Syamsurizal,. Ardi. (2019). Analysis of the Creative Thinking Ability of Class VII Students in Biology Learning at SMPN 25 Padang. Biology Education Atria. 4(4). Cite this Article: Tika Apriani Risdiyanto, Syamsurizal, Abdul Razak, Violita (2022). Development of Interactive Multimedia Assisted by Virtual Laboratory for Critical and Creative Thinking Skills in Respiration and Excretion System Materials for Class XI High School Students. 5. Husein S., Herayanti L., & Gunawan (2015) The Effect of Using Interactive Multimedia on Mastery Critical Thinking Skills of Students on Temperature and Heat Material. Physics and Technology Edu 8. Wati NN., Leny, & Saadi P (2017) improve creative thinking skills and learning outcomes through the creative problem solving learning model assisted by interactive multimedia on buffer solution material at SMAN 4 Banjarmasin. Journal of Chemistry and Education. 1(1). REFERENCES International Journal of Current Science Research and Review, 5(10), 4043-4053 4053 Corresponding Author: Syamsurizal Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 Cite this Article: Tika Apriani Risdiyanto, Syamsurizal, Abdul Razak, Violita (2022). Development of Interactive Multimedia Assisted by Virtual Laboratory for Critical and Creative Thinking Skills in Respiration and Excretion System Materials for Class XI High School Students. International Journal of Current Science Research and Review, 5(10), 4043-4053 Cite this Article: Tika Apriani Risdiyanto, Syamsurizal, Abdul Razak, Violita (2022). Development of Interactive Multimedia Assisted by Virtual Laboratory for Critical and Creative Thinking Skills in Respiration and Excretion System Materials for Class XI High School Students. International Journal of Current Science Research and Review, 5(10), 4043-4053 4053 *Corresponding Author: Syamsurizal Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053 Volume 05 Issue 10 October 2022 Volume 05 Issue 10 October 2022 Available at: ijcsrr.org Page No.-4043-4053
https://openalex.org/W4225278494	https://www.researchsquare.com/article/rs-1458211/latest.pdf	English	null	Ultra-strong comprehensive radiation effect tolerance in carbon nanotube electronics	Research Square (Research Square)	2,022	cc-by	10,286	Ultra-strong comprehensive radiation effect tolerance in carbon nanotube electronics Zhiyong Zhang (  zyzhang@pku.edu.cn ) Peking University https://orcid.org/0000-0003-1622-3447 Maguang Zhu Peking University https://orcid.org/0000-0001-7037-0136 Peng Lu Institute of Microelectronics, Chinese Academy of Sciences Xuan Wang National Space Science Center, Chinese Academy of Sciences Chen Qian National Space Science Center, Chinese Academy of Sciences Huiping Zhu Institute of Microelectronics, Chinese Academy of Sciences Yajie Zhang Peking university Jianshuo Zhou Peking University Haitao Xu Beijing Institute of Carbon-based Integrated Circuits Zhengsheng Han Institute of Microelectronics, Chinese Academy of Sciences Jianwei Han National Space Science Center, Chinese Academy of Sciences Rui Chen National Space Science Center, Chinese Academy of Sciences Bo Li Institute of Microelectronics, Chinese Academy of Sciences Lian-Mao Peng Peking University https://orcid org/0000-0003-0754-074X Ultra-strong comprehensive radiat tolerance in carbon nanotube elec Zhiyong Zhang (  zyzhang@pku.edu.cn ) Peking University https://orcid.org/0000-0003-1622-3447 Maguang Zhu Peking University https://orcid.org/0000-0001-7037-0136 Peng Lu Institute of Microelectronics, Chinese Academy of Sciences Xuan Wang National Space Science Center, Chinese Academy of Sciences Chen Qian National Space Science Center, Chinese Academy of Sciences Huiping Zhu Institute of Microelectronics, Chinese Academy of Sciences Yajie Zhang Peking university Jianshuo Zhou Peking University Haitao Xu Beijing Institute of Carbon-based Integrated Circuits Zhengsheng Han Institute of Microelectronics, Chinese Academy of Sciences Jianwei Han National Space Science Center, Chinese Academy of Sciences Rui Chen National Space Science Center, Chinese Academy of Sciences Bo Li Institute of Microelectronics, Chinese Academy of Sciences Lian-Mao Peng Peking University https://orcid.org/0000-0003-0754-074X Ultra-strong comprehensive radiation effect tolerance in carbon nanotube electronics Article Keywords: Carbon nanotube, radiation tolerance, single event effect, displacement damage, total ionizing dose Keywords: Carbon nanotube, radiation tolerance, single event effect, displacement damage, total ionizing dose Posted Date: May 2nd, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1458211/v1 carbon nanotube electronics Maguang Zhu1, †, Peng Lu2, †, Xuan Wang3,4, †, Chen Qian3,4, Huiping Zhu2, Yajie Zhang1, Jianshuo Zhou1, Haitao Xu5, Zhengsheng Han2,4, Jianwei Han3,4, Rui Chen3,4, Bo Li2, Lian-Mao Peng1,5 and Zhiyong Zhang1,5* 1 Key Laboratory for the Physics and Chemistry of Nanodevices and Center for Carbon-based Electronics, School of Electronics, Peking University, Beijing 100871, China. 1 Key Laboratory for the Physics and Chemistry of Nanodevices and Center for Carbon-based Electronics, School of Electronics, Peking University, Beijing 100871, China. 1 Key Laboratory for the Physics and Chemistry of Nanodevices and Center for Carbon-based Electronics, School of Electronics, Peking University, Beijing 100871, China. 2 Institute of Microelectronics, Chinese Academy of Sciences, Beijing 100029, China. 3 National Space Science Center, Chinese Academy of Sciences, Beijing, China. 4 University of Chinese Academy of Sciences, Beijing, China. 4 University of Chinese Academy of Sciences, Beijing, China. 4 University of Chinese Academy of Sciences, Beijing, China. 5 Beijing Institute of Carbon-based Integrated Circuits, Beijing, China. 5 Beijing Institute of Carbon-based Integrated Circuits, Beijing, China. † These authors contributed equally to this work. These authors contributed equally to this work. † These authors contributed equally to this work. Correspondence to: (R. C.) chenrui2010@nssc.ac.cn, (B. L.) libo3@ime.ac.cn,and (Z. Z.) zyzhang@pku.edu.cn. Correspondence to: (R. C.) chenrui2010@nssc.ac.cn, (B. L.) libo3@ime.ac.cn,and (Z. Z.) zyzhang@pku.edu.cn. DOI: https://doi.org/10.21203/rs.3.rs-1458211/v1 DOI: https://doi.org/10.21203/rs.3.rs-1458211/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License ABSTRACT Carbon nanotube (CNT) field-effect transistors (FETs) have been considered ideal building blocks for radiation-hardened integrated circuits (ICs), the demand for which is exponentially growing, especially in outer space exploration and the nuclear industry. Many studies on the radiation tolerance of CNT-based electronics have focused on the total ionizing dose (TID) effect, while few works have considered the single event effects (SEEs) and displacement damage (DD) effect, which are more difficult to measure but may be more important in practical applications. We first executed measurements of the SEEs and DD effect of CNT FETs and ICs and then presented a comprehensive radiation effect analysis of CNT electronics. The CNT ICs without special irradiation reinforcement technology exhibit a comprehensive radiation tolerance, including a 1 × 104 MeV∙cm2/mg level of the laser-equivalent threshold linear energy transfer (LET) for SEEs, 2.8×1013 MeV/g for DD and 2 Mrad (Si) for TID, which are at least 4 times higher than those in conventional radiation-hardened ICs. The ultrahigh intrinsic comprehensive radiation tolerance will promote the applications of CNT ICs in high-energy solar and cosmic radiation environments. Keywords: Carbon nanotube, radiation tolerance, single event effect, displacement damage, total ionizing dose Benefitting from the ultrathin body, high carrier mobility and high saturation velocity, carbon nanotubes (CNTs) have been demonstrated to be an excellent channel to construct ultra-scaled field-effect transistors (FETs) with high performance and low power dissipation1-4. Furthermore, CNT-based electronics can provide complementary metal-oxide-semiconductor (CMOS) transistors containing symmetric p- and n-type characteristics through a simple and well-compatible manufacturing process5 and have been considered promising candidates for integrated circuits (ICs), which act as the physical basis of modern information technology6 but are encountering development bottlenecks arising from the physics, power dissipation and manufacturing costs7. Subject to the immature technologies of wafer-scale material preparation, transistor fabrication and interconnection processes, the developing CNT electronics are thus far unable to meet the requirements of mainstream digital ICs, which contain tens of billions of transistors in one chip7. To promote the utility of CNT electronics as soon as possible, many researchers have explored some special purpose applications of CNT- based ICs that can take full advantage of CNT FETs while avoiding the integration density requirement. ABSTRACT As the most popular special purpose ICs, ICs radiation-hardened to high-energy solar and cosmic radiation have received increasing attention with the increasing ambition of humankind to explore outer space, ranging from launching spacecrafts to establishing space stations and exploring distant celestial objects such as the Moon or Mars. CNT FETs and ICs have shown high radiation tolerance because of the strong C-C bonds, nanoscale cross-sections, low atomic number and negligible substrate parasitic effect from CNT channels, and their first application in radiation- hardened electronics is highly expected8,9. Furthermore, two additional advantages, i.e., energy efficiency and low-temperature stability, enable CNT transistors to further meet the strict and harsh requirements for ICs used in deep space exploration10-13. Recently, great advances in radiation-hardened CNT ICs have been achieved, but these works mainly focused on the total ionizing dose (TID) effect8,9,14, which is not sufficient to characterize the comprehensive radiation tolerance performance of an IC in the real space radiation environment. Compared to the TID effect, single event effects (SEEs)15 and the displacement damage (DD) effect16 are at least equally important in practical applications but are rarely studied in CNT-based transistors and ICs because they are more difficult to measure due to the challenges from the requirements on irradiation source as well as the complex circuits and electromagnetic environment. Moreover, the radiation tolerance to the TID effect increases with shrinkage of the pitch size of transistors17, which makes the radiation tolerance to SEEs become the bottleneck of the radiation performance of ICs18. To completely evaluate the radiation tolerance properties of CNT ICs, the comprehensive radiation effects, including TID, SEEs and DD, must be studied. Here, we first executed measurements of SEEs and the DD effect of CNT FETs and ICs by using a pulse laser as the SEE irradiation source and high-energy Xe+ ions as the DD irradiation source and then presented a comprehensive radiation effect analysis of CNT electronics. Device structure optimization strategies were then proposed to improve the comprehensive radiation tolerance of CNT ICs. The CNT ICs without special irradiation reinforcement technology exhibit a comprehensive radiation tolerance, including a 1×104 MeV∙cm2/mg level of the laser-equivalent threshold linear energy transfer (LET) for SEEs, 2.8×1013 MeV/g for DD and 2 Mrad (Si) for TID, which are at least 4 times higher than those in conventional radiation-hardened ICs (100 MeV·cm2/mg for SEEs, 1011 MeV/g for DD and 500 krad (Si) for TID)12. ABSTRACT The ultrahigh intrinsic comprehensive radiation tolerance will promote the application of CNT ICs in high-energy solar and cosmic radiation environments, even in the development stage of CNT electronics. Physics of radiation-induced electronic failures in CNT FETs High-energy photons (X-rays and γ-rays) and particles interact with the space environment and may deposit energy on the materials along their incident path. For CNT FETs, radiation-induced damage predominantly occurs in the CNT films, the surrounding oxide layers (gate oxide or substrate) and the gate/contact metals and may then result in temporary or permanent effects in ICs19. Radiation-induced damage is classified into three kinds of radiation effects, as shown in Fig. 1, i.e., TID, DD and SEEs. As shown in Fig. 1a, TID-induced degradation is long-term and permanent damage that starts with ionization of atoms and then results in electron-hole (e-h) pairs in the gate oxide of the CNT FET (Fig. 1b). The electrons easily escape from the gate oxide to the CNT channel, driven by the negatively biased gate (in p-FET), and the holes are then trapped in bulk defects (Fig. 1c). The trapped charges induced by TID radiation may cause degradation of the carrier mobility and on-state current, increased leakage current and a threshold voltage shift in CNT FETs. DD is a permanent radiation effect induced by the collision of particles with the atoms of the materials in CNT FETs and may result in physical damage to the lattice of CNTs20. As shown in Fig. 1d, the collision of particles with CNTs may displace the C atoms from their initial position and create vacancies and interstitial defects in the lattice. These defects and vacancies can create deep-level traps in the bandgap of CNTs and then cause an increase in the recombination rates and a reduction in the charge carrier lifetime (Fig. 1e). As a result, the DD effect may degrade the on-state current and subthreshold swing (SS) of CNT FETs. SEEs in CNT FETs are temporary radiation effects caused by concentrated bursts of excess charges generated at random locations in the CNT channel and subsequently collected by the source/drain (Fig. 1f)21. As shown in the mechanism diagram in Fig. 1g, the SEE response starts with the creation of e-h pairs along the track of the striking particle in the CNT channel, gate oxide (HfO2) and substrate (SiO2), and is then caused by two combined effects: the radiation-sensitive volume current (Fig. 1h) and the gate electric field induced by SEEs (Fig. 1i). Physics of radiation-induced electronic failures in CNT FETs The SEE-induced transient pulse may introduce soft errors during the operation of CNT ICs (e.g., single event transients in CNT FETs and single event upsets in CNT static random-access memories (SRAMs)). Generally, radiation-hardened ICs must be simultaneously tolerant to TID, DD and SEEs. With scaling down of the technology node in Si ICs, the radiation tolerance to the TID effect increases while the tolerance to SEEs degrades. For example, CMOS ICs at 22 nm or a more advanced node exhibit TID tolerance as high as approximately 1 Mrad (Si)22, and the radiation tolerance to SEEs then becomes the bottleneck of the radiation performance of ICs18. Although some low-dimensional semiconductors have been demonstrated to be excellent materials for building radiation-hardened FETs and ICs23, almost all of these works focused on TID rather than SEEs owing to the great technology challenge in measuring SEEs. SEE Measurements in CNT electronics FETs and 6T SRAM cells are fabricated through a well-developed doping-free process on solution-derived randomly oriented CNT thin films with a semiconducting purity higher than 99.99% (see the details of the fabrication process in the Experimental Section). Two kinds of gate structures, including a local bottom gate and a top gate (Supplementary Fig. 1), are used in CNT FETs to satisfy the different irradiation sources used in this work. In particular, local bottom gate FETs and ICs are necessary to measure SEEs using pulse-laser testing technology. Although SEEs in outer space (especially the single event transient and single event upset effects) are usually caused by heavy ion irradiation, directly testing the SEEs of ICs induced by heavy ions is a major challenge due to the complex electromagnetic environment in an accelerator. Pulse-laser testing has become a valuable method to study SEEs in transistors and ICs since recent developments in terms of a laser dosimeter approach and numerical methods suggest that a quantitative correlation between the pulse laser and heavy charge deposition is possible24. Therefore, we choose a pulse laser to test the SEEs in CNT FETs and ICs (see the experimental setup in Fig. 2a and the test details in the Experimental Section). Since the bandgap (typically 0.78 eV by scanning tunnelling microscopy (STM) measurement, as shown in Supplementary Fig. 2) of CNTs in the channel is smaller than the photon energy of the laser (1064 nm, ~1.2 eV), a single event charge can be generated in the CNT films through single photon absorption (SPA). Local bottom gate CNT FETs (see the structural diagram in Fig. 2b and transfer curve in Supplementary Fig. 3) are used as the device under test (DUT) to ensure that the laser completely reaches the surface of the CNT channel. It is worth mentioning that although the SEE measurements are carried out in local bottom gate CNT FETs, the results are still available to top gate FETs owing to the superior penetration of high-energy particles. As shown in Fig. 2c, the transient source-drain current (Ids) curves are monitored by irradiating the single event-sensitive region with different laser energies (see the details to find the sensitive region in Supplementary Fig. 4). A peak current appears in the transient Ids curves as the laser pulse energy increases to 5.2 nJ/pulse, which is quantified as the threshold laser energy for single event transients (SETs) in CNT FETs. SEE Measurements in CNT electronics Beyond the threshold laser energy, the pulse laser begins to excite SET behaviour, indicated by a peak current, which increases with the laser pulse energy. Note that the transient current peak in Fig. 2c exhibits a short tail of approximately 3 ns, which is much smaller than that in a Si FinFET (approximately 70 ns)25. The short tail in the transient current mainly originates from the high carrier mobility of CNTs, which promotes diffusion of the charges induced by SETs, and indicates the low SET response of CNT FETs. We further explore how the channel length of CNT FETs affects the threshold laser energy for SETs, as shown in Fig. 2d. The threshold laser energy for SETs decreases with decreasing channel length, which is similar to that in Si FETs18. Although these results imply that the SET response of CNT FETs may increase as the channel length scales down, the threshold energy of 3.8 nJ/pulse in 0.18 µm channel length CNT FETs is still much higher than that in 0.18 µm Si MOSFETs (0.0985 nJ/pulse)26. As another SEE apart from SETs to be concerned with for electronic systems used in spacecraft, a single event upset (SEU) can also make electronic systems fail either by causing false logic functionality or scrambling storage values. Although SEU tolerance is very important for radiation-hardened ICs, there is still no study on the SEU effects in CNT ICs. To explore the radiation tolerance to SEUs of CNT ICs, we fabricate 6T SRAM cells consisting of p-type CNT FETs with a local bottom gate structure (see the schematic in Fig. 3a and equivalent circuit in Fig. 3b) and execute SEU measurements through a pulse-laser test. The transfer characteristics of CNT SRAMs can be seen in Supplementary Fig. 5. Before the SEU test, CNT SRAMs are biased to the write state, and the logic value “1” or “0” is written. Then, we monitor the transient voltage transfer curves (VQ’ or VQ) at node 2 or node 1 to record the SEU signals in an SRAM cell by irradiating a pulse laser on the most sensitive transistor (P1 or P2, respectively). When the laser pulse energy increases to the threshold laser energy for a SEU, a voltage peak appears in the transient VQ’ curve, as shown in Fig. 3c and 3d. SEE Measurements in CNT electronics The measured threshold laser energy for SEUs in SRAM is 5.2 nJ/pulse, which is strictly consistent with the threshold laser energy for SETs measured in CNT FETs with a similar gate length. Different from the SET response of an individual FET, there are two transient voltage peaks induced by one effective pulse in the SEU measurement of SRAM, and the second transient voltage peak is higher than the first one. In an SRAM cell, the logic data are stored in the two back-to-back inverters consisting of transistors P1-P4, as shown in Fig. 3b. Once a SEE causes one of the nodes (node 1 or 2 in Fig. 3b) in the inverter to flip, the disturbance may propagate forward through the inverter with a gain higher than 1 and cause a transient with an increased amplitude on the other node. As evidence, the time difference between the two peaks is approximately 1 ns (0.95 ns in Fig. 3c and 1.06 ns in Fig. 3d), which is equal to the gate propagation time of the CNT network-based inverter with a similar (1 um) gate length27. As a result, a positive feedback effect appears and causes both nodes to flip to a wrong value28, which means that an SEU occurs in the SRAM. However, the CNT SRAM must be recovered to the right state by the bitline retained in write mode during our measurements, and then, only two peaks, rather than a series As another SEE apart from SETs to be concerned with for electronic systems used in spacecraft, a single event upset (SEU) can also make electronic systems fail either by causing false logic functionality or scrambling storage values. Although SEU tolerance is very important for radiation-hardened ICs, there is still no study on the SEU effects in CNT ICs. To explore the radiation tolerance to SEUs of CNT ICs, we fabricate 6T SRAM cells consisting of p-type CNT FETs with a local bottom gate structure (see the schematic in Fig. 3a and equivalent circuit in Fig. 3b) and execute SEU measurements through a pulse-laser test. The transfer characteristics of CNT SRAMs can be seen in Supplementary Fig. 5. Before the SEU test, CNT SRAMs are biased to the write state, and the logic value “1” or “0” is written. SEE Measurements in CNT electronics Then, we monitor the transient voltage transfer curves (VQ’ or VQ) at node 2 or node 1 to record the SEU signals in an SRAM cell by irradiating a pulse laser on the most sensitive transistor (P1 or P2, respectively). When the laser pulse energy increases to the threshold laser energy for a SEU, a voltage peak appears in the transient VQ’ curve, as shown in Fig. 3c and 3d. The measured threshold laser energy for SEUs in SRAM is 5.2 nJ/pulse, which is strictly consistent with the threshold laser energy for SETs measured in CNT FETs with a similar gate length. Different from the SET response of an individual FET, there are two transient voltage peaks induced by one effective pulse in the SEU measurement of SRAM, and the second transient voltage peak is higher than the first one. In an SRAM cell, the logic data are stored in the two back-to-back inverters consisting of transistors P1-P4, as shown in Fig. 3b. Once a SEE causes one of the nodes (node 1 or 2 in Fig. 3b) in the inverter to flip, the disturbance may propagate forward through the inverter with a gain higher than 1 and cause a transient with an increased amplitude on the other node. As evidence, the time difference between the two peaks is approximately 1 ns (0.95 ns in Fig. 3c and 1.06 ns in Fig. 3d), which is equal to the gate propagation time of the CNT network-based inverter with a similar (1 um) gate length27. As a result, a positive feedback effect appears and causes both nodes to flip to a wrong value28, which means that an SEU occurs in the SRAM. However, the CNT SRAM must be recovered to the right state by the bitline retained i it d d i t d th l t k th th i of peaks, are observed in the transient voltage transfer curves for one SEU, as shown in Fig. 3c and d. If transistor P2 is struck by an effective pulse laser, then the first SEU transient voltage peak in VQ’ is negative, as shown in Fig. 3d, since the induced signal is out-of-phase amplified by the inverter consisting of P1 and P3. SEE Measurements in CNT electronics Although the pulse laser has proven to be an available and powerful tool for studying SEEs21, the measured threshold laser energy must be converted to an equivalent LET, which is the standard parameter to benchmark SEEs in the real space environment. In dosimetry, LET is the average radiation energy deposited per unit path length along the track of an ionizing particle, which largely determines the consequence of SEE radiation24. Here, we analyse the laser energy deposited on CNT devices and use the laser effective energy equivalent LET (ELET) model (referring to the Si-based devices) 21,29 to estimate the ELET from the pulse-laser energy (the details are given in the Supplementary Information). According to the measured threshold laser energy values (5.2 nJ/pulse for the SRAM in Fig. 3c and 3.8 nJ/pulse for the CNT FET in Fig. 2d), the estimated ELET is approximately 1.49×104 MeV·cm2/mg in 1 μm CNT FETs and SRAMs and declines to 1.08×104 MeV·cm2/mg in 0.18 μm CNT FETs and ICs. Therefore, CNT FETs and ICs exhibit ELETs approximately 2 orders of magnitude higher than Si-based FETs and ICs30, indicating that CNT electronics present great potential for radiation hardened properties with respect to SEEs. Mechanisms of SEEs in CNT electronics The mechanisms of SEEs in an FET can be explained as two correlated effects: (1) the radiation-sensitive volume and (2) the gate electric field induced by SEEs (Supplementary Fig. 6a)31. For Si-based devices (bulk Si, fully depleted silicon on insulator (FDSOI) or FinFET) with a large sensitive channel volume, the SEE response is mainly caused by the first mechanism. In CNT FETs with ultrathin channels and nanoscale cross sections, the SEE-sensitive channel volume is almost negligible. Therefore, the SEE response in CNT FETs mainly originates from the second mechanism, which is much more difficult to achieve than the first SEE mechanism in conventional bulk semiconductor. As a result, CNT electronics exhibit much better SEE radiation hardened properties than Si-based electronics. TCAD simulations, which can decouple these two mechanisms31,32, are used to prove this explanation. Fig. 3e shows the transient characteristics of two kinds of transistors with no physical gate. In the transistors without a gate, the SET current is only caused by the radiation-sensitive channel volume (see the details of the charge density profile at LET=104 MeV·cm2/mg with a normal incidence angle to the channel for the 180 nm node FDSOI FET and CNT FET in Supplementary Fig. 6b and Fig. 6c, respectively). Notably, compared with the FDSOI FET, the CNT FET with a similar node shows almost no SEE-sensitive channel volume current at LET=104 MeV·cm2/mg. When scaled down to the 5 nm node, the SEE-sensitive channel volume current of the CNT FET is still much lower than that of a nanosheet FET (NSFET), which has the smallest SEE-sensitive volume in Si-based transistors in theory (shown in Supplementary Fig. 6d-f). Therefore, we can conclude that the SEE response in the CNT FET is mainly caused by the gate electric field, which means that the SEE radiation hardening level can be further improved by increasing the gate efficiency. According to the experimental results shown in Fig. 3f, the threshold laser energy for SETs increases from 3.76 nJ/pulse to 8.19 nJ/pulse as the gate oxide thickness (tox) decreases from 12 nm to 6 nm. Mechanisms of SEEs in CNT electronics Not only can these experimental results further prove the SEE response mechanism of the CNT FET, but also, they indicate that we can significantly strengthen SEE radiation hardened properties in CNT electronics by scaling the thickness of the gate oxide, by which we can simultaneously strengthen the rad-hard level for TID33 and DD (we will discuss this in the next section). DD effect measurements and mechanisms in CNT transistors To systematically emulate device degradation in the space environment, analysing the DD induced by high-energy particle incidents on CNT FETs is also critical16. As the fact that the gate metal cannot resist heavy ions is well known34, 2225 MeV Xe+ ion irradiation tests are performed on top-gate CNT FETs (Fig. 4a) to investigate the DD effect (see the details of the DD experimental method in the Experimental Section and Supplementary Fig. 7). The CNT films as the channel region are first characterized by Raman tests before and after heavy ion irradiation. According to the Raman spectra of CNT films irradiated by various heavy ion fluences (Supplementary Fig. 8), Xe+ radiation has a relatively small impact on the ID/IG up to fluences (ions) of 5×1012 cm-2 (Fig. 4b), implying that few displacement defects are generated in the CNT films. Although the CNT film shows a strong tolerance against heavy ion irradiation, Xe+ radiation still causes crucial performance degradations in CNT FETs (Fig. 4c). Both the ION/IOFF and SS degrade with increasing Xe+ fluence, with a significant nonlinear correlation (Fig. 4d). The 1×1012 cm-2 Xe+ radiation leads to little degradation of ION/IOFF and SS, indicating that the CNT FETs remain intact. However, the 5×1012 cm- 2 Xe+ radiation causes significant degradation of the ION/IOFF ratio (lowers it by over 1000 times) and SS (increases it by 330 mV/dec), which indicates electronic failure in CNT FETs owing to a significant trap generation process in the dielectrics35. The statistical test results of 20 CNT FETs (Supplementary Fig. 9) further confirm that 1×1012 cm-2 Xe+ radiation induces negligible degradation, while 5×1012 cm-2 Xe+ radiation causes electronic failure. Interestingly, Xe+ ion irradiation has little impact on the gate leakage current (Supplementary Fig. 9d), indicating that the DD effect does not form a leakage path in the HfO2 gate dielectric. As a widely used key parameter, the DD dose (Dd) is employed here to benchmark the CNT FET DD tolerance34. The 1012 cm-2 Xe+ ion irradiation can induce a Dd of 2.79×1013 MeV/g, which is significantly (>102×) higher than the radiation tolerance requirement for outer space exploration missions13. We further reveal the mechanism of the DD effect in CNT FETs through Monte Carlo simulations (see the details of the SRIM simulation in the Experimental Section). Simulation results (Fig. 4e and Supplementary Fig. DD effect measurements and mechanisms in CNT transistors 10a) reflect that the damage to a transistor is approximately uniformly distributed in the CNT film and substrate, which indicates that neither the HfO2 gate dielectric nor the Pd gate metal has a shielding effect on the heavy ions. Meanwhile, few vacancies form in the CNT film (Fig. 4f and Supplementary Fig. 10b), confirming the robustness of CNTs to DD radiation, consistent with the Raman test results (Fig. 4b and Supplementary Fig. 8). Although a high concentration of vacancies appears in the gate metal, they may be shielded by the high-density electrons in the metal and do not affect the electrical properties of the CNT channel. Therefore, the gate oxide is identified as the most vulnerable component in CNT FETs, and its damage will provide a major contribution to device failure. Then, scaling tox can effectively reduce the radiation-sensitive volume and improve the DD tolerance. We also simulate the DD hardening effect of CNT FETs through TCAD simulation tools (see the details of the TCAD setup and calibration in the Experiment Section)36, and the simulated transfer curves (Fig 4g) present good agreement (error <10%) with the experimental data. As shown in Fig. 4h, tox scaling can also be utilized as an effective improvement method for DD hardening. Comprehensive radiation effects in CNT electronics Although in this work we have respectively shown that CNT FET and CNT based ICs have excellent radiation harden properties to SEEs and DD, there are multiple radiation effects combined (DD, TID and SEE) in the real space environment at the same time which may cause more severe comprehensive radiation damage to electronic systems. Here for the first time, we fabricate radiation-hardened CNT FETs and CNT SRAMs by thinning the gate oxide thickness to 8 nm and then investigate the comprehensive radiation effects, including SEEs, DD and TID. As shown in Fig. 5a, the CNT FETs and SRAMs are exposed to Xe+ DD radiation, γ-ray TID radiation (Supplementary Fig. 11) and a 1064 nm pulsed laser for SEEs in turn. Since defects induced by DD and trapped charges induced by TID will remain in FETs and then influence the following SEE response measurement11, this experimental method can be used to emulate radiation effects on CNT-based ICs in the real space environment. The transfer characteristics of CNT FETs and SRAMs with different Xe+ fluences and TIDs are shown in Supplementary Fig. 12a-d. Fig. 5b and Supplementary Fig. 12e present the SEEs of CNT FETs and SRAMs after being exposed to 1×1012 cm-2 Xe+ DD and 2 Mrad (Si) TID radiation. The threshold pulse laser energies for the SET response in CNT FETs and the SEU response in CNT SRAMs do not change with the DD and TID radiation. Although the full width at half maximum of SETs in CNT FETs slightly increases after exposure to DD and TID, the SET peak current can still be restored to 0 for approximately 500 ps. This is because the defects caused by DD and the trap charge introduced by TID can reduce the gm of CNT FETs, which may reduce the SEE diffusion current. These results demonstrate that the CNT ICs used in this work can withstand DD up to 2.79x1013 MeV/g, TID up to 2 Mrad (Si) and SEEs up to 1.78x104 MeV cm2/mg simultaneously. We benchmark the radiation tolerance of CNT ICs in this work with that of other radiation-hardened FETs37, such as reported CNT FETs8,38,39, 2-D materials40-42, metal oxide thin films43 and Si FinFETs26,44,45, based on important metrics involving TID, DD influences and the SET threshold energy, as shown in Fig. 5c. Comprehensive radiation effects in CNT electronics Compared with previously reported FETs, the CNT FETs in this work can withstand a higher threshold laser energy for SEEs and a higher heavy ion energy for DD. Although some reported works show higher radiation tolerance than this work (e.g., 15 Mrad TID for the ion gel CNT FET or 1×1014 cm-2 He+ for the MoS2 FET), they only tested a single radiation effect. In addition, considering that the radiation hardened properties of devices depend on the channel length to some extent, we compare the comprehensive radiation hardened properties of 0.18 μm CNT FETs with those of 0.18 μm Si FETs, as shown in Fig. 5d. Note that Si-based FETs require different radiation hardening methods for different irradiation effects, so we choose different reported works to compare the radiation tolerance12,26,30,46. Compared with radiation-hardened Si FETs, CNT FETs with similar gate lengths lead by more than one magnitude in DD dose, TID and LET for SEEs, indicating the huge advantage for radiation-hardened or harsh environment electronics. Conclusions In this work, we systematically demonstrate that CNT electronics are a promising radiation hardened technology for outer space exploration. We thoroughly test and analyse the SEE, DD and TID tolerance of CNT FETs and ICs and estimate their comprehensive radiation effect tolerance. By using a pulse laser as the SEE irradiation source, 2225 MeV Xe+ as the DD irradiation source and Co-60 γ-ray as the TID irradiation source to simulate the radiation environment in space, rad-hard CNT ICs are tested and exhibit a radiation tolerance level of up to 2 Mrad (Si) for TID, 2.8x1013 MeV/g for DD and threshold LET=104 MeV·cm2/mg for SEEs, which are far beyond the radiation tolerance requirements for outer space exploration (500 krad (Si) for TID, 1011 MeV/g for DD and 100 MeV·cm2/mg for SEEs). Moreover, these levels of radiation tolerance for different radiation effects can be realized on CNT FETs at the same time without special irradiation reinforcement technology. This work showcases CNT-based ICs as a promising radiation-hardened technology for application in space exploration and nuclear industry. Local bottom gate CNT FET: The local bottom gate window was patterned by electron beam lithography (EBL), followed by evaporation of 20 nm Pd and growth of 10 nm HfO2 through atomic layer deposition (ALD) with a standard lift-off process. Next, CNTs were solution-deposited and oxygen plasma etched for 60 s to define the channels of the CNT FETs. The active channel region was defined by EBL, followed by oxygen plasma etching for 60 s. The top gate window was patterned through EBL, followed by growth of 10 nm HfO2 through ALD and evaporation of 20 nm Pd with a standard lift-off process. Finally, the source and drain electrodes were patterned by EBL, followed by evaporation of 0.3 nm Ti and 70 nm Pd to fabricate a p-type contact. Top-gate CNT FET: Source and drain electrodes were patterned by EBL, followed by evaporation of 0.3 nm Ti and 70 nm Pd to fabricate a p-type contact. Next, the active channel region was defined by EBL, followed by oxygen plasma etching for 60 s. The top gate window was patterned through EBL, followed by growth of 10 nm HfO2 through ALD and evaporation of 20 nm Pd with a standard lift-off process. Finally, the upper interconnect was patterned by EBL, with deposition of 20/90 nm Ti/Au metal by electron beam evaporation (EBE) and a standard lift-off process. Preparation of CNT thin films: Uniform initially semiconducting single-walled CNT (s-SWCNT) films were prepared by deposition. Arc-discharged CNTs were purchased from Carbon Solutions Inc. Dispersants (poly[9-(1-octylonoyl)-9H-carbazole-2,7-diyl (PCz)]) were synthesized by Suzuki polycondensation. A total of 100 mg AP-SWCNTs and 100 mg PCz were added to 100 mL toluene. Then, the solution was dispersed with a top-tip dispergator (Sonics VC700) at 300 W for 30 min. The dispersed solution was centrifuged for 0.5 h at 50000 g to remove most metallic CNTs and insoluble materials. The upper 90% of the supernatant was collected and centrifuged for a second time for 2 h at 50000 g. Finally, we collected the upper 90% of the supernatant for fabrication of a thin film with the dip-coating method. The sorted CNT solution was diluted ten times with toluene. The Si/SiO2 substrate was immersed in the diluted solution for 72 h. The substrate was then removed from the solution, rinsed with toluene for 30 min, purged with 99.999% N2 and baked at 120℃ for 30 min in air. Finally, to reduce the influence of the polymer on the radiation hardened properties of CNTs, the film was annealed in a tube furnace with a forming gas (Ar/H2: 5/1) at a temperature of 600℃ for 3 h to remove the polymer. SEE pulse-laser irradiation: An oscilloscope with a 50 Ω input impedance was used to visualize and record the SETs. The laser beam was focused using a 100x microscope objective to create a charge generation spot size of approximately 2 μm. Laser pulses with a wavelength of 1064 nm and a normal pulse width of 150 fs at a repetition rate of 1 kHz were used. During the SET pulsed-laser test, the CNT FETs and CNT-based SRAMs were fixed on the x- y-z stage with a 0.1 μm resolution. Co-60 γ-ray TID irradiation: The radiation experiments were carried out by using a Co-60 source in vacuum. The radiation rate used for all samples was approximately 480 rad (Si)/s. The transistor curves were measured after each radiation exposure. Xe+ ion DD irradiation: The Xe+ ion irradiation experiment was carried out at the Heavy Ion Research Facility in Lanzhou (HIRFL) at the Institute of Modern Physics, Chinese Academy of Sciences (IMP-CAS). The Xe+ ion energy was modulated by employing both aluminium foils and air as degraders, and the value in Si was calibrated to be 27.9 MeV∙cm2/g. Tests with various ion fluences were performed at a normal angle of incidence (0°). CNT-based 6T SRAM: The local bottom gate window was patterned by EBL, followed by evaporation of 20 nm Pd and growth of 10 nm HfO2 through ALD with a standard lift-off process. Next, CNTs were solution-deposited and oxygen plasma etched for 60 s to define the channels of the CNT-based 6T SRAM. An interlayer dielectric was formed using poly(methyl methacrylate) (PMMA) 200 K via EBL with a dose of 10000 μC/cm2. Finally, the upper interconnect was patterned by EBL with 20/90 nm Ti/Au metal deposited by EBE and a standard lift-off process. The CNT-based 6T SRAMs in this work were measured using a probe station with a semiconductor analyser (Keithley 4200) in air. work were measured using a probe station with a semiconductor analyser (Keithley 4200) in air. SRIM simulation setup: The simulated Xe+ ion fluence in the SRIM simulation was set to 5×1012 cm-2, while its incident angle and energy were set according to the experimental conditions. The incident location was assumed to be uniformly distributed on the Pd surface (depth 0 Å). In the simulated 60 nm × 60 nm cross-section, the Pd gate metal electrode, HfO2 gate dielectric, CNT channel and SiO2 substrate were included. A 2 nm-thick graphene- carbon-graphene sandwich layer, featuring a middle layer with reduced carbon atom density, was used to emulate the structure of a CNT. The default material parameters of Pd, HfO2 and SiO2 were utilized for the corresponding materials. References 1 Shulaker, M. M. et al. Carbon nanotube computer. Nature 501, 526-530 (2013). 2 Liu, L. et al. Aligned, high-density semiconducting carbon nanotube arrays for high- performance electronics. Science 368, 850-856 (2020). 3 Zhong, D. et al. Gigahertz integrated circuits based on carbon nanotube films. 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To accurately calculate the CNT film carrier density, the following equations were set in the quasi-2D channel47: 2 exp F V V E E p N kT −   = −     where dCNT is the diameter of the CNT and is set to 2 nm. A modified drift-diffusion (DD) model with high field velocity saturation was used for carrier transport: where dCNT is the diameter of the CNT and is set to 2 nm. A modified drift-diffusion (DD) model with high field velocity saturation was used for carrier transport: 1 0 0 1 1 p p p satp v β β µ µ µ ε       = ×     ×   +           where ε is the lateral electric field, μ0p is the low-field hole mobility, vsatp is the saturation velocity and β is an empirical parameter, set to 1.4. The low-field mobility empirically captures the scattering term induced by various components of the CNT FET: ( ) 1 1 0 DD p CNT µ µ µ − − = + ( ) 1 1 0 DD p CNT µ µ µ − − = + where μCNT is extracted from the pristine device performance and μDD represents the where μCNT is extracted from the pristine device performance and μDD represents the scattering caused by DD (extracted to be 1.1 cm2/V-s after 5×1012 cm-2 Xe+ ion irradiation). Meanwhile, both the interface states and fixed charges in the dielectrics were extracted through threshold voltage shift and subthreshold performance analysis. The Pd S/D metals were modelled as ohmic contacts. Since the off-state leakage is strongly affected by the surface contamination, which varies between samples and cannot be effectively calibrated, no leakage model was included in the simulations. References Low-power electronic technologies for harsh radiation environments. 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Transient Response of 0.18-${\mu} $ m SOI MOSFETs and SRAM Bit-Cells to Heavy-Ion Irradiation for Variable SOI Film Thickness. IEEE Transactions on Electron Devices 65, 4826-4833 (2018). 31 Kim, J., Lee, J.-S., Han, J.-W. & Meyyappan, M. Single-event transient in FinFETs and nanosheet FETs. IEEE Electron Device Letters 39, 1840-1843 (2018). 32 Han, J.-W., Kim, J., Moon, D.-I., Lee, J.-S. & Meyyappan, M. Soft error in saddle fin based DRAM. IEEE Electron Device Letters 40, 494-497 (2019). 33 Zhu, M., Zhou, J., Sun, P., Peng, L.-M. & Zhang, Z. Analyzing Gamma-Ray Irradiation Effects on Carbon Nanotube Top-Gated Field-Effect Transistors. ACS Applied Materials & Interfaces (2021). 34 Srour, J. a. & Palko, J. Author contributions Z. Z. proposed and supervised the project. M. Z. fabricated CNT FETs and SRAM cells for radiation tests. P. L. set up the numerical simulation and interpreted the governing mechanism of the radiation effects. M. Z., X. W. and C. Q. carried out the laser single event effect experiments. Y. Z. performed the STM tests of CNTs. M. Z., H. X. and J. Z. performed the total ionizing dose tests. M. Z., P. L. and H. Z performed the displacement damage irradiation test. M. Z. performed the Raman spectroscopy characterization before and after irradiation. P. L. and H. Z. performed the SRIM Monte Carlo calculation. J. H. and R. C. supervised the radiation experiments. B. L. and Z. H. supervised the theoretical comparison between CNT FETs and Si-based transistors. M. Z., P. L., R. C., B. L. and Z. Z. analysed the data. M. Z., P. L. and Z. Z. cowrote the manuscript. All authors discussed the results and commented on the manuscript. Competing interests The authors declare no competing financial interest. References Displacement damage effects in irradiated semiconductor devices. IEEE Transactions on Nuclear Science 60, 1740-1766 (2013). 35 Schwank, J. R. et al. Radiation Effects in MOS Oxides. 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Laser-Induced Single-Event Transients in Black Phosphorus MOSFETs. IEEE Transactions on Nuclear Science 66, 384-388 (2018). 42 Liang, C. et al. Defects and low-frequency noise in irradiated black phosphorus MOSFETs with HfO2 gate dielectrics. IEEE Transactions on Nuclear Science 65, 1227-1238 (2018). 43 Park, B. et al. Solution‐Processed Rad‐Hard Amorphous Metal‐Oxide Thin‐Film Transistors. Advanced Functional Materials 28, 1802717 (2018). 44 El Mamouni, F. et al. Fin-width dependence of ionizing radiation-induced subthreshold-swing degradation in 100-nm-gate-length FinFETs. IEEE Transactions on Nuclear Science 56, 3250- 3255 (2009). 45 Paillet, P. et al. Total ionizing dose effects on deca-nanometer fully depleted SOI devices. IEEE transactions on nuclear science 52, 2345-2352 (2005). 46 Tan, F. et al. Effect of Heavy Ion Irradiation on the RF Performance of 0.18 μm Bulk Si MOSFETs. ECS Transactions 44, 141 (2012). 47 Liang, J., Akinwande, D. & Wong, H.-S. P. Carrier density and quantum capacitance for semiconducting carbon nanotubes. Journal of Applied Physics 104, 064515 (2008). Acknowledgements This work is supported by the Beijing Municipal Science and Technology Commission (Grant No. Z191100007019001-3), Natural Science Foundation of China (U21A6004), and China Postdoctoral Science Foundation (Grant No. 2021M700203). Additional Information Supplementary Information is available for this paper at https: Reprints and permissions information is available at www.nature.com/reprints. Correspondence and requests for materials should be addressed to Z.Z. Correspondence and requests for materials should be addressed to Z.Z. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Fig. 1\| Radiation damage mechanisms (TID, DD and SEEs) in CNT FETs and ICs. a, Schematic of the TID degradation mechanism of a CNT FET. b, TID-induced e-h pairs generated in the oxide. c, TID-induced holes trapped at the interface. d, Schematic of the DD degradation mechanism of a CNT FET. e, DD in a CNT FET can affect carrier recombination, trapping and concentration. f, Typical single event transient current of a CNT FET. g, Schematic of the SEE degradation mechanism of a CNT FET. h, Schematic of the radiation-sensitive channel volume current induced by SEEs. i, Schematic of the gate electric field induced by SEEs. Fig. 1\| Radiation damage mechanisms (TID, DD and SEEs) in CNT FETs and ICs. a, Schematic g \| g ( , ) , of the TID degradation mechanism of a CNT FET. b, TID-induced e-h pairs generated in the oxide. c, TID-induced holes trapped at the interface. d, Schematic of the DD degradation mechanism of a CNT FET. e, DD in a CNT FET can affect carrier recombination, trapping and concentration. f, Typical single event transient current of a CNT FET. g, Schematic of the SEE degradation mechanism of a CNT FET. h, Schematic of the radiation-sensitive channel volume current induced by SEEs. i, Schematic of the gate electric field induced by SEEs. Fig. 2\| SEEs in CNT FETs. a, Simplified block diagram of the SPA test setup. In the figure, L represents a lens, M represents a mirror, S represents a shutter, P represents a polarizer, and BS represents a beam splitter. b, Schematic of a local bottom gate CNT FET exposed to a 1064 nm laser to test the SET effects. The CNT FET presents a channel length/width of 1 μm/10 μm and is measured under a bias condition of Vds= -1 V and Vgs= 0 V. c, SETs of a CNT FET when irradiating the single event-sensitive region with different laser energies. d, Threshold SET energy for CNT FETs with different channel lengths. Correspondence and requests for materials should be addressed to Z.Z. (a) (b) (c) (d) -3 -2 -1 0 1 2 3 -0.1 0 0.1 0.2 0.3 0.4 SET current (mA) time (ns) 5.0 nJ 5.2 nJ 5.4 nJ 250 ps Vds = -1 V Vgs = 0 V λ =1064 nm short tail ~3 ns 0.2 0.4 0.6 0.8 1 3.5 4 4.5 5 5.5 SET energy (nJ) Channel length (μm) Vds = -1 V Vgs = 0 V λ =1064 nm (a) (b) (c) (d) (a) (c) (b) (d) ) 0.2 0.4 0.6 0.8 1 3.5 4 4.5 5 5.5 SET energy (nJ) Channel length (μm) Vds = -1 V Vgs = 0 V λ =1064 nm -3 -2 -1 0 1 2 3 -0.1 0 0.1 0.2 0.3 0.4 SET current (mA) time (ns) 5.0 nJ 5.2 nJ 5.4 nJ 250 ps Vds = -1 V Vgs = 0 V λ =1064 nm short tail ~3 ns Fig. 2\| SEEs in CNT FETs. a, Simplified block diagram of the SPA test setup. In the figure, L represents a lens, M represents a mirror, S represents a shutter, P represents a polarizer, and BS represents a beam splitter. b, Schematic of a local bottom gate CNT FET exposed to a 1064 nm laser to test the SET effects. The CNT FET presents a channel length/width of 1 μm/10 μm and is measured under a bias condition of Vds= -1 V and Vgs= 0 V. c, SETs of a CNT FET when irradiating the single event-sensitive region with different laser energies. d, Threshold SET energy for CNT FETs with different channel lengths. Fig. 3\| SEEs in CNT SRAMs. a, Schematic of a local bottom gate CNT SRAM being exposed a 1064 nm laser to test the SEU effects. b, Equivalent circuit diagram of the CNT SRAM. c, SE effect for the CNT SRAM being exposed to a 1064 nm laser at P1. d, SEU effect for the CNT SRA being exposed to a 1064 nm laser at P2. e, Simulated SET current of a 180 nm FDSOI FET and CNT network FET. f, Threshold laser energy for SETs in CNT FETs with different gate oxi thicknesses. Correspondence and requests for materials should be addressed to Z.Z. (a) (c) (d) (b) (e) (f) -6 -4 -2 0 2 4 6 0 5 10 15 20 VQ’ (mV) time(ns) Vdd= 2 V BL = 1 V λ = 1064 nm E0 = 5.2 nJ -6 -4 -2 0 2 4 6 -15 -10 -5 0 5 10 15 20 VQ’ (mV) time(ns) Vdd= 2 V BL = 1 V λ = 1064 nm E0 = 5.2 nJ 0 50 100 150 200 0 0.001 0.002 0.003 0.004 0.005 SET current (A) Time (ps) FDSOI CNT network LET=104 MeV·cm2/mg 180nm node p-FET Vds= -1V, Vgs= 0 V 6 7 8 9 10 11 12 3 4 5 6 7 8 9 Laser energy(nJ) tox(nm) Vds= -1 V Vgs= 0 V λ = 1064 nm (a) (b) (a) (b) (d) -6 -4 -2 0 2 4 6 -15 -10 -5 0 5 10 15 20 VQ’ (mV) time(ns) Vdd= 2 V BL = 1 V λ = 1064 nm E0 = 5.2 nJ (c) ( ) -6 -4 -2 0 2 4 6 0 5 10 15 20 VQ’ (mV) time(ns) Vdd= 2 V BL = 1 V λ = 1064 nm E0 = 5.2 nJ (c) (d) (e) 6 7 8 9 10 11 12 3 4 5 6 7 8 9 Laser energy(nJ) tox(nm) Vds= -1 V Vgs= 0 V λ = 1064 nm (e) (f 0 50 100 150 200 0 0.001 0.002 0.003 0.004 0.005 SET current (A) Time (ps) FDSOI CNT network LET=104 MeV·cm2/mg 180nm node p-FET Vds= -1V, Vgs= 0 V (f) Fig. 3\| SEEs in CNT SRAMs. a, Schematic of a local bottom gate CNT SRAM being exposed to Fig. 3\| SEEs in CNT SRAMs. a, Schematic of a local bottom gate CNT SRAM being exposed to a 1064 nm laser to test the SEU effects. b, Equivalent circuit diagram of the CNT SRAM. c, SEU effect for the CNT SRAM being exposed to a 1064 nm laser at P1. d, SEU effect for the CNT SRAM being exposed to a 1064 nm laser at P2. e, Simulated SET current of a 180 nm FDSOI FET and a CNT network FET. f, Threshold laser energy for SETs in CNT FETs with different gate oxide thicknesses. Fig. 3\| SEEs in CNT SRAMs. a, Schematic of a local bottom gate CNT SRAM being exposed to a 1064 nm laser to test the SEU effects. Correspondence and requests for materials should be addressed to Z.Z. b, Equivalent circuit diagram of the CNT SRAM. c, SEU effect for the CNT SRAM being exposed to a 1064 nm laser at P1. d, SEU effect for the CNT SRAM being exposed to a 1064 nm laser at P2. e, Simulated SET current of a 180 nm FDSOI FET and a CNT network FET. f, Threshold laser energy for SETs in CNT FETs with different gate oxide thicknesses. Fig. 4\| DD radiation effects in radiation-hardened CNT FETs. a, False-coloured SEM image of an as-fabricated top-gated CNT FET. b, The statistical study on the ratio of the D peak intensity to the G peak intensity (ID/IG) with different heavy-ion fluences. Raman spectra of top-gated CNT FETs before and after Xe+ heavy-ion irradiation with different ion fluences excited by a 785 nm laser. c, Transfer characteristics of CNT FETs with different heavy ion fluences. d, ION/IOFF and SS degradation trends in CNT FETs after irradiation with various Xe+ fluences. e, Simulation results of the radiation-induced vacancy distribution in the channel region. f, Vacancy densities in the depth direction. g, Simulated transfer characteristics of CNT FETs before and after 5×1012 cm-2 Xe+ ion irradiation. Correspondence and requests for materials should be addressed to Z.Z. h, Performance predictions of CNT FETs with various HfO2 dielectric thicknesses after 5×1012/cm2 Xe+ ion irradiation (g) (a) -2 -1.5 -1 -0.5 0 10-10 10-9 10-8 10-7 10-6 10-5 10-4 Ids(A) Vgs(V) Vds= -1 V Ions= 0/cm2 Ions= 1e12/cm2 Ions= 5e12/cm2 (b) (c) (d) (f) (e) (h) 0 5 10 15 20 25 30 0 0.01 0.02 0.03 0.04 Vacancies/(Angstrom-Ion) Depth (nm) Pd O Hf C Si O CNT Ion strike on Top gate -2 -1.5 -1 -0.5 0 10-9 10-8 10-7 10-6 10-5 10-4 10-3 Ids(A/μm) Vgs(V) Vds=-1 V Line：experiment Circle：simulation Ions= 0/cm2 Ions= 5e12/cm2 -3 -2 -1 0 10-8 10-7 10-6 Ids(A/μm) Vgs (V) Vds= -1 V tox = 5 nm tox = 10 nm tox = 20 nm 0 1 2 3 4 5 100 101 102 103 104 105 106 ION\IOFF Xe+ ion fluence (×1012 cm-2) 100 200 300 400 500 600 SS (mV/dec) 0 0.01 0.02 0.03 0.04 0.05 0.06 Xe (Ions/cm2) ID/IG (a.u.) 0 5e10 1e11 5e11 1e12 2e12 5e12 (b) 0 0.01 0.02 0.03 0.04 0.05 0.06 Xe (Ions/cm2) ID/IG (a.u.) 0 5e10 1e11 5e11 1e12 2e12 5e12 (a) (c) (d) (b) (a) (d) (e) 0 1 2 3 4 5 100 101 102 103 104 105 106 ION\IOFF Xe+ ion fluence (×1012 cm-2) 100 200 300 400 500 600 SS (mV/dec) (d) (e) 0 1 2 3 4 5 100 101 102 103 104 105 106 ION\IOFF Xe+ ion fluence (×1012 cm-2) 100 200 300 400 500 600 SS (mV/dec) (c) e) (d) (e) -2 -1.5 -1 -0.5 0 10-10 10-9 10-8 10-7 10-6 10-5 10-4 Ids(A) Vgs(V) Vds= -1 V Ions= 0/cm2 Ions= 1e12/cm2 Ions= 5e12/cm2 (f) 0 5 10 15 20 25 30 0 0.01 0.02 0.03 0.04 Vacancies/(Angstrom-Ion) Depth (nm) Pd O Hf C Si O CNT Ion strike on Top gate (g) -2 -1.5 -1 -0.5 0 10-9 10-8 10-7 10-6 10-5 10-4 10-3 Ids(A/μm) Vgs(V) Vds=-1 V Line：experiment Circle：simulation Ions= 0/cm2 Ions= 5e12/cm2 (g) (h) -2 -1.5 -1 -0.5 0 10-9 10-8 10-7 10-6 10-5 10-4 10-3 Ids(A/μm) Vgs(V) Vds=-1 V Line：experiment Circle：simulation Ions= 0/cm2 Ions= 5e12/cm2 -3 -2 -1 0 10-8 10-7 10-6 Ids(A/μm) Vgs (V) Vds= -1 V tox = 5 nm tox = 10 nm tox = 20 nm (h) -3 -2 -1 0 10-8 10-7 10-6 Ids(A/μm) Vgs (V) Vds= -1 V tox = 5 nm tox = 10 nm tox = 20 nm (h) (g) Fig. Correspondence and requests for materials should be addressed to Z.Z. 4\| DD radiation effects in radiation-hardened CNT FETs. a, False-coloured SEM image o Fig. 4\| DD radiation effects in radiation hardened CNT FETs. a, False coloured SEM image of an as-fabricated top-gated CNT FET. b, The statistical study on the ratio of the D peak intensity to the G peak intensity (ID/IG) with different heavy-ion fluences. Raman spectra of top-gated CNT FETs before and after Xe+ heavy-ion irradiation with different ion fluences excited by a 785 nm laser. c, Transfer characteristics of CNT FETs with different heavy ion fluences. d, ION/IOFF and SS degradation trends in CNT FETs after irradiation with various Xe+ fluences. e, Simulation results of the radiation-induced vacancy distribution in the channel region. f, Vacancy densities in the depth direction. g, Simulated transfer characteristics of CNT FETs before and after 5×1012 cm-2 Xe+ ion irradiation. h, Performance predictions of CNT FETs with various HfO2 dielectric thicknesses after 5×1012/cm2 Xe+ ion irradiation. Fig. 5\| Comprehensive radiation effects in rad-hard CNT FETs and SRAMs. a, Schematic diagram of the experimental method to test the comprehensive radiation effects of CNT FETs and ICs. b, SETs of a CNT FET after being exposed to Xe+ and γ-ray irradiation. c, Comparisons of the radiation hardness properties of reported advanced technology devices with those of our CNT FETs and ICs. d, Benchmarking of the comprehensive radiation hardness properties of 0.18 μm CNT FETs with those of reported rad-hard Si MOS FETs. Correspondence and requests for materials should be addressed to Z.Z. (a) (c) (d) 101 102 103 104 200 400 600 500 1000 1500 2000 2500 1010 1011 1012 1013 1014 10-2 10-1 100 101 100 101 102 103 104 105 Dd (MeV/g) Ion strike energy (MeV) Dose rate (rad(Si)/s) TID(krad) 0.18 μm rad-hard Si FET 0.18 μm rad-hard CNT FET Laser energy (nJ) LET(MeV·cm2/mg) SEE：0.18 μm SOI MOSFET TID: 0.18 μm SRAM IMEC DD：0.18 μm Bulk Si MOSFET (b) -6 -4 -2 0 2 4 6 -0.1 0 0.1 0.2 0.3 SET current (mA) time(ns) Ions=0/cm2, @ 0 Mrad(Si) Ions=1e12/cm2, @ 2Mrad (Si) Vgs= 0 V Vds= -1 V λ = 1064 nm E0 = 6.2 nJ (b) -6 -4 -2 0 2 4 6 -0.1 0 0.1 0.2 0.3 SET current (mA) time(ns) Ions=0/cm2, @ 0 Mrad(Si) Ions=1e12/cm2, @ 2Mrad (Si) Vgs= 0 V Vds= -1 V λ = 1064 nm E0 = 6.2 nJ (a) (c) (d) (b) (a) (d) 101 102 103 104 200 400 600 500 1000 1500 2000 2500 1010 1011 1012 1013 1014 10-2 10-1 100 101 100 101 102 103 104 105 Dd (MeV/g) Ion strike energy (MeV) Dose rate (rad(Si)/s) TID(krad) 0.18 μm rad-hard Si FET 0.18 μm rad-hard CNT FET Laser energy (nJ) LET(MeV·cm2/mg) SEE：0.18 μm SOI MOSFET TID: 0.18 μm SRAM IMEC DD：0.18 μm Bulk Si MOSFET (d) (c) 10 Dd (MeV/g) 10 Dd (MeV/g) Fig. 5\| Comprehensive radiation effects in rad-hard CNT FETs and SRAMs. a, Schematic diagram of the experimental method to test the comprehensive radiation effects of CNT FETs and ICs. b, SETs of a CNT FET after being exposed to Xe+ and γ-ray irradiation. c, Comparisons of the radiation hardness properties of reported advanced technology devices with those of our CNT FETs and ICs. d, Benchmarking of the comprehensive radiation hardness properties of 0.18 μm CNT FETs with those of reported rad-hard Si MOS FETs. 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https://openalex.org/W3011303883	https://ieeexplore.ieee.org/ielx7/6287639/8948470/09031387.pdf	English	null	A Secure Remote Mutual Authentication Scheme Based on Chaotic Map for Underwater Acoustic Networks	IEEE access	2,020	cc-by	12,348	Received February 16, 2020, accepted February 29, 2020, date of publication March 10, 2020, date of current version March 18, 2020. Received February 16, 2020, accepted February 29, 2020, date of publication March 10, 2020, date of current version March 18, 2020. Received February 16, 2020, accepted February 29, 2020, date of publication March 10, 2020, date of current version March 18, 2020. Digital Object Identifier 10.1109/ACCESS.2020.2979906 A Secure Remote Mutual Authentication Scheme Based on Chaotic Map for Underwater Acoustic Networks This work was supported in part by the National Natural Science Foundation of China under Grant 61962052 and Grant 61902273, in part by the Innovation Team Foundation of Qinghai Ofﬁce of Science and Technology under Grant 2020-ZJ-903, in part by the Key Laboratory of IoT of Qinghai under Grant 2020-ZJ-Y16, in part by the Hebei IoT Monitoring Center under Grant 3142016020, and in part by the Research Fund for the Chunhui Program of Ministry of Education of China. ABSTRACT Underwater acoustic networks (UANs) have emerged as a new wireless sensor network structure widely used in many applications. Sensor nodes are usually placed in a hostile and unattended underwater environment to gather information with limited resource. Since the underwater information is sensitive and special, only authenticated users have rights to get the information. The existing secure resource-constrained authentication schemes are not inapplicable for underwater acoustic networks, so a lightweight authentication scheme is the primarily task in underwater acoustic networks. In this paper, we present a chaotic maps remote user authentication and key agreement scheme for underwater acoustic networks based on the DLP and DHP, in which only authenticated users have rights to obtain the information. The proposed scheme applies the lightweight cryptographic primitives, such as one-way hash function and chaotic maps to accomplish mutual authentication and key agreement for underwater acoustic networks. The security of the proposed scheme is certiﬁed by applying the BAN logic and Random Oracle Model. Security analysis shows that our proposed scheme is safe and can meet ten security requirements and seven security goals. Performance analysis shows that our proposed scheme is more efﬁcient compared with other resource-constrained schemes. INDEX TERMS Authentication, chaotic maps, DHP, DLP, lightweight. is work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ I. INTRODUCTION [11] carried out mutual authentication among the user, the gateway and the sensor node for secure detection in coal mines. 1) Our proposed scheme accomplishes mutual authentica- tion among the user, the gateway and the sensor node by conducting two full rounds of four messages ﬂows. The ﬁrst message is from the user to the gateway node, the second is from the gateway node to the sensor node, the third is from the sensor node to the gateway node and the last message is from the gateway node to the user. 2) In our proposed scheme, we devise a smart card pre-authentication mechanism to inspect the legitimacy of the user at the login terminal, we use random num- ber mechanism to protect the user anonymity and we employ the timestamp mechanism to guarantee the freshness of the messages. 3) The security of the proposed scheme is certiﬁed by applying the BAN logic and Random Oracle Model. Security analysis shows that our proposed scheme is safe and can meet ten security requirements and seven security goals. Performance analysis shows that our proposed scheme is more efﬁcient and robust compared with other resource-constrained schemes. Lin [12] presented an improved authentication scheme by applying the property of Chebyshev chaotic maps to achieve user anonymity and good efﬁciency. Zhu [13] pointed out that static ID is unable to protect user anonymity in client–server environment and dynamic ID in the authenti- cation scheme based on Chebyshev chaotic maps can resist active attacks as well as has better computational efﬁciency. However, Truong et al. [14] showed the scheme is vulnerable to malicious user attack and common session key attack. Truong et al. [14] also showed the scheme [13] is not able to achieve perfect forward security. The anonymity protection is very vital in three-party communication wireless sensor network [15], medical information networks [16], [17], and roaming authentication networks [18]. Xie et al. [19] ﬁrst 4) As far as we know, we are the ﬁrst to apply Chebyshev chaotic maps to underwater environments and design a remote user authentication mechanism suitable for underwater acoustic networks communication. The rest of this paper is presented as follows. In Section II, we discuss some basic mathematical preliminaries needed for describing and analyzing our scheme. In Section III, we present the underwater model and requirements. In Section IV, we introduce our proposed scheme in detail. I. INTRODUCTION Besides, the underwater acoustic channel is a public cir- cumstance, which makes underwater acoustic communica- tion more vulnerable to various attacks [1]. Recent studies on UANs mainly focus on network construction or protocol management [2], while only a few studies have been done on network safety [1]–[5]. Consequently, we chieﬂy study the safety matters for UANs in this scheme. Authentication is the ﬁrst line of defense to achieve secure communication. Since the information gathered by underwater sensor nodes is sensitive and protected, only authenticated users have rights to get the information. With the increasing frequency of human activities in the ocean, more and more information need to be transmitted from the underwater environment, and the underwater acous- tic networks (UANs) communication technology has been widely applied in scientiﬁc expedition, marine engineering construction, investigation and development of submarine mineral resources, and military ﬁelds. These applications have also put forward higher requirements for the reliability and safety of underwater acoustic network communications. The sensor nodes are often placed in a hostile and unattended underwater environment to gather information, so it is necessary to guarantee the secure communication among the sensor node, the user and the gateway node. Despite UANs possess certain analogous characteristics with other constrained resource wireless sensor networks, for instance, nodes are placed in an unattended environ- ment and nodes are battery-powered [4], [17], UANs are fully distinct from other wireless senor networks in a great many of respects: absorption and attenuation, multi-channel The associate editor coordinating the review of this manuscript and approving it for publication was Javed Iqbal . 48285 48285 S. Zhang et al.: Secure Remote Mutual Authentication Scheme Based on Chaotic Map for UANs carried out three-party password authenticated scheme with- out utilizing timestamp based on chaotic maps instead of modular exponentiation and scalar multiplication on an ellip- tic curve. Their protocol is robust enough to withstand various attacks. Lee et al. [20] proposed a new three-party authen- tication scheme on the foundation of chaotic maps without using password, which had the ability to boycott the pass- word guessing attack and protect user from forgery attack. In the opinion of Lee et al. [20], there are three problems in the scheme [19], which are anonymity of users, on-line password guessing attack and password table maintenance problem. Unfortunately, Jabbari and Mohaseﬁ[21] proved that scheme [20] fails to guarantee user anonymity. I. INTRODUCTION propagation, Doppler frequency shift, time-varying, environ- mental noise. In addition, the construction cost of under- water sensor nodes is higher than other conventional sensor nodes [2]. Therefore, due to these differences, the existing secure authentication schemes with constrained resources are not inapplicable for underwater acoustic networks. Underwa- ter acoustic networks communication requires a new authen- tication scheme. An excellent authentication and key agreement scheme enables two or more parties to transmit data safely over a public channel, and communication parties can encrypt and decrypt the information through the negotiated session key [21]. The sensor node has constrained resource and weak calculation and storage abilities, so a lightweight authen- tication and key agreement scheme is necessary in such a network. Based on the analysis of some previous literatures, we come to a conclusion that lightweight authentication methods are roughly divided into three types. The ﬁrst type is merely based on the one-way hash function [6]–[11]. The second type applies the Elliptic Curve to accomplish the authentication [22]–[27]. The last type, which is also adopted in this scheme, is the chaotic maps [12]–[21]. Elliptic curves are also used in many environments to implement authentication, such as multi-server envi- ronment [22], agriculture monitoring environment [23], IOT environments [24], [25], medical information sys- tem environment [26], cloud computing environment [27]. Compared with other lightweight authentication schemes, Chebyshev chaotic maps have higher level of efﬁciency and security [1], [6], [18], [20], [28]. The chief contributions of this scheme are as follows: Next, we give a brief explanation of the three types of lightweight authentication schemes. Das [6] proposed the robust password-based remote user authentication scheme using smart card for the Integrated EPR Information System, and Li et al. [7] also introduced secure remote user authen- tication and key agreement scheme for the Integrated EPR Information System at the same year. They both applied the lightweight hash function to fulﬁll mutual authentication in their schemes. Nevertheless, Jung et al. [8] showed that Das [6] and Li et al. [7] suffered from off-line password guessing attack, user impersonate attack, could not protect user anonymity, and did not provide password change section. Koya and Deepthi [9] presented a hybrid anonymous authen- tication scheme using the physiological signal to overcome sensor node impersonation attack and Chang and Le [10] came up with an efﬁcient and ﬂexible authentication scheme, which provides perfect forward and backward secrecy. Kumar et al. Tn(x) = cos(n · arccos(x)), x ∈[−1, 1] We calculate the ﬁrst six terms: T0(x) = 1 T1(x) = x T2(x) = 2x2 −1 T3(x) = 4x3 −3x T4(x) = 8x4 −8x2 + 1 T5(x) = 16x5 −20x3 + 5x The gateway node is a trustful party with larger memory and higher security, which is used to validate the user and the sensor node by swapping messages between the user and the sensor nodes, perform mutual authentication and negotiate session key. From ﬁrst six recurrent terms, Chebyshev polynomial is expressed as: The sensor node is a speciﬁc sensor device with restricted resource, which is often placed in a hostile and unattended underwater environment to gather data. Tn(x) = 2xTn(x) −Tn−2(x), (n ≥2) The user ﬁrst needs to register at the gateway node and then becomes a valid user through mutual authentication and ﬁnally gets the information gathered by the sensor nodes. The semi group property and chaos property are two primary properties possessed in Chebyshev polynomial. The semi group property and chaos property are two primary properties possessed in Chebyshev polynomial. Deﬁnition 2 (Semi Group Property): I. INTRODUCTION In Section V and Section VI, we demonstrate the safety and feasibility of our proposed scheme through formal and informal analysis. In Section VII, we make a comparison 48286 VOLUME 8, 2020 VOLUME 8, 2020 S. Zhang et al.: Secure Remote Mutual Authentication Scheme Based on Chaotic Map for UANs among our proposed scheme with other schemes. Finally, we make a conclusion in Section VIII. where q is a large prime and x ∈(−∞, +∞) [19]. The enhanced Chebyshev chaotic maps still has semi group prop- erty and chaos property. B. COMMUNICATION MODEL Tg(Th(x)) = cos(g · arccos(cos(h · arccos(x)))) Th(Tg(x)) = cos(h · arccos(cos(g · arccos(x)))) Tg(Th(x)) = Th(Tg(x)) We consider an underwater acoustic communication model with four messages interchange in our proposed scheme as shown in ﬁgure 1. By conducting two full rounds of four messages ﬂows [33], the user, the gateway node and the sensor node are able to successfully validate each other. The Deﬁnition 3 (Chao Property): The Chebyshev polynomials map Tn(x) : [−1, 1] →[−1, 1] of degree n > 1 is a chaotic map with invariant density as: ∂(x) = 1/π √ 1 −x2 for the Lyapunov exponent λ = ln n. FIGURE 1. Communication model. 48287 II. PRELIMINARIES Deﬁnition 2 (DLP): Given α and β, it is unable to compute an integer λ to compute Tλ(α) mod q = β. In this section, we will chieﬂy present some deﬁnitions about the Chebyshev chaotic maps and one-way hash function. Some notations and abbreviations used in this paper are brieﬂy presented in table 1. Deﬁnition 3 (DHP): Given α, Tλ(α) and Tβ(α), it is unable to compute Tλβ(α). III. UNDERWATER MODEL AND REQUIREMENTS In this section, we ﬁrst present network model, communica- tion model and threat model. Next, we propose the security requirements and goals. TABLE 1. Notations and abbreviations. TABLE 1. Notations and abbreviations. Hash function plays a very important role in the ﬁeld of mod- ern cryptography and information security. Messages of arbi- trary length are compacted into a ﬁxed-length bit string by a speciﬁc hash function. Let the function H : {0, 1}α →{0, 1}n is a hash function with an output length of n, and then the function H satisﬁes hashing, resistance to weak (strong) col- lision, one-way and validity, details see references [29], [30]. I. UNDERWATER MODEL AND REQUIREMENTS A. NETWORK MODEL Deﬁnition 1: Chebyshev polynomial is presented as: There are four parties in our proposed scheme that the RC(Registration Center), the gateway node(GWN), the user and the sensor nodes(SNs). The registration center is only for off-line registration of the sensor nodes and the gateway nodes, and does not participate in the communication process. In fact, only three parties that the sensor nodes, the gateway nodes and the users send messages to each other. Tn(x) = cos(n · arccos(x)), x ∈[−1, 1] C. HASH FUNCTION TABLE 1. Notations and abbreviations. TABLE 1. Notations and abbreviations. TABLE 1. Notations and abbreviations. Step2: The RC sends (SNIDj, Sj) to the sensor node. The registration of the sensor node and the gateway node is executed in a safe off-line environment prior to their deploy- ment in the target topology area. After completing the above tasks, the RC sends the information (SNIDj, Sj) to the gateway node according to topological relationship. The gateway node stores {GWIDk, Gwk, SNIDj, Sj} in the memory safely. user dispatches the login request message {LH6, LH10, T1} to the GWN. The GWN dispatches the message {LH15, T2} to the sensor node. The sensor node dispatches the message {LH17, LH19, LH20, T3} to the GWN. The GWN dispatches the message {LH21, LH22, LH17, T4} to the user. IV. OUR PROPORSED SCHEME There are eight sections in our scheme: initialization section, gateway node registration section, sensor node registration section, user registration section, login section, authentication and key management section, sensor node section, password change section. p Step3: The SC inspects that the equation LH∗ 5 = LH5 is true or not. If the equation is true, the identity and pass- word are validated successfully. If not, terminate this section instantaneously. B. ENHANCED CHEBYSHEV CHAOTIC MAPS Deﬁnition 1 (Enhanced Chebyshev Polynomial is Expressed as): Expressed as): Tn(x) = 2xTn(x) −Tn−2(x) mod q FIGURE 1. Communication model. VOLUME 8, 2020 S. Zhang et al.: Secure Remote Mutual Authentication Scheme Based on Chaotic Map for UANs C. THREAT MODEL We consider the threat model for designing our scheme sim- ilar to that in [32]. Step3: After getting the request message, the gate- way node generates a random number g and computes LH0 = Tg(x) mod q, LH1 = h(RIDi\|\|GWIDk) ⊕h(RPWi\|\|g), LH2 = h(Gwk) ⊕h(RIDi\|\|RPWi). The gateway node stores {h(), LH1, LH2} in a smart card(SC) and issues SC to the user safely. D. SECURITY REQUIREMENTS AND GOALS In 2012, Madhusudhan and Mittal [31] advanced a new stan- dard of nine security requirements and ten goals for a secure remote user authentication mechanism. In 2016, Wang and Wang [32] indicated that though the standard advanced in lit- erature [31] is superior to the others, it still has redundancies and ignores some intrinsic collisions among the standard. The author then proposed twelve independent evaluation criteria. In 2018, Wang et al. [33] made some simple modiﬁcations on these twelve criteria. We refer to the above standards and according to the characteristics of underwater acoustic com- munication, the evaluation standards in our proposed scheme should meet the following security requirements and goals. Step4: After getting the SC, the user computes LH3 = ri ⊕ h(IDi\|\|PWi), LH4 = rp ⊕h(IDi\|\|PWi), LH5 = h(RIDi\|\|ri) ⊕ h(RPWi\|\|rp). The user issues {LH3, LH4, LH5} into smart card. Finally, SC includes {h(), LH1, LH2, LH3, LH4, LH5}. D. USER REGISTRATION Step1: The user freely chooses his identity IDi, one random number ri, his password PWi, and the other random num- ber rp. p Step2: The user computes RIDi = h(IDi\|\|ri), RPWi = h(PWi\|\|rp) and sends the request (RIDi, RPWi) to the gateway node via a safe channel. A. INITIALIZATION SECTION Step4: The SC produces a random digit rsc and computes LH6 = Trsc(x) mod q, KU−G = TrscTg(x) mod q, LH7 = LH1 ⊕h(T1), LH8 = LH2 ⊕h(RIDi\|\|RPWi), LH9 = h(T1\|\|LH1\|\|LH2), LH10 = EKU−G(LH7\|\|LH8\|\|LH9), where T1 is the current timestamp. Step1: The RC(Registration Center) freely chooses a high entropy integer x. Step2: The RC randomly choose c as its private key, and computes QRC = Tc(x) mod q as its public key, where q is a large prime number. Step5: The SC dispatches the login request message {LH6, LH10, T1} to the gateway node. Step3: The RC chooses a hash function h() and keeps c secretly. Then, RC publishes parameter {q, x, QRC, h()}. Sj = h(SNIDj\|\|GWIDk\|\|c). Step2: The RC sends (SNIDj, Sj) to the sensor node. C. SENSOR NODE REGISTRATION TABLE 2. Security requirements and goals. TABLE 2. Security requirements and goals. Step1: The RC chooses SNIDj as an identity for the sensor node, and according to topological relationship, computes E. LOGIN SECTION Step1: After inserting SC into the card reader of a speciﬁc terminal device, the user inputs his identity IDi and password PWi. Step2: The SC computes: r∗ i = LH3 ⊕h(IDi\|\|PWi), r∗ p = LH4⊕h(IDi\|\|PWi), RID∗ i = h(IDi\|\|r∗ i ), RPW ∗ i = h(PWi\|\|r∗ p ), LH∗ 5 = h(RID∗ i \|\|r∗ i ) ⊕h(RPW ∗ i \|\|r∗ p ). IV. OUR PROPORSED SCHEME B. GATEWAY NODE REGISTRATION Step1: After getting the login request message {LH6, LH10, T1}, the gateway node ﬁrst computes whether T2 −T1 ≤1T holds, where T2 is the time when the gateway node gets the request message, and 1T is the allowable maximum Step1: The RC chooses GWIDk as an identity for the gateway node, and computes Gwk = h(GWIDk\|\|c). Step1: The RC chooses GWIDk as an identity for the gateway node, and computes Gwk = h(GWIDk\|\|c). Step1: The RC chooses GWIDk as an identity for the gateway node, and computes Gwk = h(GWIDk\|\|c). Step2: The RC sends (GWIDk, Gwk) to the gateway node. Step2: The RC sends (GWIDk, Gwk) to the gateway node. Step2: The RC sends (GWIDk, Gwk) to the gateway node. 48288 VOLUME 8, 2020 VOLUME 8, 2020 S. Zhang et al.: Secure Remote Mutual Authentication Scheme Based on Chaotic Map for UANs TABLE 3. Login section. val If hold proceed to the step2 if not TABLE 4. The message from the gateway to the sensor node. TABLE 3. Login section. TABLE 4. The message from the gateway to the sensor node. TABLE 3. Login section. transmission time interval. If hold, proceed to the step2, if not, terminate this section instantaneously. TABLE 5. The message from the sensor node to the gateway. TABLE 5. The message from the sensor node to the gateway. TABLE 5. The message from the sensor node to the gateway. Step2: The gateway node computes KU−G = LH6 · LH0 = TrscTg(x) mod q, LH11 = DKU−G(LH10), decrypts LH10 with KU−G, and then obtains {LH7, LH8, LH9}. Step3: The gateway node computes LH∗ 1 = LH7 ⊕h(T1), LH∗ 2 = LH8 ⊕h(RIDi\|\|RPWi), LH∗ 9 = h(T1\|\|LH∗ 1 \|\|LH∗ 2 ). Step4: The gateway node inspects that the equation LH∗ 9 = LH9 is true or not. If the equation holds, the user is validated successfully. If not, terminate this section instantaneously. Step5: The gateway node computes KG−S = TSjTg(x) mod q, LH12 = h(Sj) ⊕h(RIDi\|\|RPWi), LH13 = h(SNIDj) ⊕ h(Gwk), LH14 = LH2 ⊕h(T2), LH15 = EKG−S(LH12 \|\|LH13\|\|LH14), and dispatches the message {LH15, T2} to the sensor node. Step6: Upon getting the message {LH15, T2} at time T3, the sensor node checks the freshness of time T2 by the means of T3 −T2 ≤1T. If hold, proceed to the step7, if not, terminate this section instantaneously. H. PASSWORD MODIFICATION SECTION When the user is prepared to change his primitive password PWi to a new password PW new i , the user executes the follow- ing steps. Step12: The gateway node computes h(SNIDj\|\|Sj)∗ = LH20 ⊕h(T3), LH∗ 18 = TrsnTrsc(h(SNIDj\|\|Sj)∗\|\|h(Gwk)\|\| h(RIDi\|\|RPWi)) mod q, LH∗ 19 = h(LH∗ 18\|\|h(SNIDj\|\|Sj)∗\|\| h(T2)). Step12: The gateway node computes h(SNIDj\|\|Sj)∗ = LH20 ⊕h(T3), LH∗ 18 = TrsnTrsc(h(SNIDj\|\|Sj)∗\|\|h(Gwk)\|\| h(RIDi\|\|RPWi)) mod q, LH∗ 19 = h(LH∗ 18\|\|h(SNIDj\|\|Sj)∗\|\| h(T2)). Step1: After inserting SC into the card reader of a speciﬁc end, the user inputs his identity IDi and password PWi. Step13: The gateway node inspects that the equation LH∗ 19 = LH19 is true or not. If the equation is true, the sensor node is validated successfully. If not, terminate this section instantaneously. Step2: The SC computes: r∗ i = LH3 ⊕h(IDi\|\|PWi), r∗ p = LH4⊕h(IDi\|\|PWi), RID∗ i = h(IDi\|\|r∗ i ), RPW ∗ i = h(PWi\|\|r∗ p ), LH∗ 5 = h(RID∗ i \|\|r∗ i ) ⊕h(RPW ∗ i \|\|r∗ i ). Step2: The SC computes: r∗ i = LH3 ⊕h(IDi\|\|PWi), r∗ p = LH4⊕h(IDi\|\|PWi), RID∗ i = h(IDi\|\|r∗ i ), RPW ∗ i = h(PWi\|\|r∗ p ), LH∗ 5 = h(RID∗ i \|\|r∗ i ) ⊕h(RPW ∗ i \|\|r∗ i ). 5 i i i i Step3: The SC inspects that the equation LH∗ 5 = LH5 is true or not. If the equation is true, the identity and pass- word are validated successfully. If not, terminate this section instantaneously. 5 Step3: The SC inspects that the equation LH∗ 5 = LH5 is true or not. If the equation is true, the identity and pass- word are validated successfully. If not, terminate this section instantaneously. Step14: The gateway node computes LH21=h(SNIDj\|\|Sj)⊕ h(T4), LH22 = h(h(SNIDj\|\|Sj)\|\|h(T1)\|\|LH18\|\|h(Gwk)). The gateway node dispatches the message {LH21, LH22, LH17, T4} to the user. Step4: The user inputs a new password PW new i and random digit rmew p . Next, The SC computes RPW new i = h(PW new i \|\|rnew p ), LHnew 1 = LH1 ⊕h(RPWi\|\|g) ⊕h(RPW new i \|\|g), LHnew 2 = LH2 ⊕h(RIDi\|\|RPWi) ⊕h(RIDi\|\|RPW new i ), LHnew 3 = LH3 ⊕h(IDi\|\|PWi) ⊕h(IDi\|\|PW new i ), LHnew 4 = rnew p ⊕h(IDi\|\|PW new i ), LHnew 5 = LH5 ⊕h(RPWi\|\|rp) ⊕ h(RPW new i \|\|rnew p ). A. SECURITY PROOF BASED ON BAN-LOGIC BAN-login is used for checking out the validity and practical- ity of the proposed scheme in tripartite mutual authentication and key agreement among the user, the gateway node and the sensor node. V. SECURITY ANALYSIS In this section, we will apply the BAN logic and Random Oracle Model as the formal analysis and informal analysis to verify that our proposed scheme is robust and secure against various attacks. B. GATEWAY NODE REGISTRATION Step7: The sensor node computes KG−S = TSjTg(x) mod q, LH16 = DKG−S(LH15), decrypts LH15 with KG−S, and then obtains {LH12, LH13, LH14, LH6}. Step8: The sensor node computes h(RIDi\|\|RPWi)∗ = h(Sj) ⊕LH12, h(Gwk)∗ = h(SNIDj) ⊕LH13, LH∗ 14 = h(Gwk)∗⊕h(RIDi\|\|RPWi)∗⊕h(T2). Step9: The sensor node inspects that the equation LH∗ 14 = LH14 is true or not. If the equation holds, the gateway node is validated successfully. If not, terminate this section instanta- neously. The sensor node dispatches the message {LH17, LH19, LH20, T3} to the gateway node. Step10: The sensor node produces a random digit rsn and computes: LH17 = Trsn(x) mod q, LH18 = TrsnTrsc (h(SNIDj\|\|Sj)\|\|h(Gwk)\|\|h(RIDi\|\|RPWi)) mod q, LH19 = h(LH18\|\|h(SNIDj\|\|Sj)\|\|h(T2)), LH20 = h(SNIDj\|\|Sj) ⊕h(T3). Step11: Upon getting the message {LH17, LH19, LH20, T3} at time T4, the gateway node checks the freshness of time T3 by the means of T4−T3 ≤1T. If hold, proceed to the step12, if not, terminate this section instantaneously. 48289 VOLUME 8, 2020 VOLUME 8, 2020 S. Zhang et al.: Secure Remote Mutual Authentication Scheme Based on Chaotic Map for UANs TABLE 7. The calculation of session key. TABLE 7. The calculation of session key. TABLE 6. The message from the gateway to the user. TABLE 6. The message from the gateway to the user. H. PASSWORD MODIFICATION SECTION Step15: Upon getting the message {LH21, LH22, LH17, T4} at time T5, the SC checks the freshness of time T4 by the means of T5−T4 ≤1T. If hold, proceed to the step16, if not, terminate this section instantaneously. Step16: The SC computes: h(SNIDj\|\|Sj)∗= LH21 ⊕h(T4), h(Gwk)∗= LH2 ⊕h(RIDi\|\|RPWi), LH∗ 18 = TrsnTrsc(h(SNIDj \|\|Sj)∗\|\|h(Gwk)∗\|\|h(RIDi\|\|RPWi)) mod q, LH∗ 22 = h(h(SNIDj \|\|Sj)∗\|\|h(T1)\|\|LH∗ 18\|\|h(Gwk)∗). i p Step5: The SC will replace the corresponding parameters. j 18 Step17: The user inspects that the equation LH∗ 22 = LH22 is true or not. If the equation is true, the user validates both the gateway node and the sensor node successfully. If not, terminate this section instantaneously. Thus, we have estab- lished mutual authentication among the user, the gateway and the sensor node in our scheme and negotiated a session key SK = LH18. G. SENSOR NODE ADDITION SECTION p p p P1: Ui\| ≡#(T1) P2: Ui\| ≡(Ui KU−G ←→GWk) P3: Ui\| ≡#(T4) P4: Ui\| ≡GWk ⇒(GWk KU−G ←→Ui) P5: GWk\| ≡#(g) P6: GWk\| ≡#(Sj) P7: GWk\| ≡#(T2) P8: GWk\| ≡(Ui Trsc(x) mod q ←→ GWk) P9: GWk\| ≡Ui ⇒(GWk KU−G ←→Ui) P10: GWk\| ≡SNj ⇒(SNj KG−S ←→GWk) P11: GWk\| ≡#(SNIDj) P12: GWk\| ≡(SNj TSj(x) mod q ←→ Gwk) P13: SNj\| ≡#(SNIDj) P14: SNj\| ≡#(Sj) P15: SNj\| ≡#(g) P16: SNj\| ≡GWk ⇒(GWk KG−S ←→SNj) P17: SNj\| ≡(GWk LH0 ←→SNj) P1: Ui\| ≡#(T1) P2: Ui\| ≡(Ui KU−G ←→GWk) P3: Ui\| ≡#(T4) P1: Ui\| ≡#(T1) P2: Ui\| ≡(Ui KU−G ←→GWk) P3: Ui\| ≡#(T4) P4: Ui\| ≡GWk ⇒(GWk KU−G ←→Ui) P5: GWk\| ≡#(g) P6: GWk\| ≡#(Sj) P7: GWk\| ≡#(T2) P8: GWk\| ≡(Ui Trsc(x) mod q ←→ GWk) P9: GWk\| ≡Ui ⇒(GWk KU−G ←→Ui) P10: GWk\| ≡SNj ⇒(SNj KG−S ←→GWk) P11: GWk\| ≡#(SNIDj) P12: GWk\| ≡(SNj TSj(x) mod q ←→ Gwk) P13: SNj\| ≡#(SNIDj) P14: SNj\| ≡#(Sj) P15: SNj\| ≡#(g) P16: SNj\| ≡GWk ⇒(GWk KG−S ←→SNj) P17: SNj\| ≡(GWk LH0 ←→SNj) h h f id li d d li S12: SNj\| ≡(GWk KG−S ←→SNj) Goal 3 S12: SNj\| ≡(GWk KG−S ←→SNj) Goal 3 M3: SNj →GWk : (LH17, LH19, LH20, T3) < {SK, (SK, S12: SNj\| ≡(GWk ←→SNj) Goal 3 M3: SNj →GWk : (LH17, LH19, LH20, T3) < {SK, (SK, j (SNIDj, Sj), T2), ((SNIDj, Sj), T3)}, Trsn(x) mod q, T3 > In the light of M3, we are able to gain S13: GWk◁< LH17, LH19, LH20, T3 > In the light of S13, P12 and R(1), we are able to gain S14: GWk\| ≡SNj ∼< LH17, LH19, LH20 > In the light of P5, P6, P7 and R(4), we are able to gain S15: GWk\| ≡#(SK, SNIDj, Sj, T2, T3, g) In the light of S14, S15 and R(2), we are able to gain S16: GWk\| ≡SNj\| ≡(SK, SNIDj, Sj, T2, T3, g) I th li ht f S16 d K T T ( ) d j j j In the light of S16 and KG−S = TgTSj(x) mod p, we are able to gain g S17: GWk\| ≡SNj\| ≡(SNj KG−S ←→GWk) Goal 6 In the light of S17, P10 and R(3), we are able to gain S18: GWk\| ≡(SNj KG−S ←→GWk) Goal 5 M4: GWk →Ui : (LH17, LH21, LH22, T4) < (Trsn(x) mod q), ((SNIDj, Sj), T4), ((SNIDj, Sj), T1, SK, Gwk), T4 > In the light of M4, we are able to gain S19: Ui◁< LH17, LH21, LH22, T4, GWk KU−G ←→Ui > In the light of S19, P2 and R(1), we are able to gain S17: GWk\| ≡SNj\| ≡(SNj KG−S ←→GWk) Goal 6 In the light of S17, P10 and R(3), we are able to gain K S17: GWk\| ≡SNj\| ≡(SNj KG−S ←→GWk) Goal 6 In the light of S17, P10 and R(3), we are able to gain K j M4: GWk →Ui : (LH17, LH21, LH22, T4) < (Trsn(x) mod q), ((SNIDj, Sj), T4), ((SNIDj, Sj), T1, SK, Gwk), T4 > In the light of M4, we are able to gain S19: Ui◁< LH17, LH21, LH22, T4, GWk KU−G ←→Ui > In the light of S19 P2 and R(1) we are able to gain j M4: GWk →Ui : (LH17, LH21, LH22, T4) < (Trsn(x) mod q), ((SNIDj, Sj), T4), ((SNIDj, Sj), T1, SK, Gwk), T4 > In the light of M4, we are able to gain j M4: GWk →Ui : (LH17, LH21, LH22, T4) < (Trsn(x) mod q), ((SNIDj, Sj), T4), ((SNIDj, Sj), T1, SK, Gwk), T4 > j j Here, we show the four idealized modality of the transmit- ted messages sequences among the user Ui, the gateway node GWk and the sensor node SNj. G. SENSOR NODE ADDITION SECTION 1, 2)}LH6, , 1 In the light of M1, we are able to gain S1: GWk◁< LH6, LH10, T1 > In the light of S1, P8 and R(1), we are able to gain S2: GWk\| ≡Ui ∼< LH7, LH8, LH9 > In the light of P5, R(4), we are able to gain S3: GWk\| ≡# (RIDi, GWIDk, RPWi, g, T1, Gwk) In the light of S3, S2 and R(2), we are able to gain S4: GWk\| ≡Ui\| ≡(RIDi, GWIDk, RPWi, g, T1, Gwk) In the light of S4 and KU−G = TrscTg(x) mod p, we are able to gain In the light of M1, we are able to gain 7. {N}K: Encrypt N with key K. 8. < N >R: The combination of N and R. Then, we introduce some signiﬁcant BAN logic postulates rules as below: K Rule(1). Message-meaning rule: ρ\|≡ρ K ←→β,ρ◁{N}K ρ\|≡ρ∼N . Rule(2). Nonce-veriﬁcation rule: ρ\|≡#N,ρ\|≡β∼N ρ\|≡β\|≡N Rule(3). Jurisdiction rule: ρ\|≡β\|≡N,ρ\|≡β⇒N ρ\|≡N Rule(4). Freshness rule: ρ\|≡# N ρ\|≡#(N,R) Rule(5). Belief rule: ρ\|≡β\|≡(N,R) ρ\|≡β\|≡N O h b i f BAN l i h hi Rule(1). Message-meaning rule: ρ\|≡ρ K ←→β,ρ◁{N}K ρ\|≡ρ∼N . In the light of S4 and KU−G = TrscTg(x) mod p, we are able to gain S5: GWk\| ≡Ui\| ≡(Ui KU−G ←→GWk) Goal 2 In the light of S5, P9 and R(3), we are able to gain S5: GWk\| ≡Ui\| ≡(Ui KU−G ←→GWk) Goal 2 In the light of S5, P9 and R(3), we are able to gain S5: GWk\| ≡Ui\| ≡(Ui KU−G ←→GWk) Goal 2 In the light of S5, P9 and R(3), we are able to gain Rule(5). G. SENSOR NODE ADDITION SECTION After forming the underwater wireless sensor network, a new node is likely to be putted in the target topology area because the sensor nodes are small devices with conﬁning energy and memory [4], [12]. For the sake of adding new nodes to the target topology area, the RC executes the 4.3 section in a safe off-line environment prior to their deployment in the target topology area. Therefore, the RC can successfully put the new sensor nodes into the target topology area after carrying out the sensor node registration section. Some symbols needed in the BAN-logic are represented as follows, where N and R denote statements, ρ denotes a principal, and Kx−y is a notation for cryptographic encryption key. 1. ρ\| ≡N: Principal ρ believes N is genuine. 2. ρ ◁N: Principal ρ receives a message containing N, that is, principal β sends a message containing N to ρ. 2. ρ ◁N: Principal ρ receives a message containing N, that is, principal β sends a message containing N to ρ. 48290 VOLUME 8, 2020 VOLUME 8, 2020 S. Zhang et al.: Secure Remote Mutual Authentication Scheme Based on Chaotic Map for UANs 3. ρ\| ∼N: Principal ρ has sent a message containing N. 3. ρ\| ∼N: Principal ρ has sent a message containing N. 3. ρ\| ∼N: Principal ρ has sent a message containing N. gateway node. M2 is from the gateway node to the sensor node. M3 is from the sensor node to the gateway node. M4 is from the gateway node to the user. 4. ρ\| ⇒N: Principal ρ has jurisdiction over N. 4. ρ\| ⇒N: Principal ρ has jurisdiction over N. 4. ρ\| ⇒N: Principal ρ has jurisdiction over N. 5. #(N): N is fresh, that is, N has not been sent as part of a message before the current round, where N is generally a temporary value. M1: Ui →GWk : (LH6, LH10, T1) < {((RIDi, GWIDk), (RPWi, g), T1), (((RIDi, RPWi), Gwk), (RIDi, RPWi)), (T1, LH1, LH2)}LH6, LH6, T1 > 6. ρ K ←→β: K is the shared key between principal ρ and principal β, and no one knows K except principal ρ and principal β and their trust principals. 6. ρ K ←→β: K is the shared key between principal ρ and principal β, and no one knows K except principal ρ and principal β and their trust principals. G. SENSOR NODE ADDITION SECTION Belief rule: ρ\|≡β\|≡(N,R) ρ\|≡β\|≡N S6: GWk\| ≡(Ui KU−G ←→GWk) Goal 1 M2: GWk →SNj : (LH15, T2) < {((RIDi, RPWi), Sj), (SNIDj, Gwk), (((RIDi, RPWi), Gwk), T2)} > In the light of M2, we are able to gain S7: SNj◁< LH15, T2 > In the light of S7, P17 and R(1), we are able to gain S8: SNj\| ≡GWk ∼< LH12, LH13, LH14, LH6 > In the light of P13, P14, P15 and R(4) we are able to gain S9: SNj\| ≡#(RIDi, RPWi, Sj, SNIDj, Gwk, g, T2) In the light of S9, S3 and R(2), we are able to gain S10: SNj\| ≡GWk\| ≡(RIDi, RPWi, Sj, SNIDj, Gwk, g, T2) In the light of S10 and KG−S = TgTSj(x) mod p, we are able to gain On the basis of BAN logic, we ought to achieve eight authentication goals to verify that our scheme is safe. Goa1: GWk\| ≡(Ui KU−G ←→GWk) Goa2: GWk\| ≡Ui\| ≡(Ui KU−G ←→GWk) Goa3: SNj\| ≡(GWk KG−S ←→SNj) Goa4: SNj\| ≡GWk\| ≡(GWk KG−S ←→SNj) Goa5: GWk\| ≡(SNj KG−S ←→GWk) Goa6: GWk\| ≡SNj\| ≡(SNj KG−S ←→GWk) Goa7: Ui\| ≡(GWk KU−G ←→Ui) Goa8: Ui\| ≡GWk\| ≡(GWk KU−G ←→Ui) Goa8: Ui\| ≡GWk\| ≡(GWk KU−G ←→Ui) S11: SNj\| ≡GWk\| ≡(GWk KG−S ←→SNj) Goal 4 In the light of S11, P16 and R(3), we are able to gain Next, we make some evident and essential presumptions to explicate our proposed scheme. G. SENSOR NODE ADDITION SECTION 2: Call Extract on LH6 and get rsc∗←Reveal(LH6) 3: Call Extract on LH0 and get g∗←Reveal(LH0) 4: call Reveal on input LH8 to get RIDi, RPWi as (RID∗ i \|\|RPW ∗ i ) ←Reveal(LH8) 5: if (Gw∗ k = Gwk) then 6: computes: LH∗ 2 = h(Gw∗ k) ⊕h(RID∗ i \|\|RPW ∗ i ) 7: if (GWID∗ k = GWIDk) then 8: computers: LH∗ 1 = h(RPW ∗ i \|\|g∗) ⊕h(GWID∗ k\|\|RID∗ i ) 9: if T ∗ 1 = T1 then 10: computes LH∗ 9 = h(T ∗ 1 \|\|LH∗ 1 \|\|LH∗ 2 ) 11: Call Reveal on LH12 and get S∗ j ←Reveal(LH12) 12: Call Reveal on LH13 and get SNID∗ j ←Reveal(LH13) 13: if (T ∗ 2 = T2) then 14: computes: LH∗ 14 = LH∗ 2 ⊕h(T ∗ 2 ) 15: Call Extract on LH∗ 17 and get rsn∗←Reveal(LH∗ 17) 16: compute: LH∗ 18 = TrsnTrsc(h(SNIDj\|\|Sj)∗\|\|h(Gwk)\|\|h(RIDi\|\|RPWi)) modq 17: if (LH∗ 19 = LH19) then 18: get the shared session key among user, gateway and sensor node. 19: else return 0 20: end if 21: else return 0 22: end if 23: else return 0 24: end if 25: else return 0 26: end if 27: else return 0 28: end if Algorithm 1 EXPHASH,DLP,DHP RMACCMUA SK, GWk KU−G ←→Ui) k i) In the light of S22, we are able to gain S24: Ui\| ≡(GWk KU−G ←→Ui) Goal 7 7: if (GWID∗ k = GWIDk) then G. SENSOR NODE ADDITION SECTION M1 is from the user to the S19: Ui◁< LH17, LH21, LH22, T4, GWk KU−G ←→Ui > In the light of S19, P2 and R(1), we are able to gain S19: Ui◁< LH17, LH21, LH22, T4, GWk KU−G ←→Ui > In the light of S19, P2 and R(1), we are able to gain VOLUME 8, 2020 S. Zhang et al.: Secure Remote Mutual Authentication Scheme Based on Chaotic Map for UANs S20: Ui\| ≡GWk ∼< LH17, LH21, LH22, T4, GWk KU−G ←→ S20: Ui\| ≡GWk ∼< LH17, LH21, LH22, T4, GWk KU−G ←→ Ui > In the light of P1, P3 and R(4), we are able to gain S21: Ui\| ≡ #(Trsn(x) mod q, SNIDj, Sj, T1, T4, SK, GWk KU−G ←→Ui) In the light of S22, S21and R(2), we are able to gain S22: Ui\| ≡GWk\| ≡(Trsn(x) mod q, SNIDj, Sj, T1, T4, SK, GWk KU−G ←→Ui) In the light of S22, we are able to gain S23: Ui\| ≡GWk\| ≡(GWk KU−G ←→Ui) Goal 8 In the light of S23, P4 and R(3), we are able to gain S24: Ui\| ≡(GWk KU−G ←→Ui) Goal 7 and Deﬁnition 3. Therefore, we have Adv(et, qR, qE) ≤ε. This veriﬁes that our proposed scheme is provably robust and secure against an adversary for deriving session key. g g S21: Ui\| ≡ #(Trsn(x) mod q, SNIDj, Sj, T1, T4, SK, GWk KU−G ←→Ui) Algorithm 1 EXPHASH,DLP,DHP RMACCMUA 1: Eavesdrop the message {LH6, LH10, T1}, {LH15, T2}, {LH17, LH19, LH20, T3}, {LH21, LH22, LH17, T4} during the login and authentication section. VI. INFORMAL ANALYSIS AND DISCUSSION In this section, through the informal security analysis and discussion we show that our proposed scheme is robust and is able to withstand various attacks. B. SECURITY PROOF BASED ON RANDOM ORACLE MODEL In this part, we perform the formal security analysis of our scheme applying the random oracle model to verify that our proposed scheme is robust and safe. We employ the similar method as shown in [11], [34]–[38]. We present two random oracles: Reveal: This random oracle will unconditionally output ﬁxed-length bit string µ from the designated hash function µ = h(γ ). Extract: This random oracle will unconditionally output λ from the designated chaotic map Tλ(x) mod q = β. Theorem 1: Under the assumption that the one-way hash function, DLP and DHP closely behave like a random ora- cle, then our proposed scheme is provably secure against an aggressor obtaining the session key among the user, the gate- way and the sensor node. Proof: Using the upper random oracles to construct an aggressor 0 who is able to derive the identity of a rightful user, the secret key session key among the user, the gateway and the sensor node. The aggressor 0 apply the random ora- cles to operate the experimental algorithm EXPHASH,DLP,DHP RMACCMUA for our proposed remote mutual authentication scheme based on chaotic maps for underwater acoustic networks, which is presented in Algorithm1. Let us deﬁne the success probability for EXPHASH,DLP,DHP RMACCMUA as succ = \| Pr \|EXPHASH,DLP,DHP RMACCMUA = 1\| −1\| where, Pr[E] denotes the probability of an event E. We deﬁne Adv(et, qR, qE) = maxA{succ} as the advantage function for this experiment, where the maximum is deﬁned overall 0 with execution time et, and the number of queries qR and qE made to the Reveal and Extract. Our scheme is veriﬁed robust and secure against an aggressor 0 for session key if Adv(et, qR, qE) ≤ε, for any sufﬁciently small ε > 0. In this experiment, if the aggressor 0 is able to invert the one-way hash function h() and crack DLP and DHP, then he can successfully obtain communication session key and win the game. Nevertheless, it is noticeable that the execu- tion of inverting the hash function and cracking DLP and DHP is impracticable in polynomial time. In other words, AdvHASH 0 (t1) ≤ε and AdvDLP,DHP 0 (t2) ≤ε for any sufﬁ- ciently small ε > 0 according to the section 2.3, Deﬁnition 2 VI. INFORMAL ANALYSIS AND DISCUSSION G. SENSOR NODE ADDITION SECTION 2: Call Extract on LH6 and get rsc∗←Reveal(LH6) 3: Call Extract on LH0 and get g∗←Reveal(LH0) 4: call Reveal on input LH8 to get RIDi, RPWi as (RID∗ i \|\|RPW ∗ i ) ←Reveal(LH8) 5: if (Gw∗ k = Gwk) then 6: computes: LH∗ 2 = h(Gw∗ k) ⊕h(RID∗ i \|\|RPW ∗ i ) 7: if (GWID∗ k = GWIDk) then 8: computers: LH∗ 1 = h(RPW ∗ i \|\|g∗) ⊕h(GWID∗ k\|\|RID∗ i ) 9: if T ∗ 1 = T1 then 10: computes LH∗ 9 = h(T ∗ 1 \|\|LH∗ 1 \|\|LH∗ 2 ) 11: Call Reveal on LH12 and get S∗ j ←Reveal(LH12) 12: Call Reveal on LH13 and get SNID∗ j ←Reveal(LH13) 13: if (T ∗ 2 = T2) then 14: computes: LH∗ 14 = LH∗ 2 ⊕h(T ∗ 2 ) 15: Call Extract on LH∗ 17 and get rsn∗←Reveal(LH∗ 17) 16: compute: LH∗ 18 = TrsnTrsc(h(SNIDj\|\|Sj)∗\|\|h(Gwk)\|\|h(RIDi\|\|RPWi)) modq 17: if (LH∗ 19 = LH19) then 18: get the shared session key among user, gateway and sensor node. 19: else return 0 20: end if 21: else return 0 22: end if 23: else return 0 24: end if 25: else return 0 26: end if 27: else return 0 28: end if Algorithm 1 EXPHASH,DLP,DHP RMACCMUA 1: Eavesdrop the message {LH6, LH10, T1}, {LH15, T2}, {LH17, LH19, LH20, T3}, {LH21, LH22, LH17, T4} during the login and authentication section. GWk KU−G ←→Ui) E. SESSION KEY ATTACK After ﬁnishing the mutual authentication, our scheme negoti- ates a robust session key SK = LH18 = TrsnTrsc(h(SNIDj \|\|Sj)\|\|h(Gwk)\|\|h(RIDi\|\|RPWi)) mod q to transmit informa- tion safely. As we can see, the session key is shielded by hash function, DLP and DHP. If an aggressor plans to get the session key, he is obliged to get the covert parameters {SNIDj, Sj, Gwk, RIDi, RPWi, q, rsc, rsn}. In order to get the aforementioned parameters, an aggressor must try various ways to get {SNIDj, c, GWIDk, IDi, ri, PWi, rq, q, rsc, rsn}, it is impractical to the get aforementioned parameters in polynomial time. Hence, the session key in our proposed scheme is safe enough. A. USER ANONYMITY Our proposed scheme provides the anonymity for the user identity in the exchange of transmitted information. The user computes RIDi = h(IDi\|\|ri) in the registration section and LH9 = h(T1\|\|LH1\|\|LH2) in the login section. The aggres- sor cannot get the ri because of the one-way hash function and cannot obtain the g because of the discrete logarithm. So, our proposed scheme fulﬁll the characteristics of user anonymity. 48292 VOLUME 8, 2020 S. Zhang et al.: Secure Remote Mutual Authentication Scheme Based on Chaotic Map for UANs C. MUTUAL AUTHENTICATION Mutual authentication denotes that the user, the gateway node and the sensor node in UANs are able to authenticate each other. In the user registration section, after getting the registration request message (RIDi, RPWi), the gateway node generates a random digit g and computes {LH0, LH1, LH2}. In the user login section, the SC inspects that the equation LH∗ 5 = LH5 is true or not. If the equation is true, the iden- tity and password are validated successfully. Then, the SC produces a random digit rsc and computes LH6, KU−G, LH7, LH8, LH9, LH10. The SC dispatches the login request mes- sage {LH6, LH10, T1} to the gateway node. F. REPLAY ATTACK In our proposed scheme, we apply the timestamp method to guarantee the freshness of transmitted messages. If an aggressor transmits the captured history information to the communication partner, the session will be ter- minated instantaneously, because the threshold time is greater than 1T. If an aggressor fabricates the trans- mitted information {LH17, LH19, LH20, T3} in the public channel with the current timestamps Ta, he ﬁrst gets {rsn, rsc, SNIDj, Sj, Gwk, RIDi, RPWi, T2}. Nevertheless, it is impractical to get the aforementioned parameters in polynomial time. Even though the aggressor fabricates the transmitted information {LH17, LH19, LH20, T3}, the gateway node will inspect that the equation LH∗ 19 = LH19 is true or not. Similarly, the aggressor is unable to replay or fabricate other information ﬂows. Hence, Our proposed scheme is able to boycott replay attacks. g { 6, 10, 1} g y In the authentication and key management section, the gateway node computes KU−G, decrypts LH10 with KU−G, and then obtains {LH7, LH8, LH9}. The gateway node inspects that the equation LH∗ 9 = LH9 is true or not. If the equations holds, the user is validated successfully. After that, the gateway node computes KG−S, LH12, LH13, LH14 and dispatches message {LH15, T2} to the sensor node. The sensor node computes KG−S, decrypts LH15 with KG−S, and then obtains {LH12, LH13, LH14, LH6}. The sensor node inspects that the equation LH∗ 14 = LH14 is true or not. If the equations holds, the gateway node is validated suc- cessfully. Then, the sensor node produces a random digit rsn and computes LH18, LH19, LH20. The sensor node dispatches the message {LH17, LH19, LH20, T3} to the gateway node. After getting the message, the gateway node inspects that the equation LH∗ 19 = LH19 is true or not. If the equa- tions holds, the sensor node is validated successfully. The gateway node computes LH21, LH22, and dispatches mes- sage {LH21, LH22, LH17, T4} to the user. After getting the message {LH21, LH22, LH17, T4}, the user inspects that the equation LH∗ 22 = LH22 is true or not. If the equation is true, the user validates both the gateway node and the sensor node successfully. G. SMART CARD LOSS ATTACK In our proposed scheme, we use smart card to achieve mutual authentication. So, we ought to consider the sit- uation that the smart card is lost. The SC include {h(), LH1, LH2, LH3, LH4, LH5}, where, LH1 = h(RIDi\|\| RPWi) ⊕h(RPWi\|\|g), LH2 = h(Gwk) ⊕h(RIDi\|\|RPWi), LH3 = ri ⊕h(IDi\|\|PWi), LH4 = rp ⊕h(IDi\|\|PWi), LH5 = h(RIDi\|\|ri) ⊕h(RPWi\|\|ri), ri, rp and g are the random digits produced by the user and the gateway node separately. If the parameters are divulged, the aggressor has no opportunity to get any valuable information about LH2, because he cannot crack the RC to get the private key c and also has no chance to get identity and password from LH1, because he cannot invert the hash function in a valid polynomial time. In summary, B. SENSOR NODE ANONYMITY requires to get the secret value {rsc, g, RIDi, RPWi}. Only the legal user is aware of the values {rsc, ri, rp}, and the gateway node will compute and inspect LH9. It is impractical for the inner aggressor to get the precise values {rsc, ri, rp} in polynomial time. Therefore, the aggressor is unable to imitate the user because he is unable to get the precise secret values from the transmitted information between the user and the gateway node. Similarly, the aggressor is unable to imitate other communications. Sensor node anonymity is that the sensor node’s authentic identity is concealed. In our proposed scheme, the sensor node’s real identity SNIDj does not save any information, and any aggressor is unable to get the sensor node’s real identity straight from the communication information. Because the sensor node registration is executed in a secure off-line envi- ronment prior to their deployment in the target topology area, without knowing the private key of the RC and the identity of target gateway node, it is impractical for the aggressor to get the authentic identity saved in Sj = h(SNIDj\|\|GWIDk\|\|c). So, our proposed scheme fulﬁll the characteristics of sensor node anonymity. D. INSIDER/USER IMPERSONATION ATTACK In our proposed scheme, an aggressor is unable to imitate the user. Supposing an inner aggressor intends to imitate the user, he needs to get LH9 = h(T1\|\|LH1\|\|LH2), LH10 = EKU−G(LH7\|\|LH8\|\|LH9), in other words, an inner aggressor 48293 VOLUME 8, 2020 S. Zhang et al.: Secure Remote Mutual Authentication Scheme Based on Chaotic Map for UANs K. NODE CAPTURE ATTACK even though the smart card is lost, our proposed scheme is still robust and can boycott the smart card loss attack. Supposing an aggressor succeed in catching a sensor node stochastically in the destination ﬁeld. As the sensor nodes are not loaded with tamper-resistant hardware, the aggressor is able to acquire all the secret information stored in the sensor node containing the random digit rsn, Sj and session key. In our proposed scheme, every sensor node has its own identity SNIDj and related Sj. The session key is produced by using the random digits rsn and rsc. Every session key is diverse between the user and the sensor node. The sensor node is dispatched with (SNIDj, Sj) prior to their deployment in the target topology area and the aggressor is able to acquire the secret information (SNIDj, Sj) of the captured sensor node merely. Nevertheless, Other sensor nodes that are not caught are still able to safely exchange information with the lawful users. Consequently, the caught sensor node will not divulge any useful information about the other non-caught sensor nodes. Hence, our proposed scheme can boycott the node capture attack. I. DENIAL OF SERVICE ATTACK In our proposed scheme, we devise a smart card pre- authentication mechanism during the login section to inspect the legitimacy of user at the login terminal. After inserting SC into the card reader of a speciﬁc terminal device, the user inputs his identity IDi and password PWi. The SC computes r∗ i = LH3 ⊕h(IDi\|\|PWi), r∗ p = LH4 ⊕h(IDi\|\|PWi), RID∗ i = h(IDi\|\|r∗ i ), RPW ∗ i = h(PWi\|\|r∗ p ), LH∗ 5 = h(RID∗ i \|\|r∗ i ) ⊕ h(RPW ∗ i \|\|r∗ i ) and compares the value LH∗ 5 with the locally stored value LH5. Only all the information is correct, the equation LH∗ 5 = LH5 hold. The SC can directly inspect the validity of the user without transmitting any information. Denial of service attack cannot be implemented by con- tinuously inputting false identity and password. Therefore, through the smart card pre-authentication mechanism, our protocol is able to boycott denial of service attack. L. MAN-IN-THE-MIDDL ATTACK Man-in-the-middle attack means that an aggressor masquer- ades himself as a legal participant in the process of authenti- cation. In our proposed scheme, M3 is information from the sensor node to the gateway node. If the aggressor is intends to disguises himself as a legitimate sensor node, he needs to con- struct the message {LH17, LH19, LH20, T3}, where LH20 = h(SNIDj\|\|Sj) ⊕h(T3), LH19 = h(LH18\|\|h(SNIDj\|\|Sj)\|\|h(T2)), in other words, the aggressor needs to gain initial values {SNIDj, GWIDk, c, IDi, ri, PWi, rp, rsc, rsn}. Among them, ri and rp are random digits that cannot be completed by random guessing at the same time. It is impractical for the aggressor to compute the parameters {c, rsc, rsn} in polyno- mial time according to DLP and DHP. Besides, the aggres- sor is unable to compute the SK = LH18 = TrsnTrsc (h(SNIDj\|\|Sj)\|\|h(Gwk)\|\|h(RIDi\|\|RPWi)) mod q, because the rsc and rsn are provisionally produced random digits in every session. Therefore, the aggressor cannot masquerade himself as a legal participant and our proposed scheme is able to boycott the man-in-the-middle attack. H. TRACEABILITY ATTACK Traceability is that an aggressor can follow the sensor node and the user in diverse sessions section. The sensor node produces random digit rsn and computes Trsn(x) mod q. The user produces random digit rsc and computes Trsc(x) mod q. Finally, the session key between the user and the sensor node is LH18 = TrsnTrsc(h(SNIDj\|\|Sj) \|\|h(Gwk)\|\|h(RIDi\|\|RPWi)) mod q. The diverse users have diverse RIDi, RPWi and the different sensor nodes have diverse SNIDj, Sj, which makes the session key is different in different communications. It is impractical for the aggressor to trace the user and the sensor node. Therefore, our proposed scheme can boycott the tracking attacks against the user and the sensor node. J. GATEWAY BY PASS ATTACK Supposing an aggressor succeed in catching the mes- sage {LH15, T2} transmitted from the gateway node to the sensor node in public channel, where LH15 = EKG−S(LH12\|\|LH13\|\|LH14\|\|LH6). Subsequently, the aggres- sor fabricates the other message {LH15, T2} and dispatches to the destination node. If the sensor node treats the message {LH∗ 15, T2} as legitimate information, the aggres- sor successfully masquerade himself as a legal gate- way. Nevertheless, if the aggressor wants to compute {LH∗ 15, T2}, he has to compute KG−S = TSjTg(x) mod q, LH12 = h(Sj) ⊕h(RIDi\|\|RPWi), LH13 = h(SNIDj) ⊕ h(Gwk), LH14 = LH2 ⊕h(T2). However, the calcula- tion of LH12, LH13 and LH14 must depends on param- eters {RIDi, RPWi, Sj}, {SNIDj, Gwk}, {RIDi, RPWi, Gwk} respectively. It is apparent that the aggressor is unable to acquire the aforementioned parameters because of the property of the irreversible hash function, DLP and DHP. Similarly, the aggressor is unable to fabricate other gateway nodes information ﬂows. Therefore, our proposed scheme can boycott the gateway by pass attack. M. PERFECT FORWARD SECURITY In our proposed scheme, the session key between the user and the sensor node is computed: RIDi = h(IDi\|\|ri), RPWi = h(PWi\|\|rp), Gwk = h(GWIDk\|\|c), LH6 = Trsc(x) mod q, LH17 = Trsn(x) mod q, SK = LH18 = TrsnTrsc(h(SNIDj\|\|Sj) \|\|h(Gwk)\|\|h(RIDi\|\|RPWi)) mod q. In our proposed scheme, the session key between the user and the sensor node is computed: RIDi = h(IDi\|\|ri), RPWi = h(PWi\|\|rp), Gwk = h(GWIDk\|\|c), LH6 = Trsc(x) mod q, LH17 = Trsn(x) mod q, SK = LH18 = TrsnTrsc(h(SNIDj\|\|Sj) \|\|h(Gwk)\|\|h(RIDi\|\|RPWi)) mod q. Even though the private key of gateway node g is divulged, an aggressor is unable to get rsc and rsn to calculate the session key between the user and the sensor node. The session key depends on the DLP and DHP. An aggres- sor is unable to get rsn from LH17 = Trsn(x) mod q and rsc from LH6 = Trsc(x) mod q in polynomial time. Hence, our proposed scheme is able to offer perfect forward security. 48294 VOLUME 8, 2020 VOLUME 8, 2020 S. Zhang et al.: Secure Remote Mutual Authentication Scheme Based on Chaotic Map for UANs TABLE 8. The security comparison. 5 After forming the underwater wireless sensor network, a new node is likely to be putted in the target topology area because the sensor nodes are small devices with conﬁning energy. For the sake of adding new nodes to the target topol- ogy area, the RC executes the sensor node registration section in a safe off-line environment prior to their deployment in the target topology area. Therefore, the RC can successfully put the new sensor nodes into the target topology area after carrying out the 4.3 section. In conclusion, our proposed scheme realizes the sound reparability and scalability. VII. COMPARISON This section contrasts our proposed scheme with other cor- relative schemes [15], [39], [20], [19], [40] in the aspects of security, computation and storage in the login and authentica- tion sections, which are analyzed in 7.1 security comparison, 7.2 computation comparison and 7.3 storage comparison in detail. The reason why we choose these schemes for compar- ison is that these schemes are all three-party communication based on chaotic map. N. INTEGRITY OF INFORMATION acquires the message from the gateway node, if in step15 T5 −T4 > 1T and in step18 LH∗ 22 ̸= LH22, the scheme will timely detect that the message is incomplete and terminate the communication immediately. In summary, our proposed scheme has the ability to detect errors timely. In our proposed scheme, we apply encryption and veriﬁcation to guarantee the integrity of the transmitted information. In our tripartite mutual authentication scheme, the dispatched information from the user to the gateway node is encrypted with KU−G and veriﬁed by LH9. The dispatched information from the gateway node to the sensor node is encrypted with KG−S and veriﬁed by LH14. The dispatched information from the sensor node to the gateway node is veriﬁed by LH19. The dispatched information from the gateway node to the user is veriﬁed by LH22. If any bit information is lost or wrong during transmission, the equation fails and the receiver will reject the request and terminate the session instantaneously. Therefore, our proposed scheme is able to guarantee the integrity of the transmitted information. O. SOUND REPARABILITY AND SCALABILITY In this portion, we will contrast the security of our pro- posed scheme with other schemes [15], [39], [20], [19], [40] in table 8. In our scheme, the user is able to change his primitive pass- word PWi to a new password PW new i freely and safely and the completion of password modiﬁcation does not require any aid from the gateway node. Furthermore, the aggressor has no ability to revise the password even thought he gains the smart card and the password, which is because the inexact password and random digit will be inspected the equation LH∗ 5 . P. TIMELY ERROR DETECTION In our proposed scheme, no matter what is wrong, the scheme will detect it timely. In the login section, we devise a smart card pre-authentication mechanism to inspect the legitimacy of the user at the login terminal. In the authentication and key agreement section, the ﬁrst message is from the user to the gateway node, the second is from the gateway node to the sensor node, the third is from sensor node to the gateway node and the last message is from the gateway node to the user. When the gateway node gets the message from the user, if in step1 T2 −T1 > 1T and in step4 LH∗ 9 ̸= LH9, the scheme will timely detect error and terminates communication instan- taneously. When the sensor node obtains the message from the gateway node, if in step6 T3 −T2 > 1T and in step9 LH∗ 14 ̸= LH14, the scheme will timely detect that there is something wrong and terminate the communication at once. When the gateway node receives the message from the sensor node, if in step11 T4−T3 > 1T and in step13 LH∗ 19 ̸= LH19, the scheme will timely detect that the message is not right and terminates the communication right away. When the user As shown in the table 8, our proposed scheme is able to boycott multiple attacks and meet more security goals contrasted with other previous schemes [15], [39], [20], [19], [40]. Our scheme and the other schemes both have ability to meet S8 and S15,while the other schemes all have no ability to meet S2, S12 and S13. Schemes [15], [40] cannot resist man-in-middle attack, and also schemes [20], [19], [40] will suffer from the gateway by pass attack. Our proposed scheme and scheme [2] can avoid denial of service attack, while schemes [15], [19], [40] will sustain traceability attack. Schemes [39], [20] fail to withstand replay attack and perfect forward security, while schemes [20], [19] are subjected to smart card loss attack. Schemes [15], [19] fail to resist insider 48295 VOLUME 8, 2020 S. Zhang et al.: Secure Remote Mutual Authentication Scheme Based on Chaotic Map for UANs TABLE 9. Computation comparison. TABLE 10. Storage comparison. TABLE 9. Computation comparison. TABLE 9. Computation comparison. TABLE 10. Storage comparison. TABLE 10. Storage comparison. FIGURE 2. Computation comparison. memory in win7 64-bit. C. STORAGE COMPARISION In this portion, we will contrast the storage of our proposed scheme with other schemes [15], [39], [20], [19], [40] in the login and authentication section in table 10 and ﬁgure 3. P. TIMELY ERROR DETECTION The computational times of hash function, Symmetric encryption/decryption and Chebyshev polynomial are approximately 0.033ms, 0.21ms and 0.46ms respectively. Since the scheme [39] only employ the hash function and symmetric key encryption/ decryption operations, scheme [39] takes the least time. Although the scheme [40] merely apply the hash function and chaotic maps, the computing time is still longer than scheme [15]. Schemes [15], [20], [19] and our proposed scheme all require hash function, chaotic maps and symmetric key encryption/ decryption three operations to achieve mutual authentication. Compared with the schemes [15], our scheme needs more time to complete mutual authentication. However, the number of exchange messages in our scheme is four, scheme [15] requires three messages exchange to accomplish mutual authentication. Compared with the schemes [19], [20], the computational scheme is more efﬁcient and applies UANs. FIGURE 2. Computation comparison. attack and only scheme [15] cannot withstand user imperson- ation attack. Only scheme [15] and our scheme realize timely error detection function. Only scheme [39] and our scheme achieve sensor node anonymity, while only scheme [19] do not implement user anonymity. REFERENCES [1] C. Peng, X. Du, K. Li, and M. Li, ‘‘An ultra-lightweight encryption scheme in underwater acoustic networks,’’ J. Sensors, vol. 2016, pp. 1–10, Mar. 2016. [2] G. Han, J. Jiang, N. Sun, and L. Shu, ‘‘Secure communication for under- water acoustic sensor networks,’’ IEEE Commun. Mag., vol. 53, no. 8, pp. 54–60, Aug. 2015. [3] E. Souza, H. C. Wong, I. Cunha, A. A. F. Loureiro, L. F. M. Vieira, and L. B. Oliveira, ‘‘End-to-end authentication in under-water sensor networks,’’ in Proc. IEEE Symp. Comput. Commun. (ISCC), Jul. 2013, pp. 000299–000304. [4] X. Du, C. Peng, and K. Li, ‘‘A secure routing scheme for underwater acoustic networks,’’ Int. J. Distrib. Sensor Netw., vol. 13, no. 6, pp. 1–13, Jun. 2017. [5] R. Diamant, P. Casari, and S. Tomasin, ‘‘Cooperative authentication in underwater acoustic sensor networks,’’ IEEE Trans. Wireless Commun., vol. 18, no. 2, pp. 954–968, Feb. 2019. FIGURE 3. Storage comparison. [6] A. K. Das, ‘‘A secure and robust password-based remote user authentica- tion scheme using smart cards for the integrated EPR information system,’’ J. Med. Syst., vol. 39, no. 3, p. 25, Mar. 2015. User gets the message {LH21, LH22, LH17, T4} from the gate- way node and the storage cost is 416(128 + 128 + 128 + 32) bits. User gets the message {LH21, LH22, LH17, T4} from the gate- way node and the storage cost is 416(128 + 128 + 128 + 32) bits. [7] C.-T. Li, C.-Y. Weng, C.-C. Lee, and C.-C. Wang, ‘‘A hash based remote user authentication and authenticated key agreement scheme for the inte- grated EPR information system,’’ J. Med. Syst., vol. 39, no. 11, p. 144, Nov. 2015. Gateway node dispatches the message {LH15, T2} to the sensor node and the cost is 160(128+32) bits. Gateway node dispatches the message {LH21, LH22, LH17, T4} to the user and the cost is 416(128 + 128 + 123 + 32) bits. Gateway node gets the message {LH6, LH10, T1} from user and the cost is 288(128 + 128 + 32) bits. Gateway node gets the message {LH17, LH19, LH20, T3} from sensor node and the cost is 416(128 + 128 + 128 + 32) bits. [8] J. Jung, D. Kang, D. Lee, and D. Won, ‘‘An improved and secure anony- mous biometric-based user authentication with key agreement scheme for the integrated EPR information system,’’ PLoS ONE, vol. 12, no. 1, 2017, Art. no. REFERENCES e0169414. [9] A. M. Koya and P. P. Deepthi, ‘‘Anonymous hybrid mutual authentication and key agreement scheme for wireless body area network,’’ Comput. Netw., vol. 140, pp. 138–151, Jul. 2018. [10] C.-C. Chang and H.-D. Le, ‘‘A provably secure, efﬁcient, and ﬂexible authentication scheme for ad hoc wireless sensor networks,’’ IEEE Trans. Wireless Commun., vol. 15, no. 1, pp. 357–366, Jan. 2016. [11] D. Kumar, S. Chand, and B. Kumar, ‘‘Cryptanalysis and improvement of an authentication protocol for wireless sensor networks applications like safety monitoring in coal mines,’’ J. Ambient Intell. Humanized Comput., vol. 10, no. 2, pp. 641–660, Feb. 2019. Sensor node dispatches the message {LH17, LH19, LH20, T3} to the gateway node and the cost is 416(128+128+ 128+32) bits. Sensor node gets the message {LH15, T2} from the gateway node and the cost is 160(128 + 32) bits. [12] H.-Y. Lin, ‘‘Improved chaotic maps-based password-authenticated key agreement using smart cards,’’ Commun. Nonlinear Sci. Numer. Simul., vol. 20, no. 2, pp. 482–488, Feb. 2015. Consequently, the total storage cost in our scheme is 2560 bits. We also calculate the total storage of other schemes in [15], [39], [20], [19], [40] separately. The energy is mainly consumed for dispatching/getting the mes- sage [41]. The total storage cost in our scheme is 2560bits, which is higher than schemes [15], [39], but lower than schemes [20], [19], [40]. Scheme [39] shows that scheme [15] is vulnerable to sensor node impersonation attack and man- in-the-middle attack. The number of transmitted messages in scheme [39] is three, however, our scheme demands four messages to ﬁnish mutual authentication, so our total storage is slightly higher than scheme [39]. [13] H. Zhu, ‘‘Cryptanalysis and improvement of a mobile dynamic ID authen- ticated key agreement scheme based on chaotic maps,’’ Wireless Pers. Commun., vol. 85, no. 4, pp. 2141–2156, Dec. 2015. [14] T.-T. Truong, M.-T. Tran, and A.-D. Duong, ‘‘Improved Chebyshev polynomials-based authentication scheme in client-server environment,’’ Secur. Commun. Netw., vol. 2019, pp. 1–11, Jan. 2019. [15] S. Kumari, X. Li, F. Wu, A. K. Das, H. Arshad, and M. K. Khan, ‘‘A user friendly mutual authentication and key agreement scheme for wireless sensor networks using chaotic maps,’’ Future Gener. Comput. Syst., vol. 63, pp. 56–75, Oct. 2016. [16] L. Zhang, H. Luo, L. Zhao, and Y. Zhang, ‘‘Privacy protection for point-of- care using chaotic maps-based authentication and key agreement,’’ J. Med. Syst., vol. 42, no. B. COMPUTATION COMPARISION In this portion, we will contrast the computation of our pro- posed scheme with other schemes [15], [39], [20], [19], [40] in table 9 and ﬁgure 2. Since the time for computing XOR operation and string concatenation is ignored as compared with the other operations[16], [26], [40], we only consider the time to calculate one-way hash function, Chebyshev polynomial and symmetric key encryption/ decryption. For comparing the computation cost with other schemes, all operations were implemented at IDEA jdk 1.8.0_60 using an Intel(R)Core(TM)i5-42102M CPU @ 2.60GHz with 8G We suppose that user identity and password are 64 bits, the timestamp is 32 bits, the other values are all 128 bits. In our scheme, the smart card contains the elements {h(), LH1, LH2, LH3, LH4, LH5}, so the smart card storage cost is 640(128 + 128 + 128 + 128 + 128) bits, which is the same as schemes [15], [40], but lower than schemes [39]. The schemes [19], [20] do not make use of smart card. User dispatches the message {LH6, LH10, T1} to the gate- way node and the storage cost is 288(128 + 128 + 32) bits. 48296 VOLUME 8, 2020 VOLUME 8, 2020 S. Zhang et al.: Secure Remote Mutual Authentication Scheme Based on Chaotic Map for UANs FIGURE 3. Storage comparison. FIGURE 3. Storage comparison. REFERENCES 12, p. 250, Dec. 2018. [17] G. Gao, X. Peng, Y. Tian, and Z. Qin, ‘‘A chaotic maps-based authentica- tion scheme for wireless body area networks,’’ Int. J. Distrib. Sensor Netw., vol. 12, no. 7, Jul. 2016, Art. no. 2174720. [18] Q. Xie, B. Hu, X. Tan, and D. S. Wong, ‘‘Chaotic maps-based strong anonymous authentication scheme for roaming services in global mobil- ity networks,’’ Wireless Pers. Commun., vol. 96, no. 4, pp. 5881–5896, Oct. 2017. VIII. CONCLUSION VIII. CONCLUSION In this article, we present a chaotic maps remote user authen- tication and key agreement scheme for underwater acoustic networks. Our scheme turns out to be robust and secure based on the DLP and DHP. By applying the BAN logic and random oracle model, we have certiﬁed that our scheme is able to accomplish mutual authentication and negotiate session key among the user, the gateway and the sensor node. We have showed that our proposed scheme is safe and can meet ten security requirements and seven security goals. Besides, our scheme is more efﬁcient compared with the other previous schemes. [19] Q. Xie, J. Zhao, and X. Yu, ‘‘Chaotic maps-based three-party password- authenticated key agreement scheme,’’ Nonlinear Dyn., vol. 74, no. 4, pp. 1021–1027, Dec. 2013. [20] C.-C. Lee, C.-T. Li, S.-T. Chiu, and Y.-M. Lai, ‘‘A new three-party- authenticated key agreement scheme based on chaotic maps without pass- word table,’’ Nonlinear Dyn., vol. 79, no. 4, pp. 2485–2495, Mar. 2015. [21] A. Jabbari and J. B. Mohaseﬁ, ‘‘Improvement in new three-party- authenticated key agreement scheme based on chaotic maps without pass- word table,’’ Nonlinear Dyn., vol. 95, no. 4, pp. 3177–3191, Mar. 2019. [22] M. Qi and J. Chen, ‘‘Anonymous biometrics-based authentication with key agreement scheme for multi-server environment using ECC,’’ Multimedia Tools Appl., vol. 78, no. 19, pp. 27553–27568, Oct. 2019. VOLUME 8, 2020 48297 S. Zhang et al.: Secure Remote Mutual Authentication Scheme Based on Chaotic Map for UANs [23] R. Ali, A. K. Pal, S. Kumari, M. Karuppiah, and M. Conti, ‘‘A secure user authentication and key-agreement scheme using wireless sensor net- works for agriculture monitoring,’’ Future Gener. Comput. Syst., vol. 84, pp. 200–215, Jul. 2018. [39] J. Li, W. Zhang, S. Kumari, K.-K.-R. Choo, and D. Hogrefe, ‘‘Security analysis and improvement of a mutual authentication and key agreement solution for wireless sensor networks using chaotic maps,’’ Trans. Emerg. Telecommun. Technol., vol. 29, no. 6, p. e3295, Jun. 2018. [24] X. Li, J. Niu, S. Kumari, F. Wu, A. K. Sangaiah, and K.-K.-R. Choo, ‘‘A three-factor anonymous authentication scheme for wireless sensor networks in Internet of Things environments,’’ J. Netw. Comput. Appl., vol. 103, pp. 194–204, Feb. 2018. [40] A. Irshad, S. A. Chaudhry, Q. Xie, X. Li, M. S. Farash, S. Kumari, and F. Wu, ‘‘An enhanced and provably secure chaotic map-based authenticated key agreement in multi-server architecture,’’ Arabian J. Sci. Eng., vol. VIII. CONCLUSION 43, no. 2, pp. 811–828, Feb. 2018. [25] A. Ghani, K. Mansoor, S. Mehmood, S. A. Chaudhry, A. U. Rahman, M. N. Saqib, ‘‘Security and key management in IoT-based wireless sensor networks: An authentication protocol using symmetric key,’’ Int. J. Com- mun. Syst., vol. 32, no. 16, p. e4139, 2019. [41] R. Amin and G. P. Biswas, ‘‘A secure light weight scheme for user authentication and key agreement in multi-gateway based wireless sensor networks,’’ Ad Hoc Netw., vol. 36, no. 1, pp. 58–80, 2016. [26] S. Qiu, G. Xu, H. Ahmad, and L. Wang, ‘‘A robust mutual authentication scheme based on elliptic curve cryptography for telecare medical informa- tion systems,’’ IEEE Access, vol. 6, pp. 7452–7463, 2017. SHUAILIANG ZHANG received the bachelor’s degree in computer science and technology from the Henan Institute of Science and Technology, Xinxiang, China, in 2015. He is currently pursuing the Ph.D. degree in computer science and technol- ogy with Qinghai Normal University, Xining. His research interests include network and information security, wireless sensor networks, and underwater sensor networks. [27] S. Namasudra and P. Roy, ‘‘A new secure authentication scheme for cloud computing environment,’’ Concurrency Comput., Pract. Exper., vol. 29, no. 20, p. e3864, 2017. [28] H. Lai, M. A. Orgun, J. Xiao, J. Pieprzyk, L. Xue, and Y. Yang, ‘‘Provably secure three-party key agreement protocol using Chebyshev chaotic maps in the standard model,’’ Nonlinear Dyn., vol. 77, no. 4, pp. 1427–1439, Sep. 2014. [29] M. Gohar, F. Bashir, J.-G. Choi, S.-J. Koh, and W. Ahmad, ‘‘A hash-based distributed mapping control scheme in mobile locator-identiﬁer separa- tion protocol networks,’’ Int. J. Netw. Manage., vol. 27, no. 2, p. e1961, Mar. 2017. [30] S. Jangirala, S. Mukhopadhyay, and A. K. Das, ‘‘A multi-server environ- ment with secure and efﬁcient remote user authentication scheme based on dynamic ID using smart cards,’’ Wireless Pers. Commun., vol. 95, no. 3, pp. 2735–2767, Aug. 2017. XIUJUAN DU received the M.S. degree in radio physics from Lanzhou University, Lanzhou, China, and the Ph.D. degree in computer applica- tion technology from Tianjin University, Tianjin, China. She is currently a Professor with the Provincial Key Laboratory of the Internet of Things, Qinghai Normal University, Xining, China. Her research interests include network and information security, mobile ad hoc networks, and underwater sensor networks, including network modeling, network protocol design, performance evaluation, optimization algorithms, and distributed computing and their applications. VIII. CONCLUSION She has received the New Century Excellent Talent from Education Ministry, China, in 2011. [31] R. Madhusudhan and R. C. Mittal, ‘‘Dynamic ID-based remote user password authentication schemes using smart cards: A review,’’ J. Netw. Comput. Appl., vol. 35, no. 4, pp. 1235–1248, Jul. 2012. [32] D. Wang and P. Wang, ‘‘Two birds with one stone: Two-factor authentica- tion with security beyond conventional bound,’’ IEEE Trans. Depend. Sec. Comput., vol. 15, no. 4, pp. 708–722, Jul./Aug. 2015. [33] D. Wang, W. Li, and P. Wang, ‘‘Measuring two-factor authentication schemes for real-time data access in industrial wireless sensor networks,’’ IEEE Trans. Ind. Informat., vol. 14, no. 9, pp. 4081–4092, Sep. 2018. [34] D. Mishra, A. K. Das, and S. Mukhopadhyay, ‘‘A secure user anonymity- preserving biometric-based multi-server authenticated key agreement scheme using smart cards,’’ Expert Syst. Appl., vol. 41, no. 18, pp. 8129–8143, 2014. [35] Y. Lu, L. Li, H. Peng, and Y. Yang, ‘‘A secure and efﬁcient mutual authentication scheme for session initiation protocol,’’ Peer-to-Peer Netw. Appl., vol. 9, no. 2, pp. 449–459, Mar. 2016. [36] S. A. Chaudhry, I. Khan, A. Irshad, M. U. Ashraf, M. K. Khan, and H. F. Ahmad, ‘‘A provably secure anonymous authentication scheme for session initiation protocol,’’ Secur. Commun. Netw., vol. 9, no. 18, pp. 5016–5027, 2016. XIN LIU received the bachelor’s degree in computer science and technology from Shanxi University, Taiyuan, China, in 2004. She is currently pursuing the Ph.D. degree in computer science and technology with Qinghai Normal University, Xining, China. Her research interests include wireless sensor networks and underwater sensor networks, including network modeling and network protocol design. XIN LIU received the bachelor’s degree in computer science and technology from Shanxi University, Taiyuan, China, in 2004. She is currently pursuing the Ph.D. degree in computer science and technology with Qinghai Normal University, Xining, China. Her research interests include wireless sensor networks and underwater sensor networks, including network modeling and network protocol design. [37] J. Jung, J. Moon, D. Lee, and D. Won, ‘‘Efﬁcient and security enhanced anonymous authentication with key agreement scheme in wireless sensor networks,’’ Sensors, vol. 17, no. 3, p. 644, 2017. [38] S. Kumari, M. Karuppiah, A. K. Das, X. Li, F. Wu, and V. Gupta, ‘‘Design of a secure anonymity-preserving authentication scheme for session initia- tion protocol using elliptic curve cryptography,’’ J. Ambient Intell. Human- ized Comput., vol. 9, no. 3, pp. 643–653, Jun. 2018. VIII. CONCLUSION 48298 VOLUME 8, 2020
https://openalex.org/W2159493447	https://www.sciencepubco.com/index.php/ijamr/article/download/1/6	English	null	He's variational method for a (2 + 1)-dimensional soliton equation	International journal of applied mathematical research	2,012	cc-by	1,941	Abstract In this Letter, a (2 + 1)-dimensional soliton equation is studied by He’s variational approach. The solitary solutions are obtained using the Ritz method. Keywords: Variational method, Solitary solution, Soliton equation, Ritz method, exact solution. International Journal of Applied Mathematical Research, 1 (1) (2012) 1-7 c⃝Science Publishing Corporation www.sciencepubco.com/index.php/IJAMR International Journal of Applied Mathematical Research, 1 (1) (2012) 1-7 c⃝Science Publishing Corporation www.sciencepubco.com/index.php/IJAMR International Journal of Applied Mathematical Research, 1 (1) (2012) 1-7 c⃝Science Publishing Corporation www.sciencepubco.com/index.php/IJAMR M. T. Darvishi and M. Najaﬁ M. T. Darvishi and M. Najaﬁ Department of Mathematics, Razi University, Kermanshah 67149, Iran Email:darvishimt@yahoo.com Department of Mathematics, Anar Branch, Islamic Azad University, Anar, Iran Email:m najaﬁ82@yahoo.com Department of Mathematics, Anar Branch, Islamic Azad University, Anar, Iran Email:m najaﬁ82@yahoo.com 1 Introduction In this letter, we consider the following (2 + 1) dimensions soliton equation to reveal new exact traveling wave solutions using He’s variational method i ut + uxx + u v = 0, vt + vy + (v u∗)x = 0, (1) (1) where i = √−1, u(x, y, t) is a complex function and v(x, y, t) is a real function which has studied in [1] by using the bifurcation theory. where i = √−1, u(x, y, t) is a complex function and v(x, y, t) is a real function which has studied in [1] by using the bifurcation theory. Soliton is an important feature of nonlinearity and can be found in many applications of science. Many eﬀective and reliable methods are used in the literature to investigate solitons and in particular multiple soliton solutions of completely integrable equations. Nonlinear evolution equations have been noticed in plasma physics, ﬂuid dynamics, optical ﬁbers, biological systems 2 M.T. Darvishi, M.Najaﬁ M.T. Darvishi, M.Najaﬁ 2 and other applications. In the past decades, there has been an increased inter- est on studying the nonlinear evolution equations. Exact solutions play a vital role in understanding various qualitative and quantitative features of nonlinear phenomena. There are diverse classes of interesting exact solutions, such as traveling wave solutions, but it often needs speciﬁc mathematical techniques to construct exact solutions due to the nonlinearity present in dynamics [2, 3]. It has recently become more interesting to obtain exact solutions of nonlinear partial diﬀerential equations (NPDEs) using symbolic computation softwares such as Maple, Mathematica and Matlab that facilitate complex and tedious algebraical computations. In recent years, various eﬀective methods have been developed to ﬁnd the exact solutions of NPDEs, such as tanh-function method [4, 5, 6, 7, 8, 9, 10, 11], generalized hyperbolic function method [12], homo- geneous balance method [13, 14], Jacobi-elliptic function method [15, 16, 17], exp-function method [18, 19], auxiliary equation method [20, 21, 22, 23, 24] and so on, e.g. see [25, 26, 27]. 2 He’s variational method In order to seek exact solutions of Eq. (1), we suppose that u(x, y, t) = φ(ξ) exp(i η), v(x, y, t) = v(ξ), η = k x + l y + λ t, ξ = K(x + L y −2k t), (2) (2) where φ(ξ) and v(ξ) are real functions, k, l, λ, K and L are real constants to be determined later. Substituting Eq. (2) into Eq. (1), we have where φ(ξ) and v(ξ) are real functions, k, l, λ, K and L are real constants to be determined later. Substituting Eq. (2) into Eq. (1), we have K2 φ ′′(ξ) −(λ + k2) φ(ξ) + φ(ξ) v(ξ) = 0, (3) (L −2 k)v ′(ξ) + (φ2(ξ)) ′ = 0. (4) (3) (4) where prime denotes the diﬀerentiation with respect to ξ. Integrating Eq. (4) with respect to ξ and taking the integration constant as zero yields v(ξ) = 1 2 k −L φ2(ξ), if L ̸= 2k. (5) (5) Substituting Eq. (5) into Eq. (3) yields Substituting Eq. (5) into Eq. (3) yields φ ′′(ξ) + α φ(ξ) −β φ3(ξ) = 0, (6) (6) and α = −λ −k2 K2 , β = 1 K2(L −2k), L ̸= 2k. 3 He’s variational method According to Ref. [29], upon using He’s semi-inverse method [30], we can arrive at the following variational formulation: According to Ref. [29], upon using He’s semi-inverse method [30], we can arrive at the following variational formulation: J(φ) = Z ∞ 0 ·1 2 (φ ′)2 −α 2 φ2 + β 4 φ4 ¸ dξ. (7) (7) We assume the soliton solution in the following form We assume the soliton solution in the following form φ(ξ) = Asech(ξ) (8) (8) where A is an unknown constant to be further determined. By Substituting Eq. (8) into Eq. (7) we obtain J = 1 6 β A4 + 1 6 A2 −1 2 α A2. (9) (9) For making J stationary with respect to A For making J stationary with respect to A ∂J ∂A = 2 3 β A3 + 1 3 A −α A (10) ∂J ∂A = 2 3 β A3 + 1 3 A −α A (10) from Eq. (10), we have A = ± p 2β (3 α −1) 2β (11) from Eq. 2 He’s variational method (10), we have p 2β (3 α 1) A = ± p 2β (3 α −1) 2β (11) (11) and α = −λ −k2 K2 , β = 1 K2(L −2k), L ̸= 2k. The solitary solution is, therefore, obtained as follows: The solitary solution is, therefore, obtained as follows: φ(ξ) = ± p 2β (3 α −1) 2β sech(ξ). (12) (12) By Eqs. (2) and (5), we have u(x, y, t) = Asech(ξ) ei η = ± p 2β (3 α −1) 2β sech(ξ) ei η, (13) v(ξ) = 3 α −1 2β (2 k −L) sech2(ξ), if L ̸= 2k, (13) where η = k x + l y + λ t , ξ = K(x + L y −2k t). (14) (14) We search another soliton solution in the form φ(ξ) = Dsech2(ξ) (15) φ(ξ) = Dsech2(ξ) (15) 4 4 M.T. Darvishi, M.Najaﬁ 4 M.T. Darvishi, M.Najaﬁ where D is an unknown constant to be further determined. By Substituting Eq. (15) into Eq. (7) we obtain J = 4 35 D4β −1 3 α D2 + 4 15 D2. (16) (16) For making J stationary with respect to D ∂J ∂D = 16 35 D3β −2 3 α D + 8 15 D. (17) (17) From Eq. (17), we have D = ± p 42β (5 α −4) 12β (18) D = ± p 42β (5 α −4) 12β (18) (18) and α = −λ −k2 K2 , β = 1 K2(L −2k), L ̸= 2k. The solitary solution is, therefore, obtained as follows: The solitary solution is, therefore, obtained as follows: φ(ξ) = ± p 42β (5 α −4) 12β sech2(ξ). (19) (19) By Eqs. (2) and (5), we have By Eqs. (2) and (5), we have u(x, y, t) = Dsech2(ξ) ei η = p 42β (5 α −4) 12β sech2(ξ) ei η, (20) v(ξ) = 73 α −1 24β (2 k −L) sech4(ξ), if L ̸= 2k, (20) where where η = k x + l y + λ t , ξ = K(x + L y −2k t). (21) (21) 3 Conclusions In this letter, we have used He’s variational method to search for solitary solutions. He’s variational principle is a very dominant instrument to ﬁnd the solitary solutions for various nonlinear equations. References [1] C. Ye, W. Zhang, New explicit solutions for (2 + 1)-dimensional soliton equation, Chaos Solitons Fractals, 44 (2011) 1063–1069. 5 5 He’s variational method [2] W.X. Ma, Diversity of exact solutions to a restricted Boiti-Leon- Pempinelli dispersive long-wave system, Phys. Lett. A, 319 (2003) 325– 333. [3] H.C. Hu, B. Tong, S.Y. Lou, Nonsingular positon and complexiton solu- tions for the coupled KdV system, Phys. Lett. A, 351 (2006) 403–412. [4] W. Malﬂiet, Solitary wave solutions of nonlinear wave equations, Am. J. Phys., 60 (1992) 650–654. [5] E.J. Parkes, B.R. Duﬀy, An automated tanh-function method for ﬁnding solitary wave solutions to nonlinear evolution equations, Comput. Phys. Commun., 98 (1996) 288–296. [6] E.G. 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https://openalex.org/W3139499612	http://old.scielo.br/pdf/riem/v14n3/1983-4195-riem-14-3-e14313.pdf	English	null	Behavior of CFA and H-section steel piles in lateral loading: experimental and numerical analysis	Revista IBRACON de Estruturas e Materiais	2,021	cc-by	9,947	Behavior of CFA and H-section steel piles in lateral loading: experimental and numerical analysis Behavior of CFA and H-section steel piles in lateral loading: experimental and numerical analysis Comportamento de estacas escavadas em hélice contínua (CFA) e de estacas metálicas em seção H sob carregamento lateral: análise experimental e numérica Alex Micael Dantas de Sousaa  Yuri Daniel Jatobá Costab  Arthur Gomes Dantas de Araujoc  Carina Maia Lins Costab  aUniversidade de São Paulo – USP, Escola de Engenharia de São Carlos, Departamento de Engenharia de Estruturas, São Carlos, SP, Brasil bUniversidade Federal do Rio Grande do Norte – UFRN, Departamento de Engenharia Civil, Natal, RN, Brasil cUniversidade Federal Rural do Semi-Árido – UFERSA, Departamento de Engenharia Civil, Angicos, RN, Brasil Abstract: The behavior of continuous flight auger (CFA) piles and steel H-section piles to lateral loading is investigated using numerical analyses supported by field tests. A three-dimensional finite element numerical model to lateral load is presented. The numerical model was validated with the results of twelve lateral load tests performed on CFA and steel H-section piles installed in two deposits of sandy soils. The three- dimensional approach proposed in this study is in good agreement with the response observed with the field tests, and thus represents a reliable soil-pile interaction for laterally loaded piles in sandy soil. Parametric analyses were used to assess the influence of relevant variables to lateral soil-pile interaction. Major findings of this paper indicate that the ultimate lateral load of short rigid piles is considerably more influenced by changes in soil-pile relative stiffness than that of long flexible units. Pile diameter and soil-pile interface friction are found to exert a marked effect on the lateral load of CFA piles, while soil dilatancy is found to play a minor influence on the response of CFA piles. Received 16 June 2020 Accepted 26 September 2020 Keywords: laterally loaded pile, soil-pile interaction, three-dimensional finite element method, field test, p- y curve. Resumo: O comportamento de estacas escavadas com trado em hélice contínua (CFA) e estacas metálicas de seção H sujeitas à cargas horizontais é investigado através de análises numéricas baseadas em resultados experimentais. O modelo numérico foi validado com os resultados de 12 ensaios de carregamento lateral de estacas realizados em estacas CFA e estacas metálicas de seção H instaladas em duas regiões de solos arenosos. How to cite: A. M. D. Sousa, Y. D. J. Costa, A. G. D. Araujo, and C. M. L. Costa, “Behavior of CFA and H-section steel piles in lateral loading: experimental and numerical analysis,” Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021, https://doi.org/10.1590/S1983- 41952021000300013 Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 g This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Corresponding author: Alex Micael Dantas de Sousa. E-mail: alex_dantas@usp.br Financial support: Fundação de Amparo à Pesquisa do Estado de São Paulo, Grant/Award Number: FAPESP - 2018/21573-2; Conselho Nacional de Desenvolvimento Científico e Tecnológico. Conflict of interest: Nothing to declare. This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021\| https://doi.org/10.1590/S1983-41952021000300013 1/17 Financial support: Fundação de Amparo à Pesquisa do Estado de São Paulo, Grant/Award Number: FAPESP - 2018/21573-2; Conselho Nacional de Desenvolvimento Científico e Tecnológico. Conflict of interest: Nothing to declare. This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Corresponding author: Alex Micael Dantas de Sousa. E-mail: alex_dantas@usp.br Financial support: Fundação de Amparo à Pesquisa do Estado de São Paulo, Grant/Award Number: FAPESP - 2018/21573-2; Conselho Nacional de Desenvolvimento Científico e Tecnológico. 1 INTRODUCTION 3-D FEM methods should be preferred over p–y curve methods because they are more rigorous in their analytical methodology. Many numerical studies have been extensively conducted to assess the behavior of piles under lateral loading. Although several studies used 3-D FEM analysis supported by field data [5], [12], [15], [20]–[22], some relevant aspects of the problem involving laterally loaded pile behavior have not been well understood. For instance, while the effect of the pile bending stiffness on pile response has been widely investigated in the literature [12], [23], [24], very little importance has been granted to the soil-pile relative stiffness, even though this variable is comparatively more suitable for describing the soil-pile interaction. Moreover, different studies present contradictory conclusions on the influence of pile diameter on the lateral behavior of piles, which warrants further research. Terzaghi [25] and Vesic [26] postulated that the pile diameter did not influence the subgrade reaction modulus (kh). Using numerical and large-scale tests, Ashford and Juirnarongrit [27] and Fan and Long [12] verified that the pile diameter has an insignificant influence on kh. However, other investigations involving experimental and analytical components found that kh and the ultimate lateral capacity (Hu) increase linearly with the pile diameter [5], [23], [28]. Another parameter of interest for the soil-pile interaction that needs further examination is the soil-pile interface property (interface friction for granular soils or interface adhesion for non-granular soils). From a FEM analysis validated against field tests, Choi et al. [15] found that the soil-pile interface friction coefficient had a great impact on the lateral load capacity of bored piles embedded in weathered granite soil. On the other hand, the computational results presented by Kim and Jeong [5] indicated that the interface adhesion exerts an insignificant influence on the subgrade reaction modulus obtained with large diameter piles in clay. Perceived gaps and conflicting results in the literature support the need for additional research using reliable 3-D FE models. The purpose of this study is to present the results of a 3-D FE model developed to represent the lateral load behavior of concrete flight auger (CFA) piles and steel H-section piles installed in two profiles of sandy soils. Accuracy of the proposed model was checked against the results of a total of 12 lateral load tests performed in the field with both CFA piles and steel H-section piles. 1 INTRODUCTION Soil-structure interaction has been extensively investigated regarding axial vertical loads in deep foundations [1]–[3]. At the same time, in several circumstances, piles need to be designed to support major lateral loads, as a result of the action of wind, water flow, horizontal earth pressure [4], earthquakes, and traffic movement. Bridges [5], tall buildings, transmission lines, retaining walls [4], offshore structures [6]–[8], wharfs [9], are a few examples of structures in which lateral loads assume primary significance. Among existing methods for the analysis of laterally loaded single piles, p–y curve methods are largely employed in current design practice. In the p–y curve approach, a laterally loaded pile is treated as a beam on an elastic foundation with independent springs. The soil resistance per unit length (p) is assumed to hold a non-linear relationship with the pile lateral displacement (y). The relationship between p and y is known as the subgrade reaction modulus kh (kh = p/y). The soil-pile system is assumed to reach the ultimate lateral resistance at a particular pile deflection level. The non-linearity between soil resistance and pile displacement has been substantiated by numerous full-scale tests [5], [10]–[14]. Although versatile and practical for structural design purposes, the p–y curve approach has several shortcomings, such as: (1) continuity of the soil is not accounted for, since the springs are uncoupled; (2) shearing forces in the pile- soil contact and pile toe are neglected; (3) pile diameter and bending stiffness are only considered indirectly; (4) soil dilatancy is neglected, even though experimental evidence reveals that pile-soil resistance can significantly increase with increasing soil dilatancy [12], [15]; (5) the soil-pile ultimate resistance (pu) is calculated according to Rankine´s theory, which assumes full slippage conditions between the soil and the structure [16], [17], even though perfectly smooth conditions are not found in the field; (6) the coefficient of horizontal earth pressure (K) is considered equal to the to the at-rest earth pressure coefficient (K0) rather than the post-installation earth pressure coefficient, which may result in underestimated ultimate soil resistances for piles [12], [18], [19]. An alternative to the p–y curve approach is the three-dimensional finite element method (3-D FEM) analysis. Although comparatively more complex to implement, 3-D FEM can reliably simulate several aspects of the problem involving a laterally loaded pile that are unable to be accomplished in p–y curve methods. Behavior of CFA and H-section steel piles in lateral loading: experimental and numerical analysis A abordagem tridimensional proposta neste estudo está de acordo com a resposta observada nos ensaios de campo e, portanto, representa uma interação solo-estaca confiável para estacas carregadas lateralmente em solo arenoso. Análises paramétricas foram utilizadas para avaliar a influência de variáveis relevantes para a interação lateral solo-estaca. As principais descobertas deste trabalho indicam que a capacidade de carga lateral última de estacas rígidas curtas é consideravelmente mais influenciada por mudanças na rigidez relativa do solo-estaca do que no caso de estacas flexíveis longas. O diâmetro da estaca e o atrito da interface solo-estaca tem efeito significativo na capacidade de carga lateral das estacas CFA, enquanto a dilatância do solo exerce uma menor influência na resposta destas. Palavras-chave: estacas carregadas lateralmente, interação solo-estaca, método dos elementos finitos tridimensional, ensaios de campo, curva p-y. 1/17 A. M. D. Sousa, Y. D. J. Costa, A. G. D. Araujo, and C. M. L. Costa Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 2 SITE CHARACTERISTICS The experimental component of this investigation was carried out at a site located in the City of Natal (05047’42”S, 35012’34”W), in the Northeast region of Brazil (Figure 1a). Tertiary and quaternary sediments compose the subsoil of Natal, which is typical of vast extensions of the upper Brazilian northeast region. The tertiary deposits comprise a continuous sequence of clastic sediments, ranging from clay to conglomerate, which presents lateritic features with iron oxides bonding the soil particles together [29]. Overlying the tertiary deposits are quaternary deposits of eolian (dune) and alluvial sediments, composed of quartz sands with sub-rounded fine to coarse grains. Specifically, the experimental site is in a deflation zone composed of sandy soils of eolian origin, with grain size distribution containing less than 20% of finer sediments. The field tests were conducted in two different locations within the experimental site, which are labeled in this study as testing Area A and testing Area B. The distance between both locations within the experimental site is approximately 300 m. The experimental site was characterized by field and laboratory tests. The field survey included four standard penetration tests (SPT) and one piezocone (CPTu) test in each area of investigation. Subsoil profiles of Areas A and B are schematically shown in Figure 1b and Figure 1c, respectively, which include the variation with depth of the mean SPT blow count corrected for 60% efficiency (N60), the cone tip resistance (qc), and the friction ratio (Rf). Groundwater level was not detected during borehole logging. The water table is usually encountered at great depths below the ground surface (> 50 m). Surveying in Area B was performed before the placement of the surficial backfill layer. In both areas, the surficial soil layer is composed of a 3-m-thick pure sand backfill, collected from a neighboring cut sector within site. Below the backfill sand lay natural layers of dune sediments, composed of sand and silty sand. The backfill is a medium uniformly graded quartz silica sand of eolian origin that classifies as SP, according to the Unified Soil Classification System. The grain size distribution of the backfill sand is shown in Figure 2a. The sand has an average particle size of 0.35 mm, a coefficient of uniformity of 2.2, and a coefficient of curvature of 1.07. The sand has less than 2% of the particles finer than 0.075 mm and more than 98% finer than 2 mm. 1 INTRODUCTION Parametric analyses were carried out to investigate the influence of (i) soil-pile relative stiffness, (ii) pile diameter, (iii) interface friction between the pile and the soil and (iv) soil dilatancy. Therefore, this study brings together experimental and numerical results that provide important background for the future preliminary design of piles under lateral loading in similar soil conditions. 2/17 A. M. D. Sousa, Y. D. J. Costa, A. G. D. Araujo, and C. M. L. Costa 2 SITE CHARACTERISTICS Its specific gravity is 2.59, and maximum and minimum void ratios are 0.83 and 0.60, respectively. The top sand layer was compacted with relative density (Dr) of 45% in Area A, and 70% in Area B. Controlled compaction in the field was carried out using vibrating drum rollers. N 0 300 600 km Natal BRAZIL (a) BRAZIL Northeast region (b) (c) Atlantic Ocean Backfill Sand (SP) (Dr=45%) Sand (SP) Silty Sand (SM) 0 2 4 6 8 10 12 14 16 0 20 40 60 0 2 4 6 8 10 12 14 16 0 20 40 60 0 2 4 6 8 10 12 14 16 0 1 2 3 Backfill Sand (SP) (Dr=70%) Sand (SP) Silty Sand (SM) 0 2 4 6 8 10 12 14 16 0 20 40 60 0 2 4 6 8 10 12 14 16 0 20 40 60 0 2 4 6 8 10 12 14 16 0 1 2 3 60 N ( ) cq MPa (%) f R 60 N ( ) cq MPa (%) f R Depth (m) Depth (m) Figure 1. (a) Location of test site; (b) Site investigation results in Area A; (c) Site investigation results in Area B. N 0 300 600 km Natal BRAZIL Northeast region Atlantic Ocean (a) BRAZIL ure 1. (a) Location of test site; (b) Site investigation results in Area A; (c) Site investigation results in Area B 3/17 Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 A. M. D. Sousa, Y. D. J. Costa, A. G. D. Araujo, and C. M. L. Costa Shear strength parameters of the backfill sand were obtained from two series of conventional CID triaxial compression tests carried out with confining effective stresses (σ´3) of 50 kPa, 100 kPa, and 200 kPa. One series of tests was prepared with target relative density (Dr) of 45%, and another series was prepared with Dr = 70%, which are the same densities used in the field. Peak friction angle (ϕ´p) was calculated from the Kf line of p-q diagrams of the tested soil. Values of ϕp’ equals 33.6º and 38.1º for Dr = 45% and 70%, respectively. Deviator stress-axial strain and volume change behavior for the soil at the relative density of 45% are presented in Figure 2b and Figure 2c, respectively, and the corresponding Kf line is indicated in Figure 2d. Figure 2. 2 SITE CHARACTERISTICS Properties of the backfill sand: (a) grain-size distribution; (b-c) results of CID triaxial tests performed on sand samples prepared with a relative density of 45%; (d) p-q diagram of the tested sand. Figure 2. Properties of the backfill sand: (a) grain-size distribution; (b-c) results of CID triaxial tests performed on sand samples prepared with a relative density of 45%; (d) p-q diagram of the tested sand. Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 3 FIELD TESTING PROGRAM AND INSTRUMENTATION A total of 12 field load tests were carried out to investigate the lateral behavior of single piles in the two sandy soil profiles. Continuous flight auger (CFA) and steel H-section model piles were installed in Areas A and B, as indicated in Figure 3. The tests were performed simultaneously on two piles with identical lengths and cross-sections, which reacted against each other. As shown in Figure 3, piles CFA-i and CFA-ii were tested together, and so did piles HS1-i and HS1-ii, and HS2-i and HS2-ii. A summary of the testing sequence is presented in Table 1, which indicates the pile type, the area where the test was carried out, and the test designation. The CFA piles were cast with a diameter (D) of 0.6 m and length (L) of 10 m. The integrity of the CFA piles was verified by a low-strain integrity test that uses the pulse-echo method, commonly named as pile integrity test (PIT). The reinforcement of the CFA piles consists of 10 longitudinal steel bars with 20 mm in diameter enveloped by a steel spiral with 8.0 mm in diameter and 200 mm in spacing. Concrete with characteristic compressive strength of 35 MPa at 28 days was used for the CFA piles. The initial flexural stiffness (EpIp) of the CFA pile cross-section is 220 MN m2. 4/17 Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 A. M. D. Sousa, Y. D. J. Costa, A. G. D. Araujo, and C. M. L. Costa Figure 3. Elevation view showing the position of model piles. Figure 3. Elevation view showing the position of model piles. Table 1. Testing program summary Table 1. Testing program summary Test Nº Pile type Testing Area Backfill relative density, Dr (%) Designation 1 CFA B 70 CFA-B-70-i 2 CFA B 70 CFA-B-70-ii 3 CFA A 45 CFA-A-45-i 4 CFA A 45 CFA-A-45-ii 5 HS1 A 45 HS1-A-45-i 6 HS1 A 45 HS1-A-45-ii 7 HS2 A 45 HS2-A-45-i 8 HS2 A 45 HS2-A-45-ii 9 HS2 B 70 HS2-B-70-i 10 HS2 B 70 HS2-B-70-ii 11 HS1 B 70 HS1-B-70-i 12 HS1 B 70 HS1-B-70-ii Two different steel H-section piles were used, as shown in Figure 3. One pair of steel piles had cross-section with width = 315 mm, height = 315 mm, flange thickness = 17.4 mm and web thickness = 17.4 mm (HS1-i and HS1-ii). Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 3 FIELD TESTING PROGRAM AND INSTRUMENTATION The other pair of steel piles had cross-section with width = 171 mm, height = 355 mm, flange thickness = 11.6 mm and web thickness = 7.2 mm (HS2-i and HS2-ii). All steel piles had a total length of 6 m and embedded length in the ground of 4.5 m. The initial bending stiffness (EpIp) of the cross-section of piles HS1 and HS2 are 56 MN.m2 and 29 MN.m2, respectively. In both testing areas, HS1 and HS2 piles were tested before the casting of the CFA piles. Spacing between the CFA and the HS1 pairs of piles (Figure 3) was defined to accommodate the use of these piles as a reaction for a vertical load test performed in another pile installed after completion of the lateral load tests, which is not included in the present study. The distance between HS1 and CFA piles was assumed sufficient to minimize cross-interference. Based on experimental results from physical model tests, Davisson [30] concluded there is essentially no influence of one pile on another, providing the spacing normal to the direction of loading is at least 2.5 D from the pile axis. According to results from instrumented tests on small-scale models of laterally loaded piles, at a depth of 3.45 D below the soil surface, horizontal stress changes extend to a maximum distance of 2.5 D beside the pile [31]. The layout of the installation and instrumentation of the CFA model piles is depicted in Figure 4a. A shallow pit with a depth of 0.5 m was excavated between the piles to accommodate the loading system. The load was applied in cumulative equal increments using a hydraulic cylinder with a maximum nominal capacity of 500 kN. The cylinder was positioned as close as possible to the pile head. Load measurements were obtained by a calibrated load cell with a maximum nominal capacity of 500 kN. Horizontal pile displacements (yt) were recorded at the ground surface level 5/17 A. M. D. Sousa, Y. D. J. Costa, A. G. D. Araujo, and C. M. L. Costa using dial gages with a resolution of 0.01 mm and a maximum stroke of 50 mm. The dial gauges were mounted in magnetic-articulated bases set on two steel reference beams. Similar instrumentation, as described above, was used in the tests with the steel piles, and a general scheme of its setup is illustrated in Figure 4b. 3 FIELD TESTING PROGRAM AND INSTRUMENTATION The loading system was mounted directly on the ground surface, and the horizontal displacements of the pile (yt) were recorded at 0.5 m and 1.2 m above the ground surface. All lateral loading tests were performed with static quick maintained load (QML) [32] and followed the Brazilian code ABNT NBR 12131:2006 [33]. Each load increment was held constant for 5 min, and displacement readings were taken at every minute. The unloading phase was carried out with four 5-minute-long decrements. The unloading stages were not recorded in Test 5 due to the sudden disassembling of the loading system during the last stage. Figure 4. General scheme of testing assemblage and used apparatus: (a) CFA piles; (b) steel H-section piles. Figure 4. General scheme of testing assemblage and used apparatus: (a) CFA piles; (b) steel H-section pile Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 4 FIELD TEST RESULTS Figure 5 shows the lateral applied load (H) versus measured lateral displacement (yt) curves of each individual pile, obtained from the field tests performed in Areas A and B. The displacements (yt) recorded with the upper dial gages in the tests with the steel H-section profiles are omitted from Figure 5 for clarity. By comparing the results of the same pair of piles, it is possible to notice that the curves of the steel H-section piles situate within a narrower range than those of the CFA piles. Imperfections in the shaft of the CFA piles associated with the construction process may partially explain the poorer repeatability of the tests with this pile type since no cast-in-place pile is exactly equal to another. 6/17 Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 A. M. D. Sousa, Y. D. J. Costa, A. G. D. Araujo, and C. M. L. Costa Figure 5. Results of the lateral tests: (a) CFA piles in Area A; (b) CFA piles in Area B; (c) steel H-section piles in Area A; (d) steel H-section piles in Area B. Figure 5. Results of the lateral tests: (a) CFA piles in Area A; (b) CFA piles in Area B; (c) steel H-section piles in Area A; (d) steel H-section piles in Area B. The CFA piles reached larger maximum lateral loads in comparison to the steel piles. The average value of the maximum horizontal load with the CFA piles was 480 kN in both testing areas, which is close to the limit load of 500 kN of the loading system. In Area A, the average value of the maximum horizontal load of pair HS1 was 242 kN, and of the pair HS2 was 195 kN. Slightly higher maximum lateral loads were obtained with the steel piles installed in Area B due to the greater relative density of the sand backfill: 334 kN with pair HS1 and 220 kN with pair HS2. The unloading portion of the curves reveals that the horizontal displacement recovered after unloading is significant and is larger with the steel piles than the CFA piles, due to the resilient feature of the steel. In both testing areas, the results of the HS1 and HS2 piles were close. 4 FIELD TEST RESULTS The rotation of the pile head at the ground surface was calculated from Matlock and Reese [34]: g p ( The rotation of the pile head at the ground surface was calculated from Matlock and Reese [3 . . 2 0 p p p p T T S 1 623H 1 750He E I E I = − − (4) . . 2 0 p p p p T T S 1 623H 1 750He E I E I = − − (4) This approach for the computation of nh assumes that the modulus of subgrade reaction, obtained for the soil near the surface of the terrain, varies linearly with depth and is representative of the entire ground along the pile length. However, potential inaccuracies produced by this assumption are not supposed to be significant, since it is well established that the response of a pile to lateral loading is essentially controlled by the stiffness of the soil at shallow depth [13], [35]. Zhang et al. [36] reported depths of influence between 3 and 4 times the pile diameter. Considering the soil profile of the present study, such depth of influence is within the surficial sand layer, which suggests that the pile response is ruled by this layer. Figure 6a and Figure 6b show the relationship between nh and the horizontal displacement y0 obtained for Areas A and B, respectively. The horizontal displacement y0 is normalized by dimension B, which is the diameter of the CFA pile or the width of the steel pile. The constant of horizontal subgrade reaction follows a steep decrease at small horizontal displacements and tends to stabilize with increasing displacements. Values of nh were significantly larger with the denser backfill sand of Area B. As pointed out by Prakash and Kumar [37] the modulus of horizontal reaction depends strongly on the relative density of sands. Figure 6. Relationship between the constant of subgrade reaction nh and the horizontal pile displacement at ground level: (a) Area A; (b) Area B. Figure 6. Relationship between the constant of subgrade reaction nh and the horizontal pile displacement at ground level: (a) Area A; (b) Area B. 4 FIELD TEST RESULTS The constant of horizontal subgrade reaction of the steel H-section piles was calculated by solving a system composed of Equations 1 and 3, which gives T, EpIp, and y0 for each applied load H. point of application of the load (e) equals zero. After finding the characteristic length, nh was calculated from Equation 2. The constant of horizontal subgrade reaction of the steel H-section piles was calculated by solving a system composed of Equations 1 and 3, which gives T, EpIp, and y0 for each applied load H. The constant of horizontal subgrade reaction of the steel H-section piles was calculated by solving a system composed of Equations 1 and 3, which gives T, EpIp, and y0 for each applied load H. ( ) ´ ´ 3 2 0 t 0 p p p p He He e y y S e e 3E I 2E I = + + − − (3) (3) where: yt = mean horizontal displacement of piles tested simultaneously against each other; S0 = rotation of the pile head at the ground surface; e´ = vertical distance from the axis of application of H to the position of measurement of yt. In the assemblage of the tests with the steel H-section piles, e = 0.2 m, and e´ = 0.3 m or 1 m (see Figure 4). where: yt = mean horizontal displacement of piles tested simultaneously against each other; S0 = rotation of the pile head at the ground surface; e´ = vertical distance from the axis of application of H to the position of measurement of yt. In the assemblage of the tests with the steel H-section piles, e = 0.2 m, and e´ = 0.3 m or 1 m (see Figure 4). The rotation of the pile head at the ground surface was calculated from Matlock and Reese [34]: where: yt = mean horizontal displacement of piles tested simultaneously against each other; S0 = rotation of the pile head at the ground surface; e´ = vertical distance from the axis of application of H to the position of measurement of yt. In the assemblage of the tests with the steel H-section piles, e = 0.2 m, and e´ = 0.3 m or 1 m (see Figure 4). 4 FIELD TEST RESULTS As expected, the curves of the HS1 piles showed slightly larger horizontal loads for a specific displacement, due to the higher cross-section flexural stiffness of that pile type. The constant of horizontal subgrade reaction (nh) was back calculated from the load test results of the CFA piles using the following equation [22]: ( ) , , 3 2 0 p p p p T T y 2 435H 1 623 He E I E I = + (1) ( ) , , 3 2 0 p p p p T T y 2 435H 1 623 He E I E I = + (1) where: y0 = horizontal displacement of the pile at the ground surface; H = horizontal applied load; e = position of the axis of application of H above the ground surface; Ep = Young´s modulus of pile material; Ip = moment of inertia of pile cross-section; T = pile characteristic length, given as: where: y0 = horizontal displacement of the pile at the ground surface; H = horizontal applied load; e = position of the axis of application of H above the ground surface; Ep = Young´s modulus of pile material; Ip = moment of inertia of pile cross-section; T = pile characteristic length, given as: p p 5 h E I T n = (2) 7/17 Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 A. M. D. Sousa, Y. D. J. Costa, A. G. D. Araujo, and C. M. L. Costa Since the displacements of the CFA piles were recorded at the level of the terrain, Equation 1 was solved assuming y0 = yt. The characteristic length T was obtained from Equation 1 assuming the distance from the ground surface to the point of application of the load (e) equals zero. After finding the characteristic length, nh was calculated from Equation 2. Since the displacements of the CFA piles were recorded at the level of the terrain, Equation 1 was solved assuming y0 = yt. The characteristic length T was obtained from Equation 1 assuming the distance from the ground surface to the point of application of the load (e) equals zero. After finding the characteristic length, nh was calculated from Equation 2. 5 THREE-DIMENSIONAL NUMERICAL MODELING OF LATERALLY LOADED CFA AND STEEL H- SECTION PILES The finite-element program Plaxis3D was used to simulate the response of a single pile under lateral load in the studied soil profiles. Figure 7a and Figure 7b show the typical undeformed 3-D FE mesh used in the analyses of the CFA piles. The model boundaries extended to a width of 10 m from the pile center and to a depth equal to 20 m (i.e., 8/17 Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 A. M. D. Sousa, Y. D. J. Costa, A. G. D. Araujo, and C. M. L. Costa the pile length plus 10 m below the pile-toe level). These dimensions were considered sufficient to exclude boundary effects in previous investigations. The outer vertical boundary of the mesh is fixed in the horizontal direction and free in the vertical direction. The base of the soil model is constrained in both vertical and horizontal directions. The mesh consists of 15-node wedge elements, which were applied to simulate both the soil and the CFA pile. The meshing discretization used in this study followed the approach proposed by Kim and Jeong [5]. For the proposed numerical model, the mesh was manually refined close to the pile. Although one dimension of the elements in the vicinity of the pile is considerably smaller than the other two (i.e., elements with large aspect ratio, as shown in Figure 7c), the use of wedge elements with quadratic approximation (15 nodes) reduced potential numerical problems of convergence. Moreover, since the mean size of the element cannot be freely defined in the software, refinement of the mesh close to the pile was necessary to ensure a higher number of elements and nodes in the loading direction. All numerical simulations were carried out under drained conditions. The soil behavior was modeled by the hardening soil (HS) constitutive model developed under the framework of the theory of plasticity, in which a stress- strain relationship, due to the primary loading, is assumed to be a hyperbolic curve [38]. Soil failure takes place according to the Mohr-Coulomb failure criterion. The CFA pile was considered as an isotropic linear-elastic material. The material properties of soils and piles used in this analysis are depicted in Table 2. Figure 7. Three-dimensional finite-element mesh: (a) 3D view of CFA pile model; (b) cross-section in x-y plane of CFA pile model; (c) surface view of steel H-section HS1 pile model. 5 THREE-DIMENSIONAL NUMERICAL MODELING OF LATERALLY LOADED CFA AND STEEL H- SECTION PILES Th di i l fi it l t h ( ) 3D i f CFA il d l (b) ti i l Figure 7. Three-dimensional finite-element mesh: (a) 3D view of CFA pile model; (b) cross-section in x-y plane of CFA pile model; (c) surface view of steel H-section HS1 pile model. 9/17 Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 A. M. D. Sousa, Y. D. J. Costa, A. G. D. Araujo, and C. M. L. Costa Table 2 - Material properties used in the FE analysis Table 2 - Material properties used in the FE analysis Material Parameter CFA pile Concrete unit weight, γ (kN/m3) 25 Pile Young’s modulus, Ep (MPa) 35,000 Pile coefficient of Poisson, νp 0.2 H-Steel pile Pile Young’s modulus, Ep (MPa) 207,000 Soil Backfill Sand Silty Sand Area A Soil unit weight, γ (kN/m3) 16 17.5 17 (Dr = 45%) Soil Young’s modulus, Es (MPa) 50 100 100 Soil coefficient of Poisson, νs 0.30 0.35 0.35 Peak friction angle, ϕ´p (o) 31.6 38 36.4 Soil-pile interface friction angle, δ (o) 22.3 27.5 26.2 Effective cohesion, c´ (kPa) 5 5 10 Dilatancy angle, ψ (o) 0 5 10 Area B Soil unit weight, γ (kN/m3) 18 17 18 (Dr = 70%) Soil Young’s modulus, Es (MPa) 100 100 100 Soil coefficient of Poisson, νs 0.35 0.35 0.35 Peak friction angle, ϕ´p (o) 35.1 37 40.6 Soil-pile interface friction angle, δ (o) 25.1 26.7 29.7 Effective cohesion, c´ (kPa) 5 5 10 Dilatancy angle, ψ (o) 10 5 10 The main parameter governing the soil-pile interface properties in Plaxis software is the strength reduction factor (Rinter ), which is the ratio between interface friction coefficient (μinter = tanδ) and soil friction coefficient (μ = tanϕ´) or the ratio between interface cohesion (cinter) and soil cohesion (c´). Rinter varies between 0 and 1. The baseline Rinter value used in the present study is 0.7. The steel H-section piles were modeled using plate elements with prescribed flexural and axial rigidities. The models with the steel piles were built with the same dimensions of the models with the CFA pile. A three-dimensional surface view of the model with steel profile HS1, showing the shape of the undeformed mesh around the pile, is presented in Figure 7c. Figure 8 compares the experimental and numerical results in terms of horizontal load (H) versus horizontal displacement (y0) relationships. 5 THREE-DIMENSIONAL NUMERICAL MODELING OF LATERALLY LOADED CFA AND STEEL H- SECTION PILES The lateral load capacities obtained from the field tests are compared with those predicted from the different methods in Table 3. FHWA [39] proposes tolerable horizontal movements of a single pile of less than 38 mm from a serviceability standpoint. According to AASHTO [40], tolerable displacements of a pile shall be limited to 50 mm or less where vertical displacements are small or to 25 mm or less where combined vertical and horizontal displacements are possible. The ultimate lateral load capacity obtained from the load tests is shown in Table 3 assuming an allowable horizontal displacement y0 = 20 mm as a serviceability limit for the foundation. Since the displacement level of 20 mm was not reached in the tests performed in Area B, the corresponding experimental ultimate loading capacity was obtained from the extrapolation of the curves using the method of Mazurkiewicz [41]. The three-dimensional finite element model closely matched the ultimate lateral capacities to the experimental data (Table 3). The computed ultimate lateral capacity for testing Area A approached the field results closely. The largest discrepancy between numerical and measured results was 21% for H-section pile HS1 embedded in the soil of testing Area B. The 3-D FEM analysis also closely matches the general trend of the experimental curves of CFA and steel H-section piles (Figure 8). Small differences between the three-dimensional numerical simulations and the field results were expected and may be attributed to the variability of soil properties and pile characteristics. 10/17 Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 A. M. D. Sousa, Y. D. J. Costa, A. G. D. Araujo, and C. M. L. Costa A. M. D. Sousa, Y. D. J. Costa, A. G. D. Araujo, and C. M. L. Costa Figure 8. Comparison of the response of laterally loaded piles obtained from field tests and by numerical modelling: (a) CFA pile in Area A; (b) CFA pile in Area B; (c) steel pile HS1 in Area A; (d) steel pile HS1 in Area B; (e) steel pile HS2 in area A; (f) steel pile HS2 in Area B. Figure 8. Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 11/17 5 THREE-DIMENSIONAL NUMERICAL MODELING OF LATERALLY LOADED CFA AND STEEL H- SECTION PILES Comparison of the response of laterally loaded piles obtained from field tests and by numerical modelling: (a) CFA pile in Area A; (b) CFA pile in Area B; (c) steel pile HS1 in Area A; (d) steel pile HS1 in Area B; (e) steel pile HS2 in area A; (f) steel pile HS2 in Area B. Table 3. Comparison of lateral load capacity of CFA and steel H-section piles obtained from the field tests and the numerical simulations. Testing Area Ultimate lateral capacity (kN) Pile type Field FEM A (Dr = 45%) CFA 355 355 HS1 188 187 HS2 162 157 B (Dr = 70%) CFA 6901 716 HS1 4901 385 HS2 3121 362 n of lateral load capacity of CFA and steel H-section piles obtained from the field tests and the numerical Table 3. Comparison of lateral load capacity of CFA and steel H-section piles obtained from the field tests simulations. Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 11/17 A. M. D. Sousa, Y. D. J. Costa, A. G. D. Araujo, and C. M. L. Costa Parametric studies were performed to analyze the influence of (i) the soil-pile relative stiffness, (ii) the pile diameter, (iii) the interface friction between pile and soil, (iv) and soil dilatancy (the volume change characteristics of the soil). The analysis of soil-pile relative stiffness was carried out for CFA piles and steel HS1 piles in Areas A and B. Analysis of pile diameter, soil-pile interface friction and soil dilation were developed considering CFA piles only. Figure 9 shows the response of the steel HS1 pile in Area A for different bending stiffness values (EpIp), which were varied by changing the Young's modulus of the pile (Ep) while keeping the second moment of inertia (Ip) constant. In Figure 9, EpIp is the baseline bending stiffness of profile HS1, which is equal to 56 MNm2. Results show that variations in pile stiffness drastically affects the behavior of the pile under lateral loading. Similar trends were also obtained from the parametric analysis carried out with the other pile types embedded in Areas A and B. The results are in accordance with the findings reported by Norris et al. [11], which used the strain wedge (SW) model to predict the behavior of cast- in-place and steel piles in cohesive and cohesionless soils. 5 THREE-DIMENSIONAL NUMERICAL MODELING OF LATERALLY LOADED CFA AND STEEL H- SECTION PILES At low L/T ratios, a small variation in relative stiffness causes a dramatic variation in Hu. Short rigid piles are, therefore, much more influenced by changes in relative stiffness than long flexible members. The effect of soil- pile stiffness becomes much less significant for L/T ratios above 10. L/T = 4 has been commonly accepted as the limit beyond which the pile behaves like a long flexible unit [30], [44]. Information presented in Figure 10 can be used in the design of laterally loaded piles in similar local soils for a preliminary estimation of the ultimate lateral capacity. 0 5 10 15 20 25 30 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 H u [-] Nondimensional length, L/T [-] CFA, Area A (Dr= 45%) CFA, Area B (Dr = 70%) HS1, Area A (Dr= 45%) HS1, Area B (Dr = 70%) H* u=3.78(L/T)-1.04 R2=0.84 Figure 10. Normalized ultimate lateral capacity of piles Hu* as a function of the nondimensional length L/T. Nondimensional length, L/T [-] Figure 10. Normalized ultimate lateral capacity of piles Hu* as a function of the nondimensional length L/T. Figure 10. Normalized ultimate lateral capacity of piles Hu* as a function of the nondimensional length L/ The influence of the pile diameter on pile behavior was investigated in the light of the soil-pile relative stiffness concept. The study included the modeling of CFA piles with diameters of 0.3, 0.6, 0.9, and 1.2 m, embedded in Area A. The non-dimensional length ratio (L/T) was maintained unchanged while the pile diameter was varied. Figure 11 shows the effect of the diameter of the pile on the horizontal load (H) for various non-dimensional length ratios (L/T). Linear trend lines were fitted to the data points in Figure 11. Appropriate values of horizontal load were obtained from the calculated H–y0 curve of each corresponding pile and using Equation 1 with the selected characteristic length T. The computational results shown in Figure 11 indicate that the effect exerted by the diameter was significant and became more evident with smaller non-dimensional length ratios. In addition, the relationship between the horizontal load and the pile diameter appears to be nearly linear for the range of examined diameters and non-dimensional length ratios, which agrees with the findings reported by Carter [28], Guo [23], and Kim and Jeong [5]. 5 THREE-DIMENSIONAL NUMERICAL MODELING OF LATERALLY LOADED CFA AND STEEL H- SECTION PILES 0 50 100 150 200 250 300 0 5 10 15 20 25 30 35 Measurement Horizontal displacement, y0(mm) Horizontal Load, H (kN) 5EpIp EpIp 0.6EpIp 0.3EpIp 0.1EpIp 0.01EpIp Figure 9. Response of steel H-section pile HS1 embedded in Area A, for various bending stiffness values. Figure 9. Response of steel H-section pile HS1 embedded in Area A, for various bending stiffness values. Deeper insight into the effect of pile stiffness can be attained under the framework of soil-pile relative stiffness. Within this concept, the pile and the surrounding soil behave as a single system. The characteristic pile length, T, defined by Equation 2, can be used to determine whether the soil-pile system behaves as a rigid or a flexible member [13], [42]. T is also called relative stiffness factor and expresses a relation between the stiffness of the soil, i.e., the constant of horizontal subgrade reaction (nh), and the flexural stiffness of the pile (EpIp). Figure 10 shows the normalized ultimate lateral capacity (Hu) as a function of the nondimensional length, given by the ratio between the pile length and the characteristic pile length (L/T). The nondimensional length is another way of quantifying the soil-pile relative stiffness and has the advantage of incorporating the pile length. The larger the nondimensional length, the more flexible the soil- pile system. Data points shown in Figure 10 belong to CFA pile and steel pile HS1 in Areas A and B and were computed for a constant lateral displacement y0 = 20 mm. A trend line fitting the data points is also shown in Figure 10, along with the corresponding empirical equation. Hu is expressed as: (5) where: Hu = ultimate lateral load at a 20-mm lateral displacement, as previously defined; B = pile diameter or width; γ = effective unit weight of the surficial sand layer; IR = rigidity index of the surficial sand layer, given as [43]: ´ ´ s R G I p tan = (6) 12/17 Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 A. M. D. Sousa, Y. D. J. Costa, A. G. D. Araujo, and C. M. L. Costa where: Gs = Es/2(1+νs) = shear modulus of the soil; p´ = reference pressure, assumed = 100 kPa. Figure 10 shows that the normalized ultimate lateral capacity Hu* decreases with increasing soil-pile relative stiffness. 5 THREE-DIMENSIONAL NUMERICAL MODELING OF LATERALLY LOADED CFA AND STEEL H- SECTION PILES 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 0 250 500 750 1000 1250 1500 L/T = 5 L/T = 6 Lateral load, H (kN) Pile diameter, D (m) L/T = 7 Figure 11. Relationship between lateral load and diameter of CFA piles for various non-dimensional length ratios. ( ) Figure 11. Relationship between lateral load and diameter of CFA piles for various non-dimensional length ratios. 11. Relationship between lateral load and diameter of CFA piles for various non-dimensional length ratios. Figure 11. Relationship between lateral load and diameter of CFA piles for various non-dimensional length ratios. 13/17 Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 A. M. D. Sousa, Y. D. J. Costa, A. G. D. Araujo, and C. M. L. Costa The effect of soil-pile interface friction on the CFA pile response was evaluated by varying the interface strength reduction factor (Rinter) within the backfill sand layer. As mentioned previously, Rinter is the main controlling parameter of the soil-pile interface in PLAXIS 3D software. The change of the ultimate lateral capacity (Hu) was investigated for strength reduction factors ranging from 0.1 to 0.9, as presented in Figure 12. The computed results reveal that the ultimate lateral capacity presents a non-linear increase with increasing interface property. The influence of Rinter on Hu is more relevant between Rinter = 0.1 to 0.3. Moreover, changes in interface property were comparatively more prominent in Area B, which has the denser surficial backfill sand. 0.0 0.2 0.4 0.6 0.8 1.0 0 200 400 600 800 1000 Ultimate lateral capacity, Hu (kN) Strength Reduction Factor, Rinter [-] Area A (Dr= 45%) Area B (Dr = 70%) Figure 12. Change of ultimate lateral capacity of CFA piles with interface property. Figure 12. Change of ultimate lateral capacity of CFA piles with interface property. Shear strength of dense cohesionless soils is largely dependent on volume change properties of the material [45]. The effect of soil dilatancy on soil-pile interaction was assessed by assigning dilatancy angles from 0º to 15º to the backfill sand of both testing areas. Figure 13 shows the variation of the ultimate lateral capacity (Hu) at 20-mm lateral displacement with dilatancy angle (ψ), obtained with the computed data of the CFA pile. The figure shows that soil dilatancy has an influence on pile performance. Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 5 THREE-DIMENSIONAL NUMERICAL MODELING OF LATERALLY LOADED CFA AND STEEL H- SECTION PILES An increment in the angle of dilatancy from 0º to 15º caused an average 9% increase in the lateral load capacity of the CFA pile. In addition, influence on ultimate lateral capacity is slightly more evident in Area B, which has the denser backfill sand. Comparatively, Choi et al. [15] obtained an increase of 20% in the lateral load capacity of bored piles in weathered granite soil, at a 20-mm lateral displacement, as ψ increased from 0º to 15º. 0 2 4 6 8 10 12 14 16 0 100 200 300 400 500 600 700 800 Ultimate lateral capacity, Hu(kN) Dilatancy angle, ψ (º) Area A (Dr=45%) Area B (Dr=70%) Figure 13. Ultimate lateral capacity of the CFA pile as a function of soil dilatancy. Figure 13. Ultimate lateral capacity of the CFA pile as a function of soil dilatancy. 14/17 Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 A. M. D. Sousa, Y. D. J. Costa, A. G. D. Araujo, and C. M. L. Costa 6 SUMMARY AND CONCLUSIONS An increment in the angle of dilatancy from 0º to 15º in the surficial sand layer caused an increase of about 9% in the lateral load capacity of the CFA pile for both tested relative densities of 45% and 70%. (4) Soil dilatancy causes a slight increase in the ultimate lateral capacity of the CFA piles in the sandy soil deposits. An increment in the angle of dilatancy from 0º to 15º in the surficial sand layer caused an increase of about 9% in the lateral load capacity of the CFA pile for both tested relative densities of 45% and 70%. ACKNOWLEDGEMENTS The authors gratefully acknowledge the financial support provided by the National Council for Scientific and Technological Development (CNPq) and the São Paulo Research Foundation (FAPESP - 2018/21573-2). The authors also acknowledge the contributions from João Paulo Costa and Avelino Lourenço on conducting the static lateral loading tests in the field. 6 SUMMARY AND CONCLUSIONS The response of continuous flight auger (CFA) bored piles and steel H-section driven piles to lateral loading was evaluated in this study by field tests and via a three-dimensional finite element (3-D FE) model. The proposed numerical model was substantiated with field tests performed on four CFA piles and eight steel H-section piles in two distinct sandy soil profiles. The three-dimensional finite element approach closely matched the response of CFA and steel H- section piles in terms of the relationships between the applied horizontal load and the horizontal pile displacement recorded from the field tests. A series of parametric analyses was carried out to investigate the role of relevant variables on lateral soil-pile interaction. The main findings of the study can be summarized as follows: (1) The pile ultimate lateral load is drastically affected by soil-pile system relative stiffness. Short rigid piles are much more influenced by changes in relative stiffness than long flexible units. The effect of soil-pile stiffness becomes significant for nondimensional length ratios (L/T) below 4. (1) The pile ultimate lateral load is drastically affected by soil-pile system relative stiffness. Short rigid piles are much more influenced by changes in relative stiffness than long flexible units. The effect of soil-pile stiffness becomes significant for nondimensional length ratios (L/T) below 4. (2) Pile diameter exerts a marked effect on pile lateral load, which is more prominent with stiffer soil-pile systems. Moreover, the relationship between horizontal load and pile diameter appears to be nearly linear for the range of examined diameters and nondimensional length ratios. 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[39] Federal Highway Administration, Design, Analysis, and Testing of Laterally Loaded Deep Foundations that Support Transportation Facilities. Washington, D.C.: Fed. Highw. Adm. (Rep. FHWA-HIF-18-031), 2018. 16/17 Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 A. M. D. Sousa, Y. D. J. Costa, A. G. D. Araujo, and C. M. L. Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021 Author contributions: AMDS: numerical analyses, manuscript editing and review; YDJC: conceptualization, funding acquisition, supervision, analysis of results and writing; AGDA: experimental analyses; CMLC: supervision and manuscript review. Editors: Mark Alexander, Guilherme Aris Parsekian. REFERENCES Costa [40] American Association of State Highway and Transportation Officials, Standard Specifications for Highway Bridges, 17th ed. Washington, D.C., 2002. [41] B. K. Mazurkiewicz, Test Loading of Piles According to Polish Regulations (Prel. Rep. 35). Stockholm: Swedish Academy of Eng Sciences, 1972. [42] B. B. Broms, "Lateral resistance of piles in cohesionless soils," J. Soil Mech. Found. Div., vol. 90, no. 3, pp. 123–156, 1964. [43] A. S. Vesic, “Bearing capacity of shallow foundations,” in Foundation Engineering Handbook, 1st ed., H. F. Winterkorn and H. Y. Fang, Eds., New York, NY: Van Nostrand Reinhold, 1975, pp. 121–147. [44] M. J. Tomlinson, Pile Design and Construction Practice, 4th ed. London: E & FN Spon., 1994. [45] M. D. Bolton, "The strength and dilatancy of sands," Geotechnique, vol. 36, no. 1, pp. 65–78, Mar 1986, http://dx.doi.org/10.1680/geot.1986.36.1.65. Editors: Mark Alexander, Guilherme Aris Parsekian. 17/17 Rev. IBRACON Estrut. Mater., vol. 14, no. 3, e14313, 2021
https://openalex.org/W1596412759	https://www.intechopen.com/citation-pdf-url/28887	English	null	Archaeal Diversity and Their Biotechnological Potential	InTech eBooks	2,012	cc-by	14,043	Archaeal Diversity and Their Biotechnological Potential Birgül Özcan Mustafa Kemal University, Faculty of Sciences and Letters, Biology Department, Turkey 2 Archaeal Diversity and Their Biotechnological Potential Birgül Özcan Mustafa Kemal University, Faculty of Sciences and Letters, Biology Department, Turkey 2 1. Introduction The extreme environments found on the planet are generally inhabited by microorganisms, which belong to the archaeal and bacterial domains of life. Extreme environments comprise the sites including physical variables as -20°C to +113°C (like stratosphere and hydrothermal vents), ≤120 Mpa (for hydrostatic pressures in the deep sea), aw ≈ 0.6 (for the activity of water in salt lakes) and ≈ 0.5< pH < 11 (for acidic and alkaline biotopes) (Woese et al., 1990). Archaeal ecology is generally accepted as synonymous with extreme environments in the point of the human being view. Representatives of Archaea, however, occur everywhere: in samples from ocean water, ocean sediments, freshwater lakes, soil, solid gas hydrates, tidal flat sediments, plant roots, peatlands, petroleum-contaminated aquifers, human subgingival area, skin and gastrointestinal tract and as a simbiyont within the sponge (Cavicchioli et al., 2003; Mills et al., 2005; DeLong, 2005; Knittel et al. 2005; Fierer et al., 2007; Brochier-Armanet et al. 2008; Oxley et al. 2010; Kong, 2011). The majority of extremophiles belongs to the Archaea, the third domain of the living organisms together with Eukarya and Bacteria as explained before (Woese et al., 1990). The Archaea are a prokaryotic domain known to be often associated with habitats of extreme temperature, salinity and pH, and their presence in constantly cold marine waters is also well documented (Karr et al., 2006). Archaeal 16S rRNA community analysis has demonstrated that novel groups of Archaea are also abundant in the open ocean, soil and freshwater ecosystems as well (Buckley et al., 1998; Falz et al., 1999). Archaea exist in a broad range of habitats, and as a major part of global ecosystems, may contribute up to 20% of Earth's biomass (DeLong & Pace, 2001). Archaea, the most recently recognized domain, contains cultivated members that span a fairly limited range of phenotypes, represented by extreme halophiles, Sulfate-reducers and sulfur-metabolizing thermophiles, and methanogens. The first-discovered Archaea were extremophiles, which can be divided into four main physiological groups. These are the halophiles, thermophiles, alkaliphiles, and acidophiles. Organisms from the domain Archaea differ fundamentally from Eukarya and Bacteria in several genetic, biochemical, and structural properties. Archaeal species have been classified as an early-branching evolutionary offshoot of the domain Bacteria and have long been considered to represent a primitive form of life that thrives only in extreme environments such as hot springs, salt lakes, or submarine volcanic habitats. 1. Introduction The curiosity of identifying, grouping, and naming organisms according to their established natural relationship has been the subject of a great interest since ancient times. Today, instead of the traditional rank-based biological classification, phylogenetic systematics, which aims at postulating phylogenetic trees rather than focusing on what taxa to delimit, has been used commonly. Carl Woese is the one who first realized that the ribosome, the ubiquitous molecular structure that conducts protein synthesis, offers a way to investigate systematically the relationships between all forms of life. Woese’s approach was to determine the sequences of the RNAs that make up the ribosome, particularly the small subunit of ribosomal RNA (rRNA) (Woese et al., 1990). Comparisons of nucleotide sequences of ribosomal genes from different organisms allowed understanding of the evolutionary relationships between the organisms: the higher the similarity or difference between the rRNA sequences, the more or less closely related the organisms are. Instead of the commonly accepted subdivision of living organisms into the five kingdoms: Monera, Protista, Fungi, Animalia, and Plantae (Whittaker, 1969), Woese and his colleagues proposed subdivision into three higher taxa: Archaea, Bacteria, and Eukarya, first, they called them primary kingdoms and then domains (Woese et al., 1990). The sequencing of rRNA genes became one of the main tools for the construction of phylogenetic backbone of microbial classification and today each new description of Bacteria and Archaea must be accompanied by the complete 16S rRNA sequence of the type strain (Ludwig & Klenk, 2001; Yarza et al. 2010). Although the 16S rDNA gene has been tremendously useful for establishing the molecular phylogeny of prokaryotes over the last three decades, it suffers from the same limits as any other single-gene phylogenetic approach does. The identification of microbial isolates by whole-cell mass spectrometry (WC-MS) is being recognized as one of the latest tools bringing a revolution in microbial diagnostics, with the potential of bringing to an end many of the time-consuming and man-power-intensive identification procedures that have been used for decades. Apart from applications of WC-MS in clinical diagnostics, other fields of microbiology also have adopted the technology with success. MALDI-TOF MS shows particular potential usefulness for applications in environmental microbiology, e.g., to rapidly reveal cryptic species in large batches of related isolates (Clermont et al., 2009; Welkera & Moore, 2011). www.intechopen.com 28 Genetic Diversity in Microorganisms Majority of the living beings thrive in environments having physically and geochemically temperate conditions. www.intechopen.com 1. Introduction However, recent researches have shown that Archaea are more physiologically diverse and ecologically widespread than was previously thought. Like Bacteria, Archaea are commonly mesophilic, and some members are known to be closely associated with eukaryotic hosts, including humans. For instance, high numbers of methane-producing Archaea (methanogens) have been detected in the gastrointestinal tract, vagina, and oral cavity (Belay et al., 1990; Vianna et al., 2006) and recently non-methanogenic Archaea including members of the Crenarchaeota, Thermococcales, Thermoplasmatales and Halobacteriaceae, have been detected in human faeces (Oxley et al., 2010). They are now recognized as a component of human microbiota and it is subjects of debate wheather archaea are cause of any disease in human. Although it was shown that members of the domain Archaea are found in greater abundance in dental plaque from sites with periodontal disease than in plaque from non-diseased sites (Lepp et al., 2004), it is generally assumed that archaea are not a cause of human disease (Vianna et al., 2006). The www.intechopen.com 29 Archaeal Diversity and Their Biotechnological Potential isolation of archaeal strains from root-canal samples by amplification of 16S rDNA were at first unsuccessful, however, a recent study has confirmed that members of the methanogenic archaea, similar to Methanobrevibacter oralis, can be detected in root canal samples (Vianna et al., 2006). Finally, the presence of archaea in root canals has been confirmed and this provides new insights into the polymicrobial communities in endodontic infections associated with clinical symptoms. isolation of archaeal strains from root-canal samples by amplification of 16S rDNA were at first unsuccessful, however, a recent study has confirmed that members of the methanogenic archaea, similar to Methanobrevibacter oralis, can be detected in root canal samples (Vianna et al., 2006). Finally, the presence of archaea in root canals has been confirmed and this provides new insights into the polymicrobial communities in endodontic infections associated with clinical symptoms. Archaea, one of the three domains of life on Earth, is predominantly composed of two major phyla: the Euryarchaeota, the Crenarchaeota. In addition to these two major phyla, the Korarchaeota (Elkins, 2008), the Nanoarchaeota (Huber et al., 2002), the Thaumarchaeota (Brochier-Armanet et al., 2008) and the Aigarchaeota (Nunoura et al., 2011) have been proposed to be potential phylum-level taxonomic groups within the Archaea; however, the establishment of these phyla is still controversial (Takai & Nakamura, 2011). 1. Introduction This division, based on small-subunit rRNA phylogeny, is also strongly strengthened by comparative genomics and phenotypic characteristics. The first archaeal genome was sequenced in 1996 and so far 52 genomes of Archaea have been sequenced. The cultured Crenarchaeota are composed of four orders: Caldisphaerales, Desulfurococcales, Sulfolobales and Thermoproteales (Chaban et al., 2006). Euryarchaeota are composed of nine orders Methanobacteriales, Methanococcales, Methanomicrobiales (Balch et al., 1979), Methanosarcinales (Boone et al., 2001), Halobacteriales (Grant & Larsen 1989), Thermoplasmatales (Reysenbach, 2001), Thermococcales (Zillig et al., 1987), Archaeoglobales and Methanopyrales (Huber & Stetter, 2001). The metabolic diversity of archaea is quite similar to bacteria in many aspects. Except for methanogenesis, all metabolic pathways discovered in archaea also exist among bacteria. Archaeal species can be either heterotrophs or autotrophs and use a large variety of electron donors and acceptors. Photosynthesis based on chlorophyll has not been found in Archaea, whereas photosynthesis based on bacteriorhodopsin, once believed unique to halophilic archaea, has been recently found in planktonic bacteria as well (Forterre, 2002). Beside their specific rRNA, archaea can be distinguished from bacteria by the nature of their membrane glycerolipids that are ethers of glycerol and isoprenol, whereas bacterial and eukaryal lipids are characterized as esters of glycerol and fatty acids (Kates, 1993). Archaeal glycerolipids are also ‘reverse lipids’, since the enantiomeric configuration of their glycerophosphate backbone is the mirror image of the configuration found in bacterial and eukaryal lipids. Another difference between Archaea and Bacteria is the absence of murein in archaea, whereas this compound is present in the cell wall of most bacteria. Archaea exhibits a great diversity of cell envelopes (Kandler & Konig, 1998), most archaea have a simple S-layer of glycoproteins covering the cytoplasmic membrane, whereas a few of them (Thermoplasmatales) only have a cytoplasmic membrane containing glycoproteins. The difference between Archaea and Bacteria at the molecular level is exemplified by the resistance of archaea to most antibiotics active on bacteria. Early studies on the molecular biology of archaea have shown that this resistance was due indeed to critical differences in the antibiotic targets (Zillig, 1991). Ammonia oxidation carried out by microorganisms has global importance in nitrogen cycling and is often thought to be driven only by ammonia-oxidizing bacteria; however, the recent finding of new ammonia-oxidizing organisms belonging to the archaeal domain challenges this notion. Two major microbial groups are now considered to be involved in ammonia oxidation. 2. Halophilic Archaea Halophilic archaea (also called halobacteria or haloarchaea) are present in high abundance and are often the dominant prokaryotes in hypersaline environments on the Earth such as solar salterns, hypersaline lakes, the Dead Sea, hypersaline microbial mats and underground salt deposits (Oren, 2002). Most known halophiles are relatively easy to grow, and genera such as Halobacterium, Haloferax, and Haloarcula have become well known models for studies of the archaeal domain because they are much simpler to handle than methanogenic and hyperthermophilic archaea (Ma et al., 2010). The haloarchaea are a monophyletic group including all known aerobic, obligate halophilic archaea. All are chemoorganotrophs; most utilize carbohydrates or amino acids and grow optimally between 3.4-4.5 M NaCl and generally require a minimum of 1.5 M NaCl for growth, some even grow well in saturated (>5 M) NaCl (Grant et al., 2001). In this respect, many haloarchaea appear ecologically equivalent in terms of resource and physiological requirements and therefore exhibit considerable overlap in their fundamental niches, defined as the combination of conditions and resources which allow a species to maintain a viable population (Begon et al., 1986). However, there are notable exceptions to this observation. For example, while most halophilic archaea exhibit optimal growth at near neutral pH, many are alkaliphilic and require at least pH 8.5 for growth (Grant et al., 2001). Recent molecular studies have revealed the presence of halophilic archaea in several low salinity environments (Elshahed et al., 2004). In addition, Purdy and colleagues (2004) isolated haloarchaea from a coastal salt marsh that exhibited optimal growth at 10% (1.7 M) NaCl but could grow slowly at 2.5% (0.43 M) NaCl. Microbial life has adapted to environments that combine high salt concentrations with extremely high pH values. Alkaline soda lakes in Africa, India, China, and elsewhere with pH values of 11 and higher and salt concentrations exceeding 300 g/l (5.1 M) are teeming with life (Oren, 2002). Hypersaline environments are found in a wide variety of aquatic and terrestrial ecosystems. These environments are inhabited by halotolerant microorganisms but also halophilic microorganisms ranging from moderate halophiles with higher growth rates in media containing between 0.5 M and 2.5 M NaCl to extreme halophiles with higher growth rates in media containing over 2.5 M NaCl (Ventosa et al., 1998). 1. Introduction These are chemolithoautotrophic ammonia-oxidizing bacteria and www.intechopen.com 30 Genetic Diversity in Microorganisms ammonia-oxidizing archaea. The first isolated ammonia-oxidizing archaeon , Nitrosopumilus maritimus, from a tropical marine aquarium tank (Konneke et al., 2005), was reported to include putative genes for all three subunits (amoA, amoB, and amoC) of ammonia monooxygenase that is the key enzyme responsible for ammonia oxidation. Ammonia- oxidizing archaea are determined to thrive in various habitats including marine and fresh waters, hot/thermal springs, soils, and wastewater treatment systems (Youa et al., 2009). The fascinating ability of members of the Archaea to thrive in extremes of temperature, salt and pH as well as in other seemingly hostile niches has generated substantial interest in the molecular mechanisms responsible for mediating survival in the face of such environmental challenges. Despite the obvious potential of extremophilic archaea to yield many commercially appealing enzymes, thermostable DNA polymerases remain the only major class of molecule to have been effectively exploited in a wide range of PCR protocols. www.intechopen.com 2. Halophilic Archaea Aerobic, anaerobic, and facultative anaerobic microbes belonging to domains Archaea and Bacteria have been recovered from these extreme ecosystems, where they participate in overall organic matter oxidation (Oren, 2002; Moune et al., 2003; Hedi et al., 2009). www.intechopen.com 31 Archaeal Diversity and Their Biotechnological Potential Hypersaline habitats are a kind of extreme environment dominated by halophilic archaea, which require a minimum of 9% (w/v) (1.5 M) NaCl for growth (Grant et al., 2001). In general, haloarchaeal strains require high salt concentration for growth and cell integrity. They, with some exceptions, lyse or lose viability in low salt concentrations or distilled water, and water sensitivity or lysis-resistence has been a key differentiation criterion between halococci and other haloarchaea (Grant et al., 2001). The most well known haloarchaea Halobacterium salinarum, for example, requires at least 2.5 M NaCl for growth and cells lose their morphological integrity instantaneously at less than 1 M NaCl (Kushner, 1964). Another representative, Halococcus morrhuae does not lyse in distilled water (Grant et al., 2001). Over the last decade, the diversity of halophilic archaea in various hypersaline environments has been examined and more fully characterized. Included among these studies are naturally occurring salt lakes, hypersaline microbial mats and man-made solar salterns (Benlloch et al., 2001; Maturrano et al., 2006). In another study the microbiota in colonic mucosal biopsies from patients with inflammatory bowel disease, 16S rDNA sequences representing a phylogenetically rich diversity of halophilic archaea from the Halobacteriaceae were determined. The study revealed a multitude of undefined bacterial taxa and a low diversity of methanogenic archaea (Oxley et al., 2010). Representatives of Archaea, the third domain of life, were generally thought to be limited to environmental extremes of the earth. However, the discovery of archaeal 16S rRNA gene sequences in water, sediment and soil samples has called into question the idea of archaea as obligate extremophiles (Purdy et al., 2004). As mentioned above based on the relationship with NaCl, the most salt requiring archaea are found in the order Halobacteriales and Methanosarcinales. They belong to the phylum Euryarchaeota; no halophilic representatives have yet been identified within the Crenarchaeota (Oren, 2008). Within the small subunit rRNA gene sequence-based tree of life there are three groups of prokaryotes that are both phylogenetically and physiologically coherent and consist entirely or almost entirely of halophiles. Within the Euryarchaeota the order Halobacteriales occurs with a single family, the Halobacteriaceae (Oren, 2002). www.intechopen.com 2. Halophilic Archaea The numbers of genera is reached to 35 (November 2011- Euzebylist) and Salarchaeaum is the last added extremely halophilic genus (Shimane et al., 2011). Most species of Halobacteriaceae are true extreme halophiles according to Kushner's definition (Kushner, 1978), however, Halobacteriaceae contains some species which can grow in low salinity for instance, Haloferax sulfurifontis (Elshahed et al., 2004), Haladaptatus paucihalophilus “the spesific epithet refers to low salt loving”, (Savage et al., 2007) and Halosarcina pallida (Savage et al., 2008). Diverse 16S rRNA gene sequences related to haloarchaea were recovered from tidal marine and salt marsh sediments, suggesting the existence of haloarchaea capable of growth at lower salt levels. In a recent study it was reported that two of three newly isolated genotypes had lower requirements for salt than previously cultured haloarchaea and were capable of slow growth at sea-water salinity (2.5% w/v NaCl). They reported the existence of archaea that could grow in non-extreme conditions and of a diverse community of haloarchaea existing in coastal salt marsh sediments and they concluded that the ecological range of these physiologically versatile prokaryotes is much wider than previously supposed (Purdy et al., 2004). Halophilic adaptation of organisms has been the subject of the great interest. Halophilic microorganisms have developed various biochemical strategies to adapt to high saline conditions, such as compatible solute synthesis to maintain cell www.intechopen.com 32 Genetic Diversity in Microorganisms structure and function (Tehei, 2002). These solutes are clearly of industrial interest. Besides these metabolical and physiological features, halophilic microorganisms are known to play important roles in fermenting fish sauces and in transforming and degrading waste and organic pollutants in saline waste waters (Hedi et al., 2009). All halophilic archaea studied balance the high osmolarity of their environment by having an at least equimolar intracellular salt concentration, KCl instead of NaCl in well-energized cells (Sopa, 2006). It has been shown that typical haloarchaeal proteins differ from mesohalic proteins by having a high fraction of acidic residues and a reduced fraction of basic residues. The genome sequences have corroborated that result and shown that a theoretical 2D gel of a haloarchaeon differs remarkably from that of other organisms (Tebbe et al., 2005). The cytoplasmic membranes of halophilic archaea of the family Halobacteriaceae contain interesting ether lipids and often have retinal proteins (bacteriorhodopsin, halorhodopsin, and sensory rhodopsins). 2. Halophilic Archaea It is known that unsaturated ether lipids are far more common in the halophilic archaea than generally assumed. Such unsaturated diether lipids were earlier reported from the psychrotolerant haloarchaeon Halorubrum lacusprofundi (Gibson et al., 2005). Many alkaliphiles are at the same time halophilic, and many useful enzymes applied in the detergent industry (washing powders), the textile industry, and other processes were obtained from bacteria growing in saline alkaline lakes. Halophilic enzymes (typical for halophilic archaea and bacteria) are characterized by an excess of acidic amino acids and subsequent negative surface charge. This feature allows effective competition for hydration water and enables function in solutions of low water activity, including organic solvent/water mixtures. The immediate advantages for enzyme technology are as follows: higher salt and heat tolerance, a catalytic environment which enables use of less polar educts, and potential reversal of hydrolytic reactions, all of which make them powerful candidates for industrial biocatalysts (Ma et al., 2010). The increase of salinity and nitrate/nitrite concentrations in soils and ground waters in the last few decades has focused much attention on the physiological and molecular mechanisms involved in salt-stress tolerance and nitrate metabolism by microorganisms. Physiological studies carried out with Haloferax mediterranei have revealed that it is resistant to very high nitrate (up to 2 M) and nitrite (up to 50 mM) concentrations (Bonete et al., 2008). Microorganisms are in general sensitive to low nitrate and nitrite concentrations. The inhibitory effect of these nitrogen compounds is due to the extreme toxicity of nitrite and nitric oxide produced upon nitrate reduction (Bonete et al., 2008). 3. Alkaliphilic Archaea Alkaliphile are microorganisms that grow very well at pH values between 9 and 12 or grow only slowly at the near neutral pH value of 6.5 (Horikoshi, 1999). The best examples of naturally occurring alkaline environments are soda deserts and soda lakes. Extremely alkaline lakes, for example, Lake Magadi in Kenya and the Wadi Natrun in Egypt, are probably the most stable highly alkaline environments on Earth, with a consistent pH of 10.5 to 12.0 depending on the site (Horikoshi, 1999). Alkaliphilic haloarchaea are a specialized group of obligate extreme halophiles that require high salinity as well as high pH (8.5-11) and low Mg2+ for growth (Kamekura et al., 1997; Xu et al., 2001). Alkaliphilic haloarchaea are commonly isolated from soda lakes but they have also been isolated and detected from solar salterns and other hypersaline environments with acidic, neutral and alkaline pHs (Gareeb & Setati, 2009). www.intechopen.com 33 Archaeal Diversity and Their Biotechnological Potential Alkaliphilic halophilic archaea are classified in the genera Natronobacterium, Natronococcus and Natronomonas (Kamekura et al., 1997), and some recent isolates are accommodated in Halalkalicoccus (Xue et al., 2005) and Natronolimnobius (Itoh et al., 2005). These five genera are so far composed exclusively of alkaliphilic strains. On the other hand, the genera Haloarcula, Natronorubrum, Halobiforma, Natrialba, Haloterrigena, Halorubrum and Halostagnicola consist of both neutrophilic and alkaliphilic species. Halorubrum and Haloarcula spp often form the majority of isolates is inhabited neutral brines (Grant et al., 2001). In a recent study isolates belonging to the genera Natrialba, Natronococcus and Natronorubrum were recovered from brine samples at evaporator ponds in Botswana (Gareeb & Setati, 2009). Hypersaline soda lakes are mostly inhabited by alkaliphilic representatives of halophilic archaea that could be in numbers of 107 to 108 /ml in soda lake brines (Horikoshi, 1999). A novel haloalkaliphilic archaeon from Lake Magadi was isolated and characterized (McGenity & Grant, 1993). It was revealed that cells of this isolate contained large gas vacuoles in the stationary phase of growth, and colonies produced by these archaea were bright pink. Xu et al. (1999) isolated two haloalkaliphilic archaea from a soda lake in Tibet. The strains were gram negative, pleomorphic, flat, nonmotile, and strictly aerobic. Their growth required at least 12% (2 M) NaCl and occurred between pH 8.0 and 11 with an optimum at pH 9.0 to 9.5. 4. Acidophilic Archaea Both natural and man-made acidic environments on the Earth are commonly found in the sites like pyrite ores, solfatara fields and marine volcanic vents; the microorganisms that inhabited these areas are called acidophiles and have a pH optimum for growth pH <3 (Baker-Austin & Dopson, 2007). Acidophiles are most widely distributed in the bacterial and archaeal domains and contribute to numerous biogeochemical cycles including the iron and sulfur cycles. Acidophiles might have played some crucial function in the evolution because metabolic processes might have originated on the surface of sulfide minerals (Wachtershauser, 2006) and structuring of the genetic code could have occurred in an intracellular environment with acidic pH (Di Giulio, 2005). Acidophiles optimally grow in low pH and metal-rich environments which might be quite resemble to volcanic aqueous conditions during Archaean and early Proterozoic periods. Therefore, it was suggested that acidophiles could represent primordial form of life from which more complex life have evolved (Baker-Austin & Dopson, 2007). Acidophiles are mostly found in isolated and inaccessible environments like geothermal vents. These environments generally have an impassable physical barrier which reduces the growth of neutrophiles. Recent bioinformatic analysis of several thermoacidophile archaeal genomes have implied that the similarities between these organisms are greater than expected when compared with other more closely related organisms. Therefore, acidic environments could establish an old and genetically distinct niche of life in which ecological closeness disregards phylogenetic nearness (Futterer et al., 2004). The ongoing exploration of the Earth has led to continued discoveries of life in environments that were previously considered uninhabitable. Thus, interest in the biodiversity and ecology of extreme environments and their inhabitants has grown over the past several years. In this regard, the study of extremely acidic environments is taking too much attention, because environmental acidity is often a consequence of microbial activity (Hallberg & Johnson, 2001). Highly acidic environments are relatively less common on Earth and are generally associated with mining activities. One of the main sources of acidity is the natural oxidation and dissolution of sulfidic minerals exposed to oxygen and water and this process can be greatly enhanced by microbial metabolism (Nordstrom & Alpers, 1999). Jhonson (1998) claimed that microorganisms that is involved in the generation of acidic metalliferous wastes cause widespread environmental pollution. 3. Alkaliphilic Archaea It was concluded that the physiological role of this enzyme is likely related to the oxidation of ethanol to acetaldehyde. 3. Alkaliphilic Archaea DNA-DNA hybridization results suggested that the two strains belonged to different species of the same genus. Recently, A novel haloalkaliphilic archaeon from commercial rock salt imported to Japan from China was isolated and characterized (Nagaoka et al., 2011). The isolation of a haloalkaliphilic archaea, grown optimally at pH 10.0, from a saline–alkaline soil was reported (Wang et al., 2010). The isolates displayed typical haloalkaliphilic growth characteristics with optimal growth at pH 9–10. Halophilic methanogens were isolated from various neutral saline areas and natural hypersaline environments (Boone et al., 2001). These strains showed the optimal growth at temperatures near 40°C and, in medium containing 0.5 to 2.5 M NaCl, at pH values near 7. Zhilina and Zavarzin (1994) described bacterial communities which inhabited in alkaline lakes, and in particular the diversity of anaerobic bacteria developing at pH 10 was exhibited. A new obligate alkaliphilic, methylotrophic methanogen was isolated from Lake Magadi (Kevbrin et al., 1997). Based on its phenotypic and genotypic properties, the isolate found to be belonged to Methanohalophilus zhilinaeae. It was an obligate alkaliphile and grew optimally within pH 9.2. The alkaliphiles are unique microorganisms, with great potential for microbiology and biotechnological exploitation. The essential use of alkaliphilic enzymes is in the detergent industry, for example, an extracellular protease from Natrialba magadii, a haloalkaliphilic archaeon, is a solvent tolerant enzyme and suggests a potential application in aqueous- organic solvent biocatalysis (Diego et al., 2007). Recently it was reported that the gene encoding the protease Nep secreted by the Natrialba magadii was cloned and sequenced (DeCastro et al., 2008). The study was announced the molecular characterization of a halolysin-like protease from alkaliphilic haloarchaea and the description of a recombinant system that facilitated high-level secretion of a haloarchaeal protease. Alcohol dehydrogenase is a key enzyme in production and utilization of ethanol. The gene encoding for ADH of the haloalkaliphilic archaeon Natronomonas pharaonis was cloned and expressed in Escherichia coli (Cao et al., 2008). The enzyme was haloalkaliphilic and thermophilic, being most active at 5 M NaCl or 4 M KCl and 70°C, respectively. The optimal activity was www.intechopen.com 34 Genetic Diversity in Microorganisms observed at pH 9.0 and significantly inhibited by Zn2+. It was concluded that the physiological role of this enzyme is likely related to the oxidation of ethanol to acetaldehyde. observed at pH 9.0 and significantly inhibited by Zn2+. www.intechopen.com 4. Acidophilic Archaea The extremely thermoacidophilic archaea are a group of interesting microorganisms in that they have to simultaneously cope with biologically extreme pHs and temperatures in their natural environments (Auernik et al., 2008). The current studies of the thermophilic and mesophilic acidophilic archaea have implied that there might be a stronger association between tetra-ether lipids and the tolerance to acid gradients than previously thought (Macalady & Banfield, 2003). Archaeal cell membrane including tetra-ether lipids instead of bacterial ester linkages is an example of highly impermeable cell membrane. These compounds were identified in Thermoplasma acidophilum (Shimada et al., 2002). Their www.intechopen.com 35 Archaeal Diversity and Their Biotechnological Potential expanding biotechnological significance relates to their role in biomining of base and precious metals and their unique mechanisms of survival in hot acid, at both the cellular and biomolecular levels. Extreme thermoacidophiles are microorganisms that are characterized by having an optimal growth temperature ≥ 60 oC and an optimal pH of ≤4. A majority of the extremely thermoacidophilic species studied to date belongs to the archaeal orders of Sulfolobales and Thermoplasmatales (Auernik et al., 2008). Acidianus infernus which is the most thermophilic of the extreme thermoacidophiles grows at temperatures up to 95 oC and at pHs as low as 1.0. On the other hand, Picrophilus species, member of the Thermoplasmatales (euryarchaeon), are the most acidophilic organisms growing at pHs as low as 0 and at temperatures up to 65 oC (Huber & Stetter, 2006). The application of recent molecular genetic systems and genome sequence data have given new clues in understanding of heavy metal tolerance, implementation of a genetic system and discovery of a new carbon fixation pathway. As a consequence, new insights into the molecular mechanisms that define extreme thermoacidophily have been gained. Extreme thermoacidophiles have evolved mechanisms for tolerating heavy metals which are physiologically toxic to most microorganisms (Salzano et al., 2007). These mechanisms cover to their capacity to recover from metal-induced damage (similar to oxidative stress) and to stop the accumulation of effective toxic metal concentration. In some cases, extreme thermoacidophiles could reduce or oxidize metals to less toxic forms by metabolic pathways. In other cases, metal chelation or complexation can perform the same job. Other mechanisms are based on the exporting toxic metal ions via P-type ATPases, instead of direct or indirect metal transformation (Ettema et al., 2006). 4. Acidophilic Archaea Acidophiles have some important biotechnological applications like metal recovery from ores, known as biomining (Rawling, 2002). Biomining is becoming increasingly important in mining because of its lower and containable pollutant outputs comparing to thermal processes. Efficacy in biomining environments demands tolerance to high levels of toxic heavy metals and the ability to incorporate inorganic carbon, as organic carbon can be scarce in this environment (Auernik et al., 2008). The future success of the biomining industry will depend upon the cellular biocatalysts with favorable features. The knowledge coming from genomic research of extreme thermoacidophiles will open new applications in this area. www.intechopen.com 5. Methanogens Methane (CH4) is an important greenhouse gas and its atmospheric abundance has been increasing by about 0.5% per year. Methanogenesis is a common process in many anaerobic environments such as digesters, rumen, rice fields, oil wells, landfills, and a range of extreme habitats (Garcia et al., 2000). Microorganisms are considered to be responsible for about the 50% of total methane production in the world. Methanogens, CH4-producing microorganisms, are strict anaerobes in the Euryarchaeota and can produce CH4 from a limited number of substrates: CO2 and H2, formate, methanol, methylamines or acetate. As terminal oxidizers in complex microbial communities, they are vital to the anaerobic microbial degradation of organic compounds in natural environments and probably also in defined ecological niches of the human body (Cavicchioli et al., 2003). Since methanogens coexist and closely interact with anaerobic bacteria at certain sites (e.g., human colon or dental plaque) they could be implicated in mixed anaeorobic infections. In fact, www.intechopen.com 36 Genetic Diversity in Microorganisms methanogens have recently been linked to periodontal disease (Lepp et al., 2004), a polymicrobial infection that affects the gums and supporting structures of the teeth and is characterized by periodontal pockets. Methanogens are phylogenetically diverse organisms although they rely on few metabolic substrates (Yavitt et al., 2011). methanogens have recently been linked to periodontal disease (Lepp et al., 2004), a polymicrobial infection that affects the gums and supporting structures of the teeth and is characterized by periodontal pockets. Methanogens are phylogenetically diverse organisms although they rely on few metabolic substrates (Yavitt et al., 2011). Methanogens are located in the domain Archaea and the phylum Euryarchaeota. Unlike Bacteria, some methanogens have pseudomurein in their cell wall instead of peptidoglycan (Balch et al., 1979). The glycan strand of pseudomurein is composed of alternating -(1→3)- linked N-acetyl-D-glucosamine and -(1→3)-linked N-acetyl-L-talosaminuronic acid (Kandler & Konig, 1998). The most closely related bacterial homologs to pseudomurein have been found in anaerobic Gram-positive or δ-proteobacterial lineages. It is reported that these organisms share the same ecological niche, and could also be the donors of peptide ligase homologs (MurC, MurE and MurF) which are probably responsible for pseudomurein biosynthesis in the Methanobacteriales (Graham & Huse, 2008). While pseudomurein is a common cell wall component in Methanobrevibacter and Methanobacterium, heteropolysaccharide is in Methanosarcina, and protein is in Methanococcus and Methanocalculus. 5. Methanogens The www.intechopen.com 37 Archaeal Diversity and Their Biotechnological Potential expectations of Earth-like organisms that could exist on other planets has varied, with methanogens often mentioned because of their adaptation to anaerobic niches with little or no organic carbon (Moissl-Eichinger, 2011), and especially with respect to the possible biogenic formation of the methane on Mars. Methanogenesis is a process occurring in many anaerobic environments. For instance methanogenesis in cold marine sediments has a global significance by leading to methane hydrate deposits, cold seeps, physical instability of sediment, and atmospheric methane emissions. The analysis of cultivation-independent archaeal community revealed that uncultivated microbes of the kingdoms Euryarchaeota and Crenarchaeota are present and that methanogens comprised a small proportion of the archaeal community. Methanogens were cultivated from depths of 0 to 60 cm in the sediments, and several strains related to the orders Methanomicrobiales and Methanosarcinales were isolated (Kendall et al., 2007). Microbial diversity was examined in the cold marine sediments (Orphan et al., 2002), however, the function that these microbes carry out during geochemical processes is still not clear. In addition to methanogens, uncultivated lineages of other Archaea have been also identified in marine sediments (Vetriani et al., 1999). expectations of Earth-like organisms that could exist on other planets has varied, with methanogens often mentioned because of their adaptation to anaerobic niches with little or no organic carbon (Moissl-Eichinger, 2011), and especially with respect to the possible biogenic formation of the methane on Mars. Methanogenesis is a process occurring in many anaerobic environments. For instance methanogenesis in cold marine sediments has a global significance by leading to methane hydrate deposits, cold seeps, physical instability of sediment, and atmospheric methane emissions. The analysis of cultivation-independent archaeal community revealed that uncultivated microbes of the kingdoms Euryarchaeota and Crenarchaeota are present and that methanogens comprised a small proportion of the archaeal community. Methanogens were cultivated from depths of 0 to 60 cm in the sediments, and several strains related to the orders Methanomicrobiales and Methanosarcinales were isolated (Kendall et al., 2007). Microbial diversity was examined in the cold marine sediments (Orphan et al., 2002), however, the function that these microbes carry out during geochemical processes is still not clear. In addition to methanogens, uncultivated lineages of other Archaea have been also identified in marine sediments (Vetriani et al., 1999). 5. Methanogens Methanogens are also characterized by including coenzyme F420, which is a cofactor necessary for certain enzyme activities such as hydrogenase and formate dehydrogenase. Another coenzyme typical to methanogens is coenzyme M, which is either produced by the methanogens, such as Methanobacterium, or is obtained from an external source, which is the case for Methanobrevibacter ruminantium (Ashby et al., 2001). Methanogens were the first organisms to be identified as archaea and classified as a separate domain. Six orders of methanogens exist: Methanobacteriales, Methanopyrales, Methanococcales, Methanomicrobiales, Methanosarcinales and Methanocellales. All of these orders contain a wide diversity of taxa that display great variance in their morphological and physiological characteristics. However, they all retain in common an anaerobic lifestyle and the ability to produce methane metabolically (Bapteste et al., 2005). Complete genome sequences for representatives of all of these orders are available. Initial analyses of the Methanocaldococcus - formerly Methanococcus jannaschii genome have revealed that the archaea have many metabolic characteristics in common with bacteria, but that the genes used for information processing are more similar to equivalent systems in eukaryotes (Walters & Chong, 2010). Following the suggestion that the Archaea are a distinct taxonomic group, it was considered that the domain would be divided along phenotypic lines and that the methanogenic archaea would be monophyletic (Bapteste et al., 2005). The 16S rRNA gene comparisons, however, indicated that this was not the case; the Methanomicrobiales were more closely related to extremely halophilic archaea (Halobacteriales) than to other methanogens (Woese et al 1990). The isolation of Methanopyrus kandleri and the sequencing of its 16S rRNA gene sequences showed that it was unrelated to any other known methanogens and originated at the base of the euryarchaeal branch of the tree, implied that the ancestor of euryarchaeal species might have been a methanogen (Burggraf et al., 1991). Recent studies based on transcriptional and translational proteins and 16S rRNA sequences have approved the absence of monophyly of methanogens and strongly suggested a close phylogenetic relationship between Methanopyrus, Methanococcus and Methanothermobacter (Brochier et al., 2004). As most of archaea have been found living at the extreme sites on this planet, they have often been proposed to resemble the life outside the earth if exists (Jarell et al., 2011). www.intechopen.com 5. Methanogens Unlike that of bacteria, the diversity of gut methanogenic archaea seems to be well understood and limited to two species belonging to Methanobacteriales, one of the five methanogenic orders defined to date: Methanobrevibacter smithii and occasionally Methanosphaera stadtmanae (Miller & Volin, 1982). In a current study the diversity of methanogenic Archaea from the gut of humans was analyzed by targeting mcrA, a molecular metabolic marker of methanogenesis (Mihajlovski et al., 2008). They reported the presence of Methanobacteriales, Methanobrevibacter smithii, Methanosphaera stadtmanae and a distant phylotype that did not cluster with any of the methanogenic orders. Their results were also supported by 16S archaeal sequences retrieved from the same volunteer, strongly suggests there may be a sixth order and hence potential underestimation of the role of methanogens in gut physiology. There is a link between the physiology of cultured methanogenic Archaea and their phylogenetic closeness based on 16S rRNA sequences (Zinder, 1993). For example, while most species of the Methanobacteriaceae and Methanomicrobiaceae prefer H2 and CO2 (or formate) as substrates for methanogenesis, Methanosaeta, a genus within the Methanosarcinaceae, is known to generate energy only from acetate fermentation. Most of the other Methanosarcinaceae preferentially use methanol and related methyl-substrates for the generation of CH4 (Kleikemper et al., 2005). It has been considered that methanogenesis may function in the mineralization of petroleum hydrocarbons in contaminated aquifers (Chapelle et al., 2002). However, petroleum hydrocarbons can not be degraded directly by methanogenic microorganisms (Zengler et al., 1999). Methanogens metabolizing H2 and CO2 involve indirectly into PHC degradation by maintaining H2 concentrations low by that way fermentation of PHC becomes exergonic and fermenting microrganisms can grow (Garcia et al., 2000). On the other hand, methanogens metabolizing acetate or methanol can degrade PHC directly by cleaving end products of fermentation. However, the role of different metabolic groups of methanogens with respect to overall methanogenic activity in PHC-contaminated aquifers is reported to be unclear (Kleikemper et al., 2005). Aceticlastic methanogenesis was assumed to be the final step of hydrocarbon degradation in a PHC-contaminated aquifer, but this was not confirmed with activity measurements (Dojka et al., 1998). The research for alternative forms of energy, including recovery of methane via anaerobic digestion of wastes, has becoming very popular since successive petroleum crisis in 1970s. www.intechopen.com 38 Genetic Diversity in Microorganisms 6. Thermophilic Archaea In the domain Archaea, hyperthermophilic and extremely thermophilic archaea are determined extensively over the phyla Crenarchaeota and Euryarchaeota. Hyperthermophiles, growing optimally at ≥ 80 C, have been acknowledged since 1981 (Zillig et al., 1981). They represent the upper temperature limit of life and are found in environments characterized with high temperature. In their basically anaerobic environments, they generally obtain energy by inorganic redox reactions. Stetter (2006) claimed that in his lab so far about 50 new species of hyperthermophiles have been isolated and characterized, among them representatives of the novel bacterial genera Thermotoga, Thermosipho, Aquifex, Thermocrinis and archaeal genera Acidianus, Metallosphaera, Stygiolobus, Thermoproteus, Pyrobaculum, Thermofilum, Desulfurococcus, Staphylothermus, Thermosphaera, Ignicoccus, Thermodiscus, Pyrodictium, Pyrolobus, Thermococcus, Pyrococcus, Archaeoglobus, Ferroglobus, Methanothermus, Methanopyrus and Nanoarchaeum. The earliest archaeal phylogenetic lineage is represented by the extremely tiny members of the novel kingdom of Nanoarchaeota (Stetter, 2006). Hyperthermophiles occupy all the short deep branches closest to the root within the universal phylogenetic tree. In rRNA-based phylogenetic tree constructed by Woese et al. (1990), all extremely short and deeply branching-off lineages within the archaea and bacteria are exclusively represented by hyperthermophiles (Fig. 1), indicating a slow rate of evolution (Stetter, 2006). Fig. 1. Universal phylogenetic tree constructed based on rRNA sequence comparison. Hyperthermophiles represented with thick red lineages (Stetter, 2006). Fig. 1. Universal phylogenetic tree constructed based on rRNA sequence comparison. Hyperthermophiles represented with thick red lineages (Stetter, 2006). www.intechopen.com 39 Archaeal Diversity and Their Biotechnological Potential Until now, all Crenarchaeota species that have been isolated are either hyperthermophilic or extremely thermophilic, although the existence of mesophilic and psychrophilic Crenarchaeota is also suggested by culture-independent molecular phylogenetic analyses. The cultured Crenarchaeota are composed of five orders: Acidilobales, Fervidicoccales, Desulfurococcales, Sulfolobales and Thermoproteales, which are well-supported by 16S rDNA sequence data and by phenotypic properties, such as cell morphology and lipid composition (Burggraf et al., 1997; Reysenbach, 2001; Chaban et al., 2006; Prokofeva et al., 2009). Some of the crenarchaeotas are sequenced from low-temperature environments (Dawson et al., 2001). So far only one member of the groups, Crenarchaeum symbiosum, from non- thermophilic environments has been cultivated in axenic culture (Preston et al., 1996). The information about non-thermophilic Crenarchaeota is, therefore, solely based on sequence data collected from various low temperature environments like soil, freshwater, deep drillings, and seawater. The analyses about the energy sources of hyperthermophiles have revealed that most species are chemolithoautotroph (Amend & Shock, 2001; Stetter, 2006). 6. Thermophilic Archaea Mode of Respiration is anaerobic -nitrate, sulphate, sulphur and carbon dioxide respiration- and aerobic, CO2 is the solely carbon source required to synthesize organic cell material. While molecular hydrogen generally serves as main electron donor, sulphide, sulphur, and ferrous iron are other electron donors. Oxygen also may serve as an electron acceptor in some hyperthermophiles which are usually microaerophilic. Anaerobic respiration forms are the nitrate-, sulphate-, sulphur- and carbon dioxide respirations. While chemolithoautotrophic hyperthermophiles synthesize organic matter, some obligate heterotrophic hyperthermophiles depend on organic material as energy- and carbon-sources. In addition, a few chemolithoautotrophic hyperthermophiles are opportunistic heterotrophs. They obtain energy either by aerobic or anaerobic respiration or by fermentation (Stetter, 2006). Recently a novel phylum of Archaea, called Nanoarchaeota, was discovered. The phylum is currently represented by a single species named as Nanoarchaeum equitans. The species is a nano-sized hyperthermophilic symbiont that grows attached to the surface of an Ignicoccus species (Huber et al., 2002). It has a cell diameter of only 400 nm and grows under strictly anaerobic conditions at temperatures between 75 °C and 98 °C. The N. equitans has a genome of only 490.885 bp, it is the smallest genome of any Archaea, and the most compact, with 95% of the DNA predicted to encode proteins or stable RNAs. Its DNA encodes the complete machinery for information processing and repair, but lacks genes for lipid, cofactor, amino acid, and nucleotide biosynthesis. The inadequate biosynthetic and catabolic capacity of N. equitans considers its symbiotic relationship to its Ignicoccus host as a parasitic relationship, and this makes N. equitans the only known archaeal parasite (Waters et al., 2003). 7. Biotechnological importance of Archaea Industries such as food processing, cleaning, biosynthetic processes and environmental bioremediation need efficient biocatalysts which can operate in harsh environments. The sources for these biocatalysts have been animals, plants, fungi and mostly bacteria. Recently, the extremophilic bacteria and archaea have become more popular since the enzymes of these organisms are able to remain catalytically active under extremes of temperature, salinity, pH and pressure (Synowiecki et al., 2006; Ozcan et al., 2009). In biotechnology for www.intechopen.com 40 Genetic Diversity in Microorganisms an efficient application, one has to determine the most suitable enzymes and best reaction conditions. Nowadays there are two main strategies for obtaining enzymes with desired properties, namely the genetic engineering of currently known enzymes and the search for new activities in previously uncharacterized microorganisms. Within the second approach, the search for enzymes in extremophiles (called extremozymes) seems to be particularly promising since the enzymes of these organisms have particular adaptations to increase their stability in adverse environments, which can potentially also increase their stability in the harsh environments in which they are to be applied in biotechnology (Oren, 2002). The domain archaea is particularly under extensive research for their potential biotechnological applications. The domain includes the extreme halophiles, the hyperthermophiles, the thermoacidophilic archaea and the psychrophiles that live in the cold waters of the Antarctic. But the most important value of these organisms (along with some of the bacteria that also tolerate extreme environments) is that their enzyme systems work at harsh conditions. For example, many of the restriction enzymes used in gene splicing and cloning are products of extremophiles. In 1993 a report from US National Academy of Sciences noted that world enzyme sales equaled to US$1 billion and it is a market that has been expected to grow about 10% per year. It is expected that enzymes from extremophiles will constitute an important part of this market. Recently genes encoding several enzymes from extremophiles have been cloned in mesophilic hosts, with the objective of overproducing the enzyme and altering its properties to suit commercial applications (Alqueres et al., 2007). It has been revealed that enzymes derived from extremophilic archaea are in many cases superior to bacterial homologs. They have higher stability towards heat, pressure, detergents and solvents (Egorova & Antranikian, 2005). The archaeal hyperthermophiles, known as heat-stable prokaryotes, have exceptionally high growth temperature limits and unique ether-linked lipids as well as eukaryotic transcription and translation factors. www.intechopen.com 7. Biotechnological importance of Archaea It has been proposed that heat shock proteins which are inducible by supraoptimal temperatures (Kagawa et al., 1995) and reverse gyrase which installs positive supercoils in DNA (Guipaud et al., 1997) could have been involved in unusual adaptive mechanisms of the hyperthermophiles. DNA polymerase I from the bacterium Thermus aquaticus, Taq polymerase, is the first thermostable DNA polymerases in biotechnology and has an extensive use in PCR. For PCR applications, the archaeal DNA polymerases have been found to have some superior features to bacterial ones. For instance, Pwo from Pyrococcus woesei, Pfu from P. furiosus, Deep Vent polymerase from the Pyrococcus strain GB-D and Vent polymerase from Thermococcus litoralis, reported to have an error rate that is much lower than that of Taq polymerase. The starch-processing industry needs thermostable enzymes in order to convert starch into more valuable products such as dextrins, glucose, fructose and trehalose, (Egorova & Antranikian, 2005). It is well established that in all starch-converting processes, high temperatures are required to liquefy starch and to make it accessible to enzymatic hydrolysis. The synergetic action of thermostable amylases, pullulanases and ǂ-glucosidases include the advantage of lowering the cost of making sugar syrup, production of new starch-based materials that have gelatin-like characteristics and defined linear dextrins that can be used as fat substitutes, texturizers, aroma stabilizers and prebiotics. Recently a variety of starch-degrading enzymes from extremophilic archaea has been published. They www.intechopen.com 41 Archaeal Diversity and Their Biotechnological Potential determined the optimal temperatures for the activity of the archaeal amylases in a range between 80 C and 100 C. Especially, the high thermostability of the extracellular ǂ-amylase from Pyrococcus sp. (retain its activity even at 130oC) makes these enzymes ideal candidates for industrial application (Egorova & Antranikian, 2005). determined the optimal temperatures for the activity of the archaeal amylases in a range between 80 C and 100 C. Especially, the high thermostability of the extracellular ǂ-amylase from Pyrococcus sp. (retain its activity even at 130oC) makes these enzymes ideal candidates for industrial application (Egorova & Antranikian, 2005). Lipases (carboxyl ester hydrolases) are ubiquitous in nature, produced by animals, plants, and fungi, as well as bacteria. Recently halophilic archaeal organisms are taking more attention due to their lipolytic enzymes. For instance, Haloarcula marismortui, a halophilic archaeon whose genome was sequenced contains genes encoding for putative esterase and lipase. These genomic predictions have been verified recently (Camacho et al. 2009). 7. Biotechnological importance of Archaea They reported that Haloarcula marismortui displays esterase and lipase activity intracellularly and extracellularly. While lipase was accumulated mainly extracellular, esterase was generally accommodated intracellular (Camacho et al., 2009). The esterase and lipase genes of Pyrococcus furiosus, a hyperthermophilic archaeon, have been cloned in E. coli and functional properties have been determined. The archaeal enzyme reported as the most thermostable and thermoactive esterase known to date (Ikeda and Clark 1998). In our laboratory, the halophilic archaeal isolates, grown best in a range of 2-5 M NaCl, produced high lipolytic activity in the range of 3-4.5 M NaCl (Ozcan et al., 2009). It was found that the lipolytic activity dropped at 5 M NaCl. Therefore these enzymes can be classified as not only salt dependent but many can be also thermostable (Oren, 2002; Ozcan et al., 2009). Heat-stable proteases have some utilities in biotechnology, especially in the detergent industry. It has been revealed that most proteases from extremophilic Archaea belong to the serine type and are stable at high temperatures, even in the presence of high concentrations of detergents and denaturing agents (Antranikian et al., 2005). Proteases are also applied for peptide synthesis using their reverse reaction, mainly because of their compatibility with organic solvents (Egorova & Antranikian, 2005). Recently a protease from Thermococcus kodakarensis has been characterized (Foophow et al., 2010). It has been shown that a ǃ-jelly roll domain is not directly involved in proteolytic activity, however, takes role in the extreme thermostability of the enzyme. One of the important strategies in searching the alternative energy sources could be the conversion of biomass to fuel products (biofuels). Both chemical and biological processes are being explored for the production of bioethanol, biodiesel, biobutanol, biomethane and biohydrogen. For the production of biohydrogen some heterotrophic hyperthermophiles particularly have been found useful because of their abilities to produce molecular hydrogen (Atomi et al., 2011). It is covalently linked to Lys216 in the chromophore by Schiff base action. Habitats with harsh environmental conditions are often populated by archaea that are specialized to live in water near salt saturation also specialized to utilize solar energy. Light of distinct wavelengths is used to fuel primary transport of both protons and chloride ions as well as for phototaxis. The halophilic archaeon makes use of light for both energy and sensory transduction by exploiting a family of light-sensitive proteins called rhodopsin. 7. Biotechnological importance of Archaea Bacteriorhodopsin is a 25-kDa integral membrane protein that carries a retinal group (cromophore) and it is covalently linked to lysine-216 by Schiff-base action. Its function was discovered in the early 1970s during studies of the purple membrane, patches of membrane that contain only bacteriorhodopsin and lipids, found within the cell membrane of Halobacterium salinarum (Oren, 2008). The excellent thermodynamic and photochemical www.intechopen.com 42 Genetic Diversity in Microorganisms stability of bacteriorhodopsin has led to many uses in technical applications like holography, spatial light modulators, artificial retina, neural network optical computing, and volumetric and associative optical memories (Alqueres et al., 2007). stability of bacteriorhodopsin has led to many uses in technical applications like holography, spatial light modulators, artificial retina, neural network optical computing, and volumetric and associative optical memories (Alqueres et al., 2007). Archaeosomes, a kind of liposomes, are produced from natural lipids found in Archaea or from synthetically synthesized compounds that share the unique structural features of archaeal lipids. The isoprenoid glycerolipid membrane of archaeosomes can develop into a bilayer, a monolayer, or a combination of mono- and bilayers made from bipolar and monopolar archaeal lipids (Jacquemet et al., 2009). The archaeal lipids formulations have shown to exhibited relatively higher physico-chemical stabilities to oxidative stress, high temperature, alkaline pH, action of phospholipases, bile salts and serum media (Brard et al., 2007). These properties contribute to their efficacy as self-adjuvant vaccine delivery vesicles. Additionally, archaesomes were found to be safe and not toxic in mice both in-vitro and - vivo studies (Omri et al., 2003). Thus, it has been claimed that the biocompatibility and the superior stability properties of archaeosomes in several conditions give advantages over conventional liposomes in the manufacture and the use in biotechnology including vaccine and drug delivery (Jacquemet et al., 2009). www.intechopen.com 8. Conclusion On the other hand alkalithermophilic members of the Archaea were isolated besides halophilic. These oorganisms have economic value because alkaliphilic enzymes have been used in different industries (Horikoshi, 1999). and ATP generation), novel extracellular polysaccharides, exoenzymes (amylase, cellulase, xylanase, lipase and protease) and poly-hydroxyalkanoate (used in biodegradable plastic production), and a protein from Halobacterium salinarum has significance in cancer research. Some members are extremely alkaliphilic as well as being halophilic. They grow optimally in alkaline environment with pH values above 9 such as soda lakes and carbonate-rich soils but cannot grow or grow only slowly at the near-neutral pH value (6.5). On the other hand alkalithermophilic members of the Archaea were isolated besides halophilic. These oorganisms have economic value because alkaliphilic enzymes have been used in different industries (Horikoshi, 1999). Acidophilic archaea have a pH optimum for growth of less than pH 3. They contribute to numerous biogeochemical cycles including the iron and sulfur cycles. Acidophiles have different biotechnological applications. Firstly metal extraction from ores and this sustainable biotechnological process is becoming increasingly important because of its reduced and containable pollutant outputs. Acidophiles could also be a source of gene products; for example, acid-stable enzymes with applications as lubricants and catalysts (Baker-Austin and Dopson, 2007). Metabolism of methanogens is unique in that energy is obtained by the production of methane (natural gas). Biological methanogenesis is applied to the anaerobic treatment of sewage sludge, and agricultural, municipal and industrial wastes, where the maintenance of a desired methanogenic flora is achieved by inoculation (Schiraldi et al., 2002). Many archaea colonize extreme environments. Because extremophilic microorganisms have unusual properties, they are a potentially valuable resource in the development of novel biotechnological processes. Especially, based on the unique stability of archaeal enzymes at high temperature, salt and extremes of pH, they are expected to be a very powerful tool in industrial biotransformation processes that run at harsh conditions. The growing demand for more effective biocatalysts has been satisfied either by improving the properties of existing proteins or by producing new enzymes. The majority of the industrial enzymes known to date have been extracted mostly from bacteria and fungi. Until now, only a few archaeal enzymes have been found to be useful in industrial applications. 8. Conclusion The majority of the living beings thrive in environments having physically and geochemically temperate conditions. The extreme environments found on the planet are generally inhabited by microorganisms, which belong to the Archaeal and Bacterial domains of life. Archaea, members of the third domain of life, are prokaryotes that harbour many unique genotypic and phenotypic properties. Two archaeal phyla are presently recognized; the Euryarchaeota and the Crenarchaeota (Woese et al., 1990). Archaea exhibit a wide diversity of phenotypes. The first phenotypes to be recognized were the methanogens which are strict anaerobes and methane producers. Many archaea are extremophiles which are capable of growth at high tempereture, salinity and extremes of pH. In common usage, cultivated Archaea were, without exception, considered to be extremophiles. Extreme environments comprise sites of extreme temperature, pH, pressure and salinity. Archaea exist in a broad range of habitats, and as a major part of global ecosystems, may contribute up to 20% of earth's biomass (Delong and Pace, 2001). Thermophilic archaea inhabit environments like hot springs, ocean vents, and geysers which are inhospitable to many other organisms. These habitats not only have extremely high temperatures but have high concentrations of dissolved minerals and low concentrations of oxygen (Egorova and Antranikian, 2005). In particular, enzymes from thermophilic and hyperthermophilic Archaea have industrial relevance. Enzymes of thermophilic archaea are superior to the traditional catalysts because they can perform industrial processes even under harsh conditions (Egorova and Antranikian, 2005). The extreme halophilic archaea require at least 1.5 M NaCl. Most strains grow best at 3.5–4.5 M NaCl. Members of the halophilic archaea are dominant microorganisms in hypersaline environments worldwide including salt lakes, crystallizer ponds of solar salterns, salt mines, as well as hypersaline soda lakes (Oren, 2002). Halophilic archaea have a number of useful applications in biotechnological processes and potential new applications are being investigated. For instance, they produce bacteriorhodopsin (used in information processing www.intechopen.com 43 Archaeal Diversity and Their Biotechnological Potential and ATP generation), novel extracellular polysaccharides, exoenzymes (amylase, cellulase, xylanase, lipase and protease) and poly-hydroxyalkanoate (used in biodegradable plastic production), and a protein from Halobacterium salinarum has significance in cancer research. Some members are extremely alkaliphilic as well as being halophilic. They grow optimally in alkaline environment with pH values above 9 such as soda lakes and carbonate-rich soils but cannot grow or grow only slowly at the near-neutral pH value (6.5). 8. Conclusion Based on the unique stability of archaeal enzymes at high temperature, salt and extremes of pH, they are expected to be a very powerful tool in industrial biotransformation processes that run at harsh conditions. Owing to the unique features of Archaea, their potential applications in biotechnology are far reaching, ranging from bioremediation potential of nitrite and nitrate from groundwater, oil pollutions, toxic compounds, metal polluted sites and biomining and nitrate removing from brines (Schiraldi et al., 2002). 9. References Alquéres, S.M.C., Almeida, R.V., Clementino, M.M., Vieira, R.P., Almeida, W.I., Cardoso, A.M., Martins, O.B. (2007) Exploring the biotechnologial applications in the archaeal domain Brazilian Journal of Microbiology 38:398-405 Amend, J. P., Everett, L. (2001) Shock Energetics of overall metabolic reactions of thermophilic and hyperthermophilic Archaea and Bacteria. FEMS Microbiology Reviews. 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Sci. 361, 1787–1806. www.intechopen.com 51 Archaeal Diversity and Their Biotechnological Potential Walters, A. D. and Chong, J. P. J. (2010) An archaeal order with multiple minichromosome maintenance genes. Microbiol. Vol. 9. References Biogeochemistry. Vol. (24 September), pp. 1-15 Youa, J., Dasa, A., Dolanb, E.M., and Hua, Z. (2009) Ammonia-oxidizing archaea involved in nitrogen removal. Water Research, Vol. 43(7), pp.1801-1809 g ( ) pp Zhilina, T. N., and Zavarzin, G. A. (1994) Alkaliphilic anaerobic community at pH 10. Curr. Microbiol. Vol. 29, pp. 109–112 Zillig, W., Holz, I., Klenk, H. P., Trent, J., Wunderl, S., Janekovıc, D., Imsel, E. and Haas, B. (1987) Pyrococcus woesei, sp. nov., an ultra-thermophilic marine Archaebacterium, representing a novel order, Thermococcales. Syst. Appl. Microbiol.,Vol.9, pp. 62-70 www.intechopen.com www.intechopen.com 52 Genetic Diversity in Microorganisms Zillig, W. (1991) Comparative biochemistry of Archaea and Bacteria, Curr. Opin. Genet. Dev Vol.1, pp.544–551 Zillig, W. (1991) Comparative biochemistry of Archaea and Bacteria, Curr. Opin. Genet. Dev. Vol.1, pp.544–551 Zillig. W., Stetter. K. O., Schäfer, W., Janekovic, D., Wunderl. S., Holz, f., and Palm, P.: (1981) Thermoproteales: a novel type of extremely thermoacidophilic anaerobic archaebacteria isolated from Icelandic solfataras. Zbl. Bakt. Hyg. I. Abt. Orig. Vol.C2, pp. 205–227 pp Zinder, S. H. (1993) Physiological ecology of methanogens, In: Methanogenesis: ecology, physiology, biochemistry and genetics, Ferry, J. G., (Ed), pp. 128–206, Chapman & Hall, Inc., New York. www.intechopen.com p y gy, y g , y, J , ( ), pp , p Hall, Inc., New York. www.intechopen.com www.intechopen.com Genetic Diversity in Microorganisms Edited by Prof. Mahmut Caliskan ISBN 978-953-51-0064-5 Hard cover, 382 pages Publisher InTech Published online 24, February, 2012 Published in print edition February, 2012 Genetic Diversity in Microorganisms presents chapters revealing the magnitude of genetic diversity of microorganisms living in different environmental conditions. The complexity and diversity of microbial populations is by far the highest among all living organisms. The diversity of microbial communities and their ecologic roles are being explored in soil, water, on plants and in animals, and in extreme environments such as the arctic deep-sea vents or high saline lakes. The increasing availability of PCR-based molecular markers allows the detailed analyses and evaluation of genetic diversity in microorganisms. The purpose of the book is to provide a glimpse into the dynamic process of genetic diversity of microorganisms by presenting the thoughts of scientists who are engaged in the generation of new ideas and techniques employed for the assessment of genetic diversity, often from very different perspectives. The book should prove useful to students, researchers, and experts in the area of microbial phylogeny, genetic diversity, and molecular biology. How to reference In order to correctly reference this scholarly work, feel free to copy and paste the following: Birgül Özcan (2012). Archaeal Diversity and Their Biotechnological Potential, Genetic Diversity in Microorganisms, Prof. Mahmut Caliskan (Ed.), ISBN: 978-953-51-0064-5, InTech, Available from: http://www.intechopen.com/books/genetic-diversity-in-microorganisms/archaeal-diversity-and-their- biotechnological-potential InTech Europe University Campus STeP Ri Slavka Krautzeka 83/A 51000 Rijeka, Croatia Phone: +385 (51) 770 447 Fax: +385 (51) 686 166 www.intechopen.com InTech China Unit 405, Office Block, Hotel Equatorial Shanghai No.65, Yan An Road (West), Shanghai, 200040, China Phone: +86-21-62489820 Fax: +86-21-62489821 InTech China Unit 405, Office Block, Hotel Equatorial Shanghai No.65, Yan An Road (West), Shanghai, 200040, China Phone: +86-21-62489820 Fax: +86-21-62489821 InTech Europe University Campus STeP Ri Slavka Krautzeka 83/A 51000 Rijeka, Croatia Phone: +385 (51) 770 447 Fax: +385 (51) 686 166 www.intechopen.com © 2012 The Author(s). Licensee IntechOpen. This is an open access artic stributed under the terms of the Creative Commons Attribution 3.0 cense, which permits unrestricted use, distribution, and reproduction in ny medium, provided the original work is properly cited. © 2012 The Author(s). Licensee IntechOpen. This is an open access article distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
W3133613871.txt	https://link.springer.com/content/pdf/10.1007/s12054-021-00361-3.pdf	de		Kinder- und Jugendhilfe und LGBTIQ*	Sozial extra	2,021	cc-by	2,673	Sozial Extra 2 2021: 80–84 https://doi.org/10.1007/s12054-021-00361-3 © Der/die Autor(en) 2021 Online publiziert: 26. Februar 2021 Extrablick: Kinder- und Jugendhilfe und LGBTIQ* Kinder- und Jugendhilfe und LGBTIQ* Eine Einführung in den Schwerpunkt Wie finden in Angeboten der Kinder- und Jugendhilfe queere Jugendliche Raum? Wie werden sie berücksichtigt – sichtbar oder unsichtbar gemacht? Wie werden junge Menschen, die sich abseits von heteronormativen Wertvorstellungen positionieren, in der Kinder- und Jugendhilfe adressiert? Mit diesen Fragen setzt sich der Schwerpunkt „Kinder- und Jugendhilfe und LGBTIQ“ auseinander und möchte die unterschiedlichen Felder der Kinder- und Jugendhilfe für geschlechtliche und sexuelle Vielfalt sensibilisieren. S o wurde der Themenschwerpunkt „Kinder- und Jugendhilfe und LGBTIQ“ angekündigt und wir freuen uns, dass wir mit diesem Schwerpunkt ein Thema umsetzen können, das empirisch längst überall gegenwärtig ist, mit dem jedoch nach wie vor fachliche Unsicherheiten und Fragen einhergehen. Aussagen wie „Bei uns im Jugendtreff gibt es keine schwulen Jugendlichen“, die wir auf einem Fachtag zum Thema „LGBTIQ* und Kinder- und Jugendhilfe“ hörten, lassen uns nachdenklich zurück. Empirisch kann diese Aussage nicht zutreffen, da sich fünf bis zehn Prozent aller Menschen als homosexuell positionieren. Es handelt sich also möglicherweise vielmehr um ein Unsichtbarmachen und mangelnde Sensibilität. Oder aber die Aussage ist so zu lesen, dass queere Jugendliche Angebote der Kinder- und Jugendarbeit meiden, wenn es ihKatharina Mangold Universität Hildesheim Hildesheim, Deutschland 1979; Dr., Diplom-Pädagogin, wissenschaftliche Mitarbeiterin am Institut für Sozial- und Organisationspädagogik der Universität Hildesheim. katharina.mangold@uni-hildesheim.de Angela Rein Muttenz, Schweiz 1979; Dr., Diplom-Pädagogin, Professorin am Institut Kinderund Jugendhilfe der Hochschule für Soziale Arbeit, Fachhochschule Nordwestschweiz (FHNW). angela.rein@fhnw.ch Zusammenfassung Der Beitrag führt in den Schwerpunkt ein und skizziert dabei dessen Zielsetzung, indem er die Texte und deren Akzentuierung kurz vorstellt. Schlüsselwörter LGBTIQ, Kinder- und Jugendhilfe, Verbesonderung, Homogenisierung 80 nen möglich ist, weil sie sich dort nicht verstanden und angenommen fühlen. Begrifflichkeiten und Glossar Mit LGBTIQ fokussieren wir auf Menschen, die sich als lesbisch, gay (schwul), bisexuell, trans/transgeschlechtlich, intergeschlechtlich und/oder queer positionieren. Mit dem Sternchen „“ wird darauf hingewiesen, dass auch diese Positionierungen stets heterogen sind und hier nicht von einer Ausschließlichkeit, Abgeschlossenheit oder einer Homogenisierung einer realen Gruppe ausgegangen werden darf. In der Thematisierung und Auseinandersetzung mit LGBTIQ* wird häufig deutlich, dass „I“, also intergeschlechtlich, zwar in der Abkürzung Eingang findet, in den jeweiligen Studien oder Argumentationen wird die Lebenssituation von intergeschlechtlichen Menschen dann aber wenig beleuchtet und diskutiert. Daher freuen wir uns besonders über den Beitrag von Kerstin Schumann, der sich mit der Notwendigkeit der Einbeziehung von Inter* in die Debatte um Vielfalt in der Kinder- und Jugendhilfe beschäftigt und dies in Bezug auf die Frage von Kindeswohl diskutiert. Damit verbunden ist das notwendige Hinterfragen von Zweigeschlechtlichkeit. Seit 2018 ist es im deutschen Personenstandsrecht möglich, neben „männlich“ und „weiblich“ die Kategorie „divers“ eintragen zu lassen. Dennoch sind heteronormativen Vorstellungen fest in unserer Gesellschaft verwoben und es bedarf gezielter Aktivitäten, daran zu rütteln und das Denken in bipolaren Kategorien (Mann – Frau) aufzubrechen. Geschlecht ist weder eindeutig noch unveränderbar. Dasselbe gilt für die Begehrenspositionen. © Ruth Hebler Die Bezeichnungen im Detail: • Cis-Geschlechtlichkeit bezeichnet Menschen, die sich mit dem Geschlecht identifizieren, welches ihnen bei der Geburt zugewiesen wurde. • Transgeschlechtlichkeit bezeichnet Menschen, die in einem anderen Geschlecht leben, als ihnen bei der Geburt zugewiesen wurde, aber auch Menschen, die sich gar nicht einer Geschlechterkategorie zuordnen, die Geschlechter wechseln oder sich mehreren Geschlechtern zugehörig fühlen. • Intergeschlechtlichkeit (oder Inter) bezeichnet Menschen, deren genetische, hormonelle oder körperliche Merkmale weder ausschließlich männlich noch ausschließlich weiblich sind. Heteronormativität in pädagogischen Feldern Schmidt und Schondelmeyer (2015) zeigen auf, dass sexuelle und geschlechtliche Vielfalt in der pädagogischen Praxis kein oder wenig Thema ist. Elisabeth Tuider macht deutlich, dass pädagogische Arbeit – ob innerhalb oder außerhalb der Schule – nach wie vor auf kaum hinterfragten Annahmen von eindeutigen, kohärenten und identitären Zugehörigkeiten hinsichtlich Geschlecht, Sexualität, aber auch Nationalität und körperliche und geistige ‚Unversehrtheit‘ basiert (Tuider 2016, S. 57). Damit wird sowohl deutlich, dass Schule nach wie vor eine Arena darstellt, welche durch Heteronormativität gekennzeichnet ist. Darüber hinaus wird der Blick aber auch auf pädagogische Angebote abseits der Schule gelenkt. Auch diese Angebote basieren auf heteronormativen Annahmen. Queere Jugendliche sind jedoch in allen Feldern der Kinder- und Jugendhilfe längst Realität, dennoch kann und muss kritisch gefragt werden, wie die jeweiligen Feldern auf queere Kinder und Jugendliche eingestellt sind, welche Angebote sie bereithalten, welche Praktiken der Unsichtbar-Machung zu beobachten sind, welche Diskriminierungen junge Menschen erleben (müssen). In unserem Schwerpunkt … • … fokussiert Mart Busche auf die Offene Kinderund Jugendarbeit und fragt am Beispiel dieses niedrigschwelligen, offenen Angebotes für alle Kinder und Jugendlichen danach, ob von einer Post-Heteronormativität zu sprechen ist. • … greifen Steffen Baer und Davina Höblich auf verschiedene Interviews mit (sozial-) pädagogischen Fachkräften zurück und machen deutlich, wie ein affirmativer Ansatz aussehen kann, bzw. wo dieser auch an Grenzen stößt. • … gehen Fabian Baier und Stephanie Nordt, die als Bildungsreferentinnen bei Queerforamt aktiv sind, auf konkrete Herausforderungen und die damit verbundenen Unsicherheiten auf Seiten der Fachpraxis ein und stellen einen konkreten Weiterentwicklungsvorschlag bereit. • … beschäftigt sich Kerstin Schumann mit der Notwendigkeit der Einbeziehung von Inter* in die Debatte um Vielfalt in der Kinder- und Jugendhilfe und diskutiert dies in Bezug auf das Kindeswohl. • … beleuchtet Angela Rein das Feld der stationären Kinder- und Jugendhilfe und fragt danach, wie queere 81 Sozial Extra 2 2021 Extrablick: Kinder- und Jugendhilfe und LGBTIQ* junge Menschen rückblickend die Zeit in der Wohngruppe erlebt haben und wie dort Heteronormativität im Alltag zum Ausdruck kommt. Gefahr der Homogenisierung – Notwendigkeit einer intersektionellen Perspektive Deutlich wird in den Beiträgen des Schwerpunkts, dass LGBTIQ-Jugendliche nicht eine Gruppe sind und es auch nicht darum geht, Menschen, die sich queer positionieren, zu homogenisieren. So wird in nahezu allen Beiträgen auf die Notwendigkeit einer intersektionellen Betrachtung hingewiesen. Mit einer solchen Perspektive, die verschiedene Differenzkategorien – wie race, gender, class, aber auch Gesundheit/Körperlichkeit – sichtbar macht und darauf verweist, dass sich Ungleichheiten durchaus in diesen Verwobenheiten verstärken oder andere auch abschwächen können. Die Perspektive der Intersektionalität ist auch bedeutsam, um rassistische Bilder zu hinterfragen, die häufig im Kontext von LGBTIQ-Jugendlichen aufgerufen werden, wenn bei Migrations-Anderen auf Probleme der Heteronormativität im familiären Kontext verwiesen wird oder im Kontext von Behinderung queere Lebensweisen de-thematisiert werden. Zwischen Verbesonderung und Ignoranz Angebote der Kinder- und Jugendhilfe haben stets mit dem grundlegenden Dilemma zu tun, wie Angebote geschaffen werden können, ohne dabei gleichzeitig Menschen zu verbesondern. Angebote wie Coming-Out-Gruppen, Mädchenarbeit oder ähnliches de-thematisieren Zwischenräume und Uneindeutigkeiten. Hierbei wird jedoch ein Spannungsfeld aufgerufen, das Christine Riegel (2017) als Verbesonderung versus Ignoranz bezeichnet. So lassen sich in der pädagogischen Arbeit insbesondere zwei Praktiken beobachten: Die Verbesonderung der jeweiligen Adressatinnen und damit verbunden Othering-Prozesse als Herstellung von „den Anderen“ und als Abweichende von der Norm und die Ignoranz, die über das Unsichtbarmachen „des Anderen“ heteronormative Deutungen stärkt und Vielfalt aberkennt. Eine einheitliche Antwort auf die Frage, wie Angebote der Kinder- und Jugendhilfe mit sexueller und geschlechtlicher Vielfalt umgehen, lässt sich freilich nicht liefern. Zu vielfältig sind die Angebote und die jeweiligen Menschen und Haltungen. Genau darin liegt möglicherweise aber eine Gefahr, weil sexuelle und geschlechtliche Vielfalt eben nicht strukturell verankert sind, sondern das Prinzip Zufall eine bedeutende Rolle spielt. So lässt sich die Angebotslandschaft eher als eine Art Kontinuum beschreiben. Dabei gibt es Angebote, die explizit die LGBTIQ-Jugendlichen adressieren und sich als Schutzräume und An82 gebote des Empowerments verstehen. Mit Riegel (2017) ist hier auch von einer Verbesonderung auszugehen, welche aber in den jeweiligen sozialpädagogischen Angeboten erforderlich ist, um die Idee eines Schutzraumes zu ermöglichen und Menschen, die in vielfältigen Angeboten häufig als „die Anderen“ gelabelt werden bzw. sich selbst als „die Anderen“ erleben müssen, weil ihre geschlechtliche Identität oder Begehrensposition nicht mitgedacht wird. Exemplarisch sei hier auf vielfältige Angebote in der Offenen Kinder- und Jugendarbeit verwiesen und das „Praxisbuch Q* – Queere Vielfalt in der Jugendarbeit“ (Landesjugendring Niedersachsen e. V. 2018). Darüber hinaus gibt es Angebote, die LGBTIQ* nicht explizit adressieren, die aber aufgrund von Mitarbeiterinnen und fachlichen Kompetenzen und Haltungen sexuelle und geschlechtliche Vielfalt stets mitdenken und -reflektieren. Interessant ist hier, dass ähnlich wie in anderen Bereichen auch, insbesondere Menschen, die sich selbst als LGBTIQ positionieren Angebote für LGBTIQ-Jugendliche machen und eine Sensibilität dafür haben. Schließlich gibt es nach wie vor Angebote, in denen sexuelle und geschlechtliche Vielfalt nicht mitgedacht werden. Um nochmals auf eine Aussage von dem bereits oben erwähnten Fachtage „LGBTIQ in der Kinder- und Jugendhilfe“, der 2018 an der Universität Hildesheim stattfand, einzugehen: Hier berichtete ein Mitarbeiter aus der stationären Kinder- und Jugendhilfe, dass in seiner Einrichtung das Prinzip der offenen Türen als sexualpädagogisches Konzept vorhanden sei. So dürfen ein Junge und ein Mädchen nur bei offener Tür gemeinsam im Zimmer sein. Zwei Jungs hingegen dürfen bei geschlossener Türe gemeinsam im Zimmer sein. Ein Studierender fasste das folgendermaßen zusammen: „Wow, endlich mal Vorteile, wenn man schwul ist“. Diese durchaus zugespitzte Aussage bringt jedoch sowohl die Kritik an einer Praktik der offenen Türen als sexualpädagogisches Konzept zum Ausdruck als auch das Unverständnis, nach wie vor junge Menschen stets als heterosexuell zu adressieren. Zwischen institutioneller Diskriminierung und individuellem Zugewandt-Sein Deutlich ist, dass die Strukturen der Kinder- und Jugendhilfe vielfältig so sind, dass LGBTIQ-Personen sich nicht immer verstanden und gesehen fühlen (Beispiele hierfür sind Toiletten für zwei Geschlechter, Formulare mit eindeutigen Geschlechtszuweisungen oder die klare Adressierung von Pflegevater und Pflegemutter in Formularen auch bei homosexuellen Pflegeeltern). Als ein Beispiel der institutionellen Diskriminierung kann die sogenannte Stiefkindadoption in Familien mit gleichgeschlechtlichen Eltern benannt werden. Wis- sen wir, dass es Kindern in queeren Familien recht gut zu gehen scheint (Rupp 2009), so wird ebenfalls sichtbar, dass die rechtliche Situation das Herstellen von Familie für queere Familien nach wie vor erschwert. Seit dem 01.01.2005 ist die Stiefkindadoption durch die Lebenspartnerin in Deutschland rechtlich möglich. Mit der Einführung der „Ehe für alle“ 2017 ist zwar das gemeinsame Adoptionsrecht verbunden, die Stiefkindadoption bleibt als Verfahren erhalten, um als gleichgeschlechtliches Paar ein gemeinsames Sorgerecht und eine gemeinsame verwandtschaftliche Beziehung zu dem – eben auch gemeinsamen – Kind herzustellen. Hier ist also ein zentraler Unterschied zu heterosexuellen Ehen zu verzeichnen, in welchen der verheiratete Partner automatisch Elternteil ist, egal ob biologisch nachweisbar oder nicht. Möglichkeiten von Mehr-Elternschaften oder auch der sofortigen Eintragung von zwei Müttern bei Geburt wie sie in Dänemark möglich sind, werden bislang in der BRD (noch) nicht weiterverfolgt. Im Lehr-Forschungs-Projekt „Queere Familien“ (2018 bis 2019) wurden queere Eltern befragt; insbesondere die Praktik der Stiefkindadoption wird von den interviewten Müttern als belastend und diskriminierend hervorgehoben. Sie stellt eine Gefahr für die Paarbeziehung dar, weil es zu ungleichen Positionen zwischen Mutter und Co-Mutter kommen, insbesondere bis die Stiefkindadoption stattgefunden habe und somit die Elternschaft rechtlich für beide Elternteile anerkannt ist. Darüber hinaus hat das Kind bis zur Stiefkindadoption rechtlich nur ein Elternteil, was ebenfalls als Gefahr und Belastung gesehen wird. Als herausfordernd thematisieren die Interviewpartnerinnen jedoch vor allem die Situation des Überprüft-Werdens und die Ungerechtigkeit, dass sie trotz Ehe und trotz der gemeinsamen Entscheidung ein Kind zu bekommen, das Verfahren der Stiefkindadoption über sich ergehen lassen müssen (Arns et al. 2019; Mangold und Schröder 2020). Wir greifen exemplarisch auf einen Interviewauszug zurück, in dem das Verhalten der Mitarbeiterin des Jugendamts von den interviewten Eltern als wertschätzend eingeordnet wird. „B: Die war auch ganz toll. Das war die hier, bei der wir hier waren zur Adoption quasi und sie uund normalerweise muss das Kind sagt man muss das Kind erst ein Jahr alt sein, damit man feststellen kann, ob irgendwie ne Bindung da ist und so [T: die Bindung steigt] B: Ja bla bla. [alle lachen] Ja aber die war halt super. Sie ist direkt zum Hausbesuch gekommen und hat dieses Gutachten total schnell geschrieben. Sie hat sich nicht mal alle Zimmer angeguckt. Wir haben nett mit ihr Kaffee getrunken. Die war wirklich toll da haben wir wirklich Glück gehabt. Und hat ein ganz tolles Gutachten geschrieben und dann ähm. Also unser Kind ist Anfang Februar geboren und am 06.12. des gleichen Jahres war dann schon gleich der Termin am Amtsgericht“ (Interview mit lesbischen Eltern B und T) In dieser Beschreibung wird deutlich, dass die Mitarbeiterin schnell handelt, um nicht zu viel Zeit bis zur Stiefkindadoption verstreichen zu lassen und dass sie nicht als kontrollierend wahrgenommen wird, dass sie nicht mal alles sehen will. Gebündelt wird dies im Bild des netten gemeinsamen Kaffeetrinkens. Hiermit wird ausgedrückt, dass die Mitarbeiterinnen Spielräume in den vorhandenen Strukturen der Stiefkindadoption haben, indem sie schnell(er) handeln, weniger kontrollieren und die Situation sozial angenehm und anerkennend gestalten. Die Frauen fühlen sich durch die Jugendamtsmitarbeiterin ernst genommen in ihrem Bedürfnis, möglichst schnell die Stiefkindadoption hinter sich zu bringen und sie fühlen sich über die geringe Kontrollwahrnehmung wertgeschätzt. Dennoch kann in dem angeführten Beispiel die strukturelle Ungerechtigkeit, die in der rechtlichen Regelung der Stiefkindadoption für queere Familien angelegt ist, nicht aufgelöst werden. Selbst ein schnelles Handeln der Jugendamtsmitarbeiterin in diesem Beispiel lässt das Verfahren der Stiefkindadoption zehn Monate dauern. So lässt sich an diesem Beispiel zeigen, dass es auf struktureller Ebene darum geht, Ungleichheiten abzubauen. Dennoch lassen sich auch im Rahmen von institutionellen Regelungen wertschätzende Formen des Umgangs finden und die Mitarbeiterinnen haben Spielräume, in denen sie sich verhalten können. Ziel des Schwerpunkts Wir möchten mit diesem Schwerpunkt dazu einladen, die Vielfältigkeiten und Machtverhältnisse von geschlechtlichen Positionierungen und Begehrenspositionen in allen Angeboten der Kinder- und Jugendhilfe zu reflektieren und die jungen Menschen ernst zu nehmen, sie in und mit ihren Bedürfnissen zu unterstützen. Damit verbunden ist für pädagogische Fachkräfte, sich mit den eigenen normativen Positionierungen auseinanderzusetzen und diese gegebenenfalls zu hinterfragen. Das bedeutet auch, die geschlechtliche und sexuelle Positionierung der jungen Menschen dort zum Thema zu machen oder ihnen dann einen Raum zu geben, wenn dies von den Jugendlichen gewünscht ist und sie nicht strukturell und stetig zu verbesondern. Die jungen Menschen in ihren Lebensentwürfen zu unterstützen, ihnen Ansprechperson und Unterstützerin zu sein in Identitätsfragen und im Coming-Out. Damit verbunden ist 83 schließlich auch das Eintreten gegen heteronormative Denkweisen und Machtstrukturen und somit eine Mitgestaltung und Einmischung weit über die individuelle s Arbeit mit dendem jeweiligen Adressatin. ∑ Angenommen. 28. Januar 2021 Funding. Open Access funding enabled and organized by Projekt DEAL. Open Access. Dieser Artikel wird unter der Creative Commons Namensnennung 4.0 International Lizenz veröffentlicht, welche die Nutzung, Vervielfältigung, Bearbeitung, Verbreitung und Wiedergabe in jeglichem Medium und Format erlaubt, sofern Sie den/die ursprünglichen Autor(en) und die Quelle ordnungsgemäß nennen, einen Link zur Creative Commons Lizenz beifügen und angeben, ob Änderungen vorgenommen wurden. Die in diesem Artikel enthaltenen Bilder und sonstiges Drittmaterial unterliegen ebenfalls der genannten Creative Commons Lizenz, sofern sich aus der Abbildungslegende nichts anderes ergibt. Sofern das betreffende Material nicht unter der genannten Creative Commons Lizenz steht und die betreffende Handlung nicht nach gesetzlichen Vorschriften erlaubt ist, ist für die oben aufgeführten Weiterverwendungen des Materials die Einwilligung des jeweiligen Rechteinhabers einzuholen. Weitere Details zur Lizenz entnehmen Sie bitte der Lizenzinformation auf http://creativecommons.org/licenses/by/4.0/deed.de Literatur Arns, M., Böttcher, N.-L., Frey, J., Lucka, M., Mangold, K., & Schröder, J. (2019). Queere Familien. Eine Broschüre für sozialpädagogische Fachkräfte und Interessierte. https://www.uni-hildesheim.de/fb1/institute/institut-fuersozial-und-organisationspaedagogik/team/mitarbeiterinnen/dr-katharinamangold/#c28588. Zugegriffen: 11. Jan. 2021. Landesjugendring Niedersachsen e. V. (2018). Juleica Praxisbuch Q. Queere Vielfalt in der Jugendarbeit. https://www.ljr.de/news/news-archiv/ detail/artikel/neuerscheinung-praxisbuch-q-queere-vielfalt-in-der-jugendarbeit.html. Zugegriffen: 11. Jan. 2021. Mangold, K., & Schröder, J. (2020). „Ganz normal und doch immer besonders“ – Kategorisierungsarbeit queerer Familien. Gender. Zeitschrift für Geschlecht, Kultur und Gesellschaft, , 124–140. Sonderheft: Elternschaft und Familie jenseits von Heteronormativität und Zweigeschlechtlichkeit.htt ps://doi.org/10.2307/j.ctv15r56vn.10. Riegel, C. (2017). Queere Familien in pädagogischen Kontexten – zwischen Ignoranz und Othering. In J. Hartmann, A. Messerschmidt & C. Thon (Hrsg.), Queertheoretische Perspektiven auf Bildung – Pädagogische Kritik der Heteronormativität. Jahrbuch Frauen- und Geschlechterforschung in der Erziehungswissenschaft, Bd. 13. Rupp, M. (2009). Die Lebenssituation von Kindern in gleichgeschlechtlichen Lebenspartnerschaften. Rechtstatsachenforschung. : Bundesanzeiger Verlag. Köln. Schmidt, F., & Schondelmayer, A.-C. (2015). Sexuelle und geschlechtliche Vielfalt – (k)ein pädagogisches Thema. In Schmidt, F., Schondelmayer, A.-C, und Schröder, U. B. (Hrsg.), Selbstbestimmung und Anerkennung sexueller und geschlechtlicher Vielfalt. Lebenswirklichkeiten, Forschungsergebnisse und Bildungsbausteine (S. 223–240). Wiesbaden: Springer VS. Tuider, E. (2016). Trans in Bildung, Pädagogik und Sozialer Arbeit. In BMFSFJ (Hrsg.), Geschlechtliche Vielfalt. Begrifflichkeiten, Definitionen und disziplinäre Zugänge zu Trans- und Intergeschlechtlichkeiten (S. 57– 62). Begleitforschung zur Interministeriellen Arbeitsgruppe Inter & Transsexualität. 84 Hier steht eine Anzeige. 123
https://openalex.org/W4388427354	https://jurnal.polkesban.ac.id/index.php/jks/article/download/1460/820	Indonesian	null	PENGARUH METODE PENGUMPULAN SALIVA SEBELUM DAN SETELAH MENYIKAT GIGI TERHADAP TITER STATUS SEKRETOR	Jurnal Kesehatan Siliwangi	2,023	cc-by	3,674	ABSTRACT Secretor status refers to individuals who secrete ABH antigens in their body fluids such as saliva. Saliva collection needs to be considered how it is collected and handled. This study aims to analyze the effect of collection methods on saliva before and after brushing teeth on secretor status titers. The type of research conducted was quasi-experimental with a pseudo research design. In this study saliva was collected from 8 healthy individuals, collection was carried out by passive drooling and spitting methods before and after brushing teeth. The study was conducted at the hematology laboratory of the Medical Laboratory Technology department of the Bandung Ministry of Health Polytechnic in May 2023. Secretor status titer was examined by hemagglutination inhibition method. The results of the study conducted Willcoxon test on the group of saliva data collected before brushing teeth with different methods obtained p value = 0.018 and the group of saliva data collected after brushing teeth with different methods p value = 0.027. Both data groups showed a sig value of p<0.05. Meanwhile, the saliva data group collected by the Passive drooling method before and after brushing teeth obtained a p value of 0.180 and the saliva group collected by the Spitting method before and after brushing teeth obtained a p value of 0.317 both data showed p>0.05. So it can be concluded that there is an effect of collecting saliva with different methods. However, there is no effect of collecting saliva taken before and after brushing teeth. Key words: Secretor Status, Passive drooling, Spitting, Tooth Brushing ABSTRAK Status sekretor mengacu pada individu yang mengeluarkan antigen ABH pada cairan tubuhnya seperti pada saliva. Pengumpulan saliva perlu diperhatikan bagaimana pengumpulan dan penanganannya. Pada penelitian ini bertujuan untuk menganalisis adanya pengaruh metode pengumpulan pada saliva sebelum dan setelah menyikat gigi terhadap titer status sekretor. Jenis penelitian yang dilakukan adalah kuarsi eksperimen dengan desain penelitian semu. Dalam penelitian ini saliva dikumpulkan dari 8 individu sehat, pengumpulan dilakukan dengan metode passive drooling dan spitting sebelum dan setelah menyikat gigi. Penelitian dilakukan di Laboratorium hematologi jurusan Teknologi Laboratorium Medis Poltekkes Kemenkes Bandung pada Mei 2023. Titer status status sekretor diperiksa dengan metode hemaglutinasi inhibisi. Hasil penelitian dilakukan uji Willcoxon pada kelompok data saliva yang dikumpulkan sebelum menyikat gigi dengan metode yang berbeda didapatkan nilai p=0,018 dan kelompok data saliva yang dikumpulkan setelah menyikat gigi dengan metode berbeda nilai p=0,027. Kedua kelompok data tersebut menunjukkan nilai sig p<0,05. Sedangkan, kelompok data saliva 251 https://doi.org/10.34011/jks.v4i1.1460 yang dikumpulkan dengan metode Passive drooling sebelum dan setelah menyikat gigi didapatkan nilai p 0.180 dan kelompok saliva yang dikumpulkan dengan metode Spitting sebelum dan setelah menyikat gigi didapatkan nilai p yaitu 0.317 kedua data menunjukkan p>0,05. Sehingga dapat disimpulkan terdapat pengaruh pengumpulan saliva dengan metode yang berbeda. Namun, tidak terdapat pengaruh pengumpulan saliva yang diambil sebelum dan setelah menyikat gigi. yang dikumpulkan dengan metode Passive drooling sebelum dan setelah menyikat gigi didapatkan nilai p 0.180 dan kelompok saliva yang dikumpulkan dengan metode Spitting sebelum dan setelah menyikat gigi didapatkan nilai p yaitu 0.317 kedua data menunjukkan p>0,05. Sehingga dapat disimpulkan terdapat pengaruh pengumpulan saliva dengan metode yang berbeda. Namun, tidak terdapat pengaruh pengumpulan saliva yang diambil sebelum dan setelah menyikat gigi. Kata kunci: Status Sekretor, Passive drooling, Spitting, Menyikat Gigi HASIL Pada penelitian ini dilakukan pemeriksaan status sekretor dengan metode pengumpulan yang berbeda yang dilakukan sebelum dan setelah menyikat gigi, kemudian dilihat titer aglutinasi yang terbentuk. Penelitian ini menggunakan 8 subjek mahasiswa Teknologi Laboratorium Medis Poltekkes Kemenkes Bandung dengan jumlah 32 unit penelitian yang terdiri dari 8 unit penelitian saliva yang dikumpulkan dengan metode Passive drooling sebelum menyikat gigi. 8 unit penelitian saliva yang dikumpulkan dengan metode Passive drooling sesudah menyikat gigi, 8 unit penelitian saliva yang dikumpulkan dengan metode Spitting sebelum menyikat gigi, dan 8 unit penelitian saliva yang dikumpulkan dengan metode Spitting setelah menyikat gigi. Kedelapan subjek ini merupakan individu yang berstatus sekretor dengan 4 orang bergolongan darah A dan 4 Orang bergolongan darah B. Proses pememeriksaan diawali dengan validasi reagen zat anti A dan zat anti B yang digunakan untuk memastikan zat anti dalam keadaan valid. Hasil validasi reagen zat anti golongan darah disajajiakn dalam tabel1. Jenis penelitian yang dilakukan adalah kuarsi eksperimen dengan desain penelitian semu. Dalam penelitian ini saliva dikumpulkan di waktu yang berbeda yaitu sebelum dan setelah menyikat gigi, setiap pengumpulan dilakukan dengan metode yang berbeda yaitu dengan Passide Drolling dan Spitting. Passive Drooling dikumpulkan dengan cara saliva dikumpulkan selama 3-5 menit tanpa adanya pergerakkan di dasar mulut (berbicara, menggerakkan lidah atau melakukan gerakan menelan) lalu secara drooling alirkan saliva melalui saluran saliva collection aid. Metode Spitting dilakukan dengan cara kumpulkan saliva didalam mulut ludahkan saliva 60 detik sekali lakukan sampai volume saliva tercukupi. Titer status status sekretor diperiksa dengan metode hemaglutinasi inhibisi. g Populasi pada penelitian ini adalah 8 individu sehat Sampel pada penelictian ini adalah individu sehat dengan golongan darah A dan B, diambil di pagi hari sebelum melakukan aktivitas makan dan minum. dengan kriteria inklusi sampel yaitu mahasiswa/i Teknologi Laboratorium Medis Poltekkes Kemenkes Bandung berjenis kelamin laki-laki atau perempuan bergolongan darah A atau B berstatus sekretor. Sedangkan untuk kriteria eksklusi penelitian ini yaitu bukan mahasiswa/I Teknologi Laboratorium Medis Poltekkes Kemenkes Bandung, bergolongan darah A atau B berstatus non sekretor, golongan darah O atau AB berstatus sekretor/ non sekretor. Tabel 1 Validasi Zat Anti Zat Anti Golongan Darah No.Lot Reagen Anti Hasil Kesimpulan Anti A AA-2210-1/4 +4 Valid Anti B BB-2201-1/1 +4 Valid Tabel 1 menunjukkan hasil validasi zat anti A dan anti B menghasilkan aglutinasi +4 yang dipastikan valid dan dapat digunakan untuk menentukan titer zat anti yang selanjutnya dilakukan dengan pemeriksaan titer hemaglutinasi inhibisi dan ditentukan status sekretornya. Bandung. Pengolahan data dilakukan uji normalitas, distribusi data tidak normal maka dilakukan uji statistic Wilcoxon. PENDAHULUAN Metode Pengumpulan lain seperti spitting dapat digunakan ketika laju aliran sangat rendah dan penguapan saliva dapat diminimalkan 7. Sayangnya, metode spitting memiliki sedikit efek stimulant dan mengandung lebih banyak bakteri yang dapat mempengaruhi analisis saliva namun metode spitting memberikan informasi yang serupa dengan whole saliva unstimulant 8 . Pemeriksaan status sekretor ini biasanya dilakukan untuk pemeriksaan serologis forensik yang berfungsi untuk menentukan golongan darah seseorang dengan menggunakan cairan tubuh.1 Selain itu, status sekretor juga dapat digunakan untuk membantu pemeriksaan pada diskrepansi ABO seperti menemukan subkelompok A dan B yang langka. Pemeriksaan status sekretor ini murah dan dapat dilakukan dipusat laboratorium manapun 2,3. Pada SalivaBio 3rd Edition tahun 2015 pengumpulan saliva disarankan untuk tidak makan 60 menit sebelum dilakukan pengumpulan saliva, hal tersebut disebakan karena mengunyah makanan mempengaruhi laju aliran saliva yang akan menyebabkan produksi volume saliva lebih banyak 6. Hal ini dibuktikan pada penelitian Nilla Marlia tahun 2018 dimana didapatkan perbedaan yang bermakna antara titer status sekretor pada pengambilan spesimen saliva pagi dan 60 menit setelah makan 9. Saliva merupakan salah satu cairan tubuh yang dapat digunakan dalam pemeriksaan status sekretor. Saliva mengandung musin glikoprotein yang memiliki peran penting sebagai pembawa antigen golongan darah dalam saliva4. pemilihan saliva sebagai spesimen untuk pemeriksaan status sekretor dikarenakan saliva merupakan cairan tubuh yang paling mudah didapatkan. p Pengumpulan whole saliva unstimulant lebih disukai karena dapat meminimalkan pengenceran analit. Metode yang menjadi gold standar dalam pengambilan sampel saliva adalah passive drooling 5. Kelebihan dari metode passive drooling yaitu volume sampel yang didapatkan cukup banyak dan pengaruh dari kontaminasi kecil. Namun, metode passive drooling tidak cocok untuk individu yang mengalami kesulitan dengan metode tersebut seperti pada anak kecil, sleepers, dan lansia yang lemah. 6 . Pendahuluan berisi latar belakang, konteks penelitian, hasil kajian pustaka, dan tujuan penelitian. 3 Selain mengunyah makanan faktor lain yang akan mempengaruhi laju aliran saliva adalah menyikat gigi. Menurut Ligtenberg tahun 2006 setelah menyikat gigi tingkat sekresi saliva meningkat secara signifikan selama 60 menit 10 sedangkang menurut Affo dan Hoek menyikat gigi merangsang produksi saliva di lima menit pertama 11,12, selain itu menurut penelitian Hoek dkk mengatakan bahwa menyikat gigi selain mempengaruhi laju aliran saliva juga memiliki signifikan efek pada komposisi protein air liur, yang mungkin bertahan setelah lebih dari 45 menit 12. Tujuan dari penelitian ini adalah untuk mengetahu pengaruh metode https://doi.org/10.34011/jks.v4i1.1460 252 HASIL Tabel 2 Hasil Titer Status Sekretor Subjek Titer Status Sekretor Passive drooling Spitting Sebelum Menyikat Gigi Setelah Menyikat Gigi Sebelum Menyikat Gigi Setelah Menyikat Gigi 1 1/512 1/512 1/512 1/512 2 1/32 1/16 1/16 1/8 3 1/16 1/16 1/4 1/4 4 1/512 1/512 1/256 1/256 5 1/64 1/32 1/32 1/32 6 1/16 1/16 1/8 1/8 7 1/256 1/256 1/128 1/128 8 1/128 1/128 1/64 1/64 Tabel 2 Hasil Titer Status Sekretor Tabel 2 Hasil Titer Status Sekretor S S Tabel 2 Hasil Titer Status Sekretor Subjek Titer Status Sekretor Passive drooling Spitting Sebelum Menyikat Gigi Setelah Menyikat Gigi Sebelum Menyikat Gigi Setelah Menyikat Gigi 1 1/512 1/512 1/512 1/512 2 1/32 1/16 1/16 1/8 3 1/16 1/16 1/4 1/4 4 1/512 1/512 1/256 1/256 5 1/64 1/32 1/32 1/32 6 1/16 1/16 1/8 1/8 7 1/256 1/256 1/128 1/128 8 1/128 1/128 1/64 1/64 Gambar 1 Grafik Hasil Pemeriksaan Pengumpulan Saliva Metode Passive drooling Sebelum Menyikat Gigi dan Setelah Menyikat Gigi 0 100 200 300 400 500 600 Sebelum Menyikat Gigi Setelah Menyikat Gigi Passive Drolling 0 100 200 300 400 500 600 Sebelum Menyikat Gigi Setelah Menyikat Gigi Spitting G b 1 G fik H il P ik P l S li M t d P i d li 0 100 200 300 400 500 600 Sebelum Menyikat Gigi Setelah Menyikat Gigi Passive Drolling https://doi.org/10.34011/jks.v4i1.1460 254 Gambar 1 Grafik Hasil Pemeriksaan Pengumpulan Saliva Metode Passive drooling Sebelum Menyikat Gigi dan Setelah Menyikat Gigi y g y g 0 100 200 300 400 500 600 Sebelum Menyikat Gigi Setelah Menyikat Gigi Spitting Gambar 1 Grafik Hasil Pemeriksaan Pengumpulan Saliva Metode Passive drooling Sebelum Menyikat Gigi dan Setelah Menyikat Gigi 0 100 200 300 400 500 600 Sebelum Menyikat Gigi Setelah Menyikat Gigi Spitting https://doi.org/10.34011/jks.v4i1.1460 254 Gambar 2 Grafik Hasil Pemeriksaan Pengumpulan Saliva Metode Spitting Sebelum Menyikat Gigi dan Setelah Menyikat Gigi Gambar 2 Grafik Hasil Pemeriksaan Pengumpulan Saliva Metode Spitting Sebelum Menyikat Gigi dan Setelah Menyikat Gigi Dari tabel 2 didapatkan rentang titer status sekretor mulai dari 1/4 – 1/512. Dimana nilai titer tertinggi ada pada setiap metode pengumpulan dengan perlakukan sebelum dan setelah menyikat gigi. Namun, nilai titer terkecil berada pada kelompok data pengumpulan saliva dengan metode spitting baik sebelum menyikat gigi maupun setelah menyikat gigi. HASIL Berikut merupakan hasil penelitian Tabel 1 Validasi Zat Anti Tabel 1 menunjukkan hasil validasi zat anti A dan anti B menghasilkan aglutinasi +4 yang dipastikan valid dan dapat digunakan untuk menentukan titer zat anti yang selanjutnya dilakukan dengan pemeriksaan titer hemaglutinasi inhibisi dan ditentukan status sekretornya. Berikut merupakan hasil penelitian Penelitian ini sudah memperoleh pernyataan layak etik dari KEPK Bandung no.43/KEPK/EC/IV/2023. Penelitian ini dilaksanakan bulan mei hingga juni di Laboratorium Hematologi Jurusan TLM Politeknik Kesehatan https://doi.org/10.34011/jks.v4i1.1460 253 pengaruh metode pegumpulan saliva sebelum dan setelah menyikat gigi terhadap titer status sekretor. pengaruh metode pegumpulan saliva sebelum dan setelah menyikat gigi terhadap titer status sekretor. HASIL Dengan demikian terdapat perbedaan bermakna antara saliva yang dikumpulkan dengan metode yang berbeda baik sebelum menyikat gigi ataupun setelah menyikat gigi terhadap titer status sekretor. Dari enam belas data titer status sekretor yang dikumpulkan sebelum dan setelah menyikat gigi hanya terdapat tiga data (30%) yang mengalami penurunan titer status sekretor yang dikumpulkan sebelum dan setelah menyikat gigi. Dua dari tiga data tersebut berasal dari kelompok data titer status sekretor yang pengambilan saliva dikumpulkan dengan metode passive drooling dan satu data berasal dari kelompok data titer status sekretor yang pengambilan saliva dikumpulkan dengan metode spitting. Pada kelompok data sebelum menyikat gigi dan setelah menyikat gigi didapatkan data dimana terdapat perbedaan titer status sekretor yang dipengaruhi oleh metode pengumpulan saliva. Pada pengumpulan saliva dengan metode Passive drooling titer status sekretor yang dihasilkan lebih tinggi dibandingkan dengan pengumpulan saliva metode spitting. Dari kelompok data sebelum menyikat gigi 87.5% atau tujuh dari data data titer status sekretor dengan pengumpulan saliva secara passive drooling menunjukkan titer status sekretor lebih tinggi dibandingan titer status sekretor saliva yang dikumpulkan dengan metode spitting. Sedangkan, pada kelompok data setelah menyikat gigi 75% atau enam dari delapan data menunjukkan titer status sekretor dengan pengumpulan saliva secara passive droling lebih tinggi dibandingkan dengan spitting. Pada kelompok data saliva yang dikumpulkan dengan metode Passive drooling sebelum dan setelah menyikat gigi didapatkan nilai p yaitu 0.180 yang menunjukkan p > 0,05 maka H0 diterima dan H1 ditolak atau tidak terdapat perbedaan titer status sekretor pada saliva yang dikumpulkan dengan metode Passive drooling sebelum dan setelah menyikat gigi. Pada kelompok saliva yang dikumpulkan dengan metode Spitting sebelum dan setelah menyikat gigi didapatkan nilai p yaitu 0.317 yang menunjukkan p > 0,05 maka H0 diterima dan H1 ditolak atau tidak terdapat perbedaan titer status sekretor pada saliva yang dikumpulkan dengan metode Spitting. sebelum dan setelah menyikat gigi. Dengan demikian tidak terdapat perbedaan bermakna antara saliva yang dikumpulkan sebelum menyikat gigi dan setelah menyikat gigi terhadap titer status sekretor. Individu golongan sekretor memiliki substansi H dalam cairan tubuhnya bersama dengan substan A dan B yang sesuai dengan golongan darahnya. Gen sekretor ini diwariskan dalam bentuk autosomal yang dominan. Dimana gen Se adalah dominan dan gen se agdalah resesif. Individu dengan status sekretor memiliki fenotif SeSe atau Sese sedangkan Individu dengan status non sekretor memiliki genotif sese 13. Gen Se mengkode enzim fucosyltransferase tertentu yang terletak HASIL Tabel 4 Uji Non Parametrc Wilcoxon Dasar pengambilan keputusan Jika p > 0,05, maka H0 diterima Jika p < 0,05, maka H0 ditolak Kelompok Data Hasil Kesimpulan Passive drooling vs Spitting Sebelum Menyikat Gigi p<0,05 Ada Perbedaan Setelah Menyikat Gigi p<0,05 Ada Perbedaan Sebelum menyikat gigi vs Setelah Menyikat gigi Passive drooling p>0,05 Tidak Ada Perbedaan Spitting p>0,05 Tidak Ada Perbedaan Tabel 4 Uji Non Parametrc Wilcoxon Data hasil penelitian pada tabel 2 dilakukan uji normalitas data menggunakan uji Shapiro-wilk karena jumlah data <30. Uji Normalitas ini untuk mengetahui apakah data penelitian terdistribusi normal atau tidak normal. Hasil uji Normalitas dapat dilihat pada tabel 3 dibawah ini. Tabel 3 Uji Normalitas Metode Penumpulan Hasil Kesimpulan Passive droolin g Sebelum Menyikat Gigi p<0,05 Distrbusi Tidak Normal Setelah Menyikat Gigi p<0,05 Distrbusi Tidak Normal Spitting Sebelum Menyikat Gigi p<0,05 Distrbusi Tidak Normal Setelah Menyikat Gigi p<0,05 Distrbusi Tidak Normal Tabel 3 Uji Normalitas Dasar pengambilan keputusan g g Dari tabel 4 dapat dilihat pengaruh metode pengumpulan saliva sebelum dan setelah menyikat gigi terhadap titer status sekretor. Hasil uji Non Parametric Wilcoxon pada kelompok data saliva yang dikumpulkan sebelum menyikat gigi dengan metode yang berbeda didapatkan nilai p yaitu 0,018 yang menunjukkan p < 0,05 maka H0 ditolak dan H1 diterima atau terdapat perbedaan titer status sekretor pada saliva yang dikumpulkan sebelum menyikat gigi dengan metode pengumpulan saliva berbeda yaitu passive drooling dan spitting. Dari tabel 3 hasi uji normalitas diatas diperoleh nilai Sig < 0,05 untuk semua kelompok data, sehingga dapat disimpulkan bahwa distribusi data pemeriksaan titer status sekretor dengan pengumpulan saliva metode berbeda sebelum dan setelah menyikat gigi tidak terdistribusi normal sehingga pengolahan data dilanjutkan dengan uji Willcoxon. Pada kelompok data saliva yang dikumpulkan setelah menyikat gigi https://doi.org/10.34011/jks.v4i1.1460 255 dengan metode yang berbeda didapatkan nilai p yaitu 0,027 yang menunjukkan p < 0,05 maka H0 ditolak dan H1 diterima atau terdapat perbedaan titer status sekretor pada saliva yang dikumpulkan setelah menyikat gigi dengan metode pengumpulan saliva berbeda yaitu passive drooling dan spitting. Dengan demikian terdapat perbedaan bermakna antara saliva yang dikumpulkan dengan metode yang berbeda baik sebelum menyikat gigi ataupun setelah menyikat gigi terhadap titer status sekretor. dengan metode yang berbeda didapatkan nilai p yaitu 0,027 yang menunjukkan p < 0,05 maka H0 ditolak dan H1 diterima atau terdapat perbedaan titer status sekretor pada saliva yang dikumpulkan setelah menyikat gigi dengan metode pengumpulan saliva berbeda yaitu passive drooling dan spitting. PEMBAHASAN Pada hasil penelitian yang disajikan pada tabel 2 diketahui terdapat perbedaan bermakna pada pemeriksaan titer status sekretor dengan saliva yang dikumpulkan dengan metode yang berbeda. Namun, tidak terdapat perbedaan bermakna titer status sekretor pada saliva yang diambil sebelum dan setelah menyikat gigi. https://doi.org/10.34011/jks.v4i1.1460 256 di dalam epitel jaringan sekretorik seperti kelenjar ludah, keringat, air mata dan sekresi lendir gastrointestinal sehingga membuat kelenjar-kelenjar tersebut menghasilkan antigen golongan darah yang sama dengan eritrosit. saliva yang sama dengan passive drooling namun pada metode spitting memiliki efek stimulasi sehingga tidak cocok digunakan untuk sampel yang membutuhkan sampel unstimulant saliva 7 . Komponen utama saliva dengan metode unstimulant saliva berasal dari kelenjar submandibular. Kelenjar ini bertanggung jawab untuk memproduksi musin dengan konsentrasi tinggi di dalam saliva. Sebaliknya, untuk saliva yang memiliki proses stimulant mekanik, saliva sebagian besar berasal dari kelenjar parotis dan sebagian besar terdiri dari air 16. Konsentrasi musin inilah yang mempengaruhi titer status sekretor di dalam saliva. Menyikat gigi merupakan kegiatan oral hygine yang juga merupakan proses penghilangan bakteri secara mekanis 14. Berdasarkan hasil penelitian mengenai pengaruh menyikat gigi terhadap titer status sekretor tidak ada perbedaan. Hal tersebut dapat disebabkan pada pengolahan saliva baik pada saliva sebelum dan setelah menyikat gigi dilakukan pemanasan terlebih dahulu yang berfungsi untuk menghilangkan bakteri pengganggu pada pemeriksaan titer status sekretor. Sehingga tidak terjadi perbedaan bermakna pada hasil titer sekretor baik sebelum menyikat gigi maupun setelah menyikat gigi. Meskipin begitu, kegiatan menyikat gigi mempengaruhi laju aliran saliva. 14 . Kelenjar submandibular merupakan kelenjar yang memproduksi air liur terbanyak yaitu sekitar 70%. Kelenjar submandibular menghasilakn 80% serous (cairan ludah yang encer) dan 20% mukosa (cairan ludah yang pada). Serous atau cairan ludah yang encer dihasilkan oleh acinous serous. Hasil sekresi dari acinous serous berisi enzim ptyalin yang bersifat jernih dan encer. Sedangkan untuk mukosa dihasilkan oleh acinous mucous dimana hasil sekresi dari sel ini adalah musin yang sangat kental 17. Perbedaan titer status sekretor yang disebabkan oleh metode pengumpulan saliva disebabkan perbedaan kandungan saliva yang dihasilkan pada kedua metode pengupulan saliva. Pada metode passive droling memberikan hasil titer status sekretor yang lebih tinggi dibandingkan metode spitting, hal tersebut disebabkan saliva yang dihasilkan dengan pengumpulan passive drooling memberikan sampel yang murni tanpa stimulasi, volume sampel yang besar dan mudah dilakukan, selain itu pengumpulan saliva dengan metode passive drooling juga mengandung musin 15. PEMBAHASAN Dimana glikoprotein musin merupakan komponen utama protein yang membawa spesifitas golongan darah berupa oligosakarida yang kandungannya terdiri dari substansi golongan darah pada saliva manusia. Sedangkan untuk metode spitting meskipun metode ini menghasilkan Berdasarkan penelitian terdahulu 16 dengan judul “Evaluation of saliva collection device for analysis of protein” dimana pada penelitian tersebut dijelaskan bahwa metode pengumpulan saliva menunjukkan perbedaan signifikan yang relevan dalam tingkat analit yang berbeda. Berdasarkan penelitian tersebut metode pengumpulan saliva memperngaruhi konsentrasi dari CRP, myoglobin dan IgE sehingga penting untuk untuk mengevaluasi dengan benar metode pengumpulan agar mendapatkan hasil interpretasi hasil yang bermakna. SIMPULAN Hasil statistil uji Willcoxon pada kelompok data saliva yang dikumpulkan https://doi.org/10.34011/jks.v4i1.1460 257 Trends Anal Chem. 2020;124. doi:10.1016/j.trac.2019.115781 sebelum menyikat gigi dengan metode yang berbeda didapatkan p 0,018 dan kelompok data saliva yang dikumpulkan setelah menyikat gigi dengan metode berbeda nilai p 0,027. Kedua kelompok data tersebut menunjukkan nilai sig p<0,05. Sedangkan, kelompok data saliva yang dikumpulkan dengan metode Passive drooling sebelum dan setelah menyikat gigi didapatkan nilai p 0.180 dan kelompok saliva yang dikumpulkan dengan metode Spitting sebelum dan setelah menyikat gigi didapatkan nilai p yaitu 0.317 kedua data menunjukkan p>0,05. Sehingga dapat disimpulkan terdapat pengaruh pengumpulan saliva dengan metode Passiver drolling dan Spitting. Namun, tidak terdapat pengaruh pengumpulan saliva yang diambil sebelum dan setelah menyikat gigi. j 6. Salimetrics, L.L.C., SalivaBio LLC. Saliva Collection and Handling Advice. Methods. 2015;44(0):1-15. www.salimetrics.com 7. Priya Y, Prathibha K M. Methods of collection of saliva-A Review. Int J Oral Heal Dent. 2017;3(3):149-153. doi:10.18231/2395-499X.2017.0032 8. Bhattarai KR, Kim HR, Chae HJ. Compliance with saliva collection protocol in healthy volunteers: Strategies for managing risk and errors. Int J Med Sci. 2018;15(8):823-831. doi:10.7150/ijms.25146 9. Malianti N. No Title. Perbandingan Titer Status Sekretor pada Pengambilan Spesimen Saliva Pagi dan 60 Menit Setelah Makan. Published online 2018:http://repository.poltekkesbdg. info/files/original. https://doi.org/10.34011/jks.v4i1.1460 DAFTAR RUJUKAN 1. Metgud R, Khajuria N, Mamta, Ramesh G. Evaluation of the secretor status of abo blood group antigens in saliva among southern rajasthan population using absorption inhibition method. J Clin Diagnostic Res. 2016;10(2):ZC01-ZC03. doi:10.7860/JCDR/2016/11598.716 1 10. 10. Ligtenberg AJ, Brand HS, Bots CP, Nieuw Amerongen A V. The effect of toothbrushing on secretion rate, pH and buffering capacity of saliva. Int J Dent Hyg. 2006;4(2):104-105. doi:10.1111/j.1601- 5037.2006.00170.x 11. Affoo RH, Trottier K, Garrick R, Mascarenhas T, Jang Y, Martin RE. The Effects of Tooth Brushing on Whole Salivary Flow Rate in Older Adults. Biomed Res Int. 2018;2018. doi:10.1155/2018/3904139 2. 2. Bharath RR, Arumugam P. The prevalence of secretor status and co- expression of lewis antigen in voluntary blood donors. Asian J Med Sci. 2016;7(5):93-96. doi:10.3126/ajms.v7i5.14848 12. 12. Hoek GH, Brand HS, Veerman ECI, Amerongen AVN. Toothbrushing affects the protein composition of whole saliva. Eur J Oral Sci. 2002;110(6):480-481. doi:10.1034/j.1600- 0722.2002.21370.x 3. Banerjee S, Alwar V, Dr., Sitalakshmi S. The importance of testing for secretor status of ABH antigens: A case series. Iraqi J Hematol. 2021;10(2):176. doi:10.4103/ijh.ijh_25_21 4. Aliviameita A, MAR MS, Yudianto A. Forensic Identification of Blood Types in Pear (Pyrus bretschneideri) Fruit Bitemark. Folia Medica Indones. 2018;54(4):269. doi:10.20473/fmi.v54i4.10710 13. Daniels G. ABO, H, and Lewis Systems. Hum Blood Groups. Published online 2013:11-95. doi:10.1002/9781118493595.ch2 14. 14. Justino AB, Teixeira RR, Peixoto LG, Jaramillo OLB, Espindola FS. Effect of saliva collection methods and oral hygiene on salivary biomarkers. Scand J Clin Lab Invest. 5. Bellagambi FG, Lomonaco T, Salvo P, et al. Saliva sampling: Methods and devices. An overview. TrAC - https://doi.org/10.34011/jks.v4i1.1460 258 Evaluation of saliva collection devices for the analysis of proteins. Clin Chim Acta. 2012;413(13- 14):1066-1070. doi:10.1016/j.cca.2012.02.020 17. Rahayu YCR, Kurniawati A. CAIRAN RONNGGA MULUT.; 2018. http://repository.unej.ac.id/bitstream /handle/123456789/65672/Ainul Latifah- 101810401034.pdf?sequence=1 Evaluation of saliva collection devices for the analysis of proteins. 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https://openalex.org/W3024908664	https://zenodo.org/record/5724816/files/source.pdf	French	null	Endemic Peperomia (Piperaceae) novelties from eastern Madagascar	Candollea	2,020	cc-by	3,879	Résumé MATHIEU, G. (2020). Nouvelles espèces endémiques de Peperomia (Piperaceae) de l’est de Madagascar. Candollea 75: 75 – anglais et français. DOI: http://dx.doi.org/10.15553/c2020v751a7 Trois endémiques nouvelles pour la science du genre Peperomia Ruiz & Pav. (Piperaceae), provenant des forêts tropicales humides de l’est de Madagascar, sont décrites, illustrées et comparées avec leurs espèces parentes. Les statuts de conser- vation respectifs sont déterminés. La présence de Peperomia irrasa G. Mathieu est apparemment restreinte au massif d’Anjanaharibe et celle de Peperomia robusta G. Mathieu est indiquée pour le Parc National de Zahamena dans l’aire protégée de Loky-Manambato (Daraina). Peperomia variilimba G. Mathieu est une espèce communément répandue du massif de Tsaratanana jusqu’à Analamazaotra. En incluant les présentes nouveautés, le nombre des espèces acceptées de Peperomia à Madagascar est de 42. Abstract MATHIEU, G. (2020). Endemic Peperomia (Piperaceae) novelties from eastern Madagascar. Candollea 75: 75 – 82. In English, English and French- abstracts. DOI: http://dx.doi.org/10.15553/c2020v751a7 Three endemic Peperomia Ruiz & Pav. species (Piperaceae) from the eastern Malagasy rainforests are presented as new to science. They are described, illustrated, compared with presumed closest relatives and their conservation status assess- ments are provided. Peperomia irrasa G. Mathieu seems to be restricted to the Anjanaharibe massif, while Peperomia robusta G. Mathieu is reported from the National Park of Zahamena to the Loky-Manambato Protected Area (Daraina). Peperomia variilimba G. Mathieu is an apparently common species occurring from the Tsaratanana massif to Anal- amazaotra. These novelties bring the number of accepted Peperomia species on Madagascar to 42. Endemic Peperomia (Piperaceae) novelties from eastern Madagascar Guido Mathieu Address of the author: www.peperomia.net. E-mail: guido.mathieu@taxa.be Submitted on December 12, 2019. Accepted on April 2, 2020. ISSN : 0373-2967 – Online ISSN : 2235-3658 – Candollea 75(1) : 75 – 82 (2020) © CONSERVATOIRE ET JARDIN BOTANIQUES DE Address of the author: www.peperomia.net. E-mail: guido.mathieu@taxa.be Submitted on December 12, 2019. Accepted on April 2, 2020. First published online ISSN : 0373-2967 – Online ISSN : 2235-3658 – Candollea 75(1) : 75 – 82 (2020) Introduction lamina succulent, obovate, (0.5 – )1 – 1.7 × (0.3 – )0.5( – 1) cm, length/width ratio (1.1 – )1.4( – 1.9), apex rounded, distinctly emarginate, base acute to cuneate, 3-palmatinerved, only main nerve distinct, adaxially nerves slightly impressed, abaxially main nerve slightly protruding basally, adaxially discretely pubescent at the main nerve (most dense in the basal part) and in a periapical zone, abaxially glabrous, sometimes except for a small part along the base of the main nerve, margin apically ciliate, bright green adaxially, whitish green abaxially. Inflorescence 1 – 3 terminal spadices, peduncle ca 1 cm long, rachis 3 – 4 cm long in anthesis. Mature fruit not seen. Peperomia Ruiz & Pav. (Piperaceae) is a giant, pantropical genus of terrestrial and epiphytic herbs. As of April 2020 it includes 1625 accepted taxa, 1455 species plus 172 forms and varieties (Mathieu, 2001 – 2020). The genus shows its highest diversity in the neotropics. In the paleotropics, its diversity density is striking on the Malagasy Island. With the three novelties here presented, there are currently 42 species rec- ognized (Madagascar Catalogue, 2020), about twice the number known from the entire African continent. However, the Malagasy Peperomia richness remained underestimated for a long time. Field work conducted in 2001 and subsequent scrutinization of herbarium collections already revealed ten new species (Mathieu, 2003a, 2003b, 2006). Three more are presented here as new to science. They are described, illus- trated, compared with morphologically close taxa and assigned to subgenus. Etymology. – The specific epithet irrasa (“unshaven”) refers to the minute pubescent indument of stem and petioles, resembling a stubble beard (Fig. 1B), a key character of the species. Distribution and habitat. – The new species is known from only three collections (Cours 3861 being probably a duplicate of one of Humbert’s), made during the same expedition in December 1951- January 1952 in the Anjanaharibe massif in the region of SAVA and has apparently not been reported since. It occurs at an altitude of 1600 – 1800 m, in the upper part of the medium altitude moist evergreen forest (sensu Gautier et al., 2018) corresponding to the “Sylve à Lichen” of Humbert (1955). It is believed to be endemic from the Anjanaharibe massif. The collections show only young inflo- rescences without mature fruits. Introduction Two of the three newly described species were observed in situ and their type collections were made during the three month Malagasy Peperomia expedition in 2001. The third species is an herbarium finding, discovered during subsequent scrutinization of about 1700 herbarium specimens of Malagasy Peperomias (at B, BM, BR, E, G, GENT, GH, HUA, JE, K, L, LY, M, MO, P, PR, S, TAN, US, W, WAG, Z). The new species have been compared macroscopically and by light microscope with similar taxa. Digital images of the types of species mentioned in this article can be accessed online by the TRGP (Taxonomic Rep- ertory of the genus Peperomia) (Mathieu, 2001 – 2020) and distribution maps by the Madagascar Catalogue (2020). Calculations of extent of occurrence (EOO) and area of occu- pancy (AOO) have been conducted with Geocat (Bachmann & Moat, 2012). Conservation status. – Peperomia irrasa is known from three (most likely two) old specimens collected most likely in a single location from Anjanaharibe Sud National Park. Despite the very restricted distribution and the absence of recent col- lection, there appear to be no current threats, and thus P. irrasa is assigned a status of “Least Concern” [LC] using the IUCN Red List Categories and Criteria (IUCN, 2012). Paratypi. – Madagascar. Reg. SAVA [Prov. Antsiranana]: Anjanaha- ribe, [14°43'S 49°30'E], 1700 m, 25.XII.1950, Cours et al. 3861 (P, TAN); ibid. loco, 1600 – 1800 m, 10.XII.1950 – 3.I.1951, Humbert et al. 24635 (BR, P). Keywords PIPERACEAE – Peperomia – Madagascar – New species – Taxonomy PIPERACEAE – Peperomia – Madagascar – New species – Taxonomy PIPERACEAE – Peperomia – Madagascar – New species – Taxonomy 76 – Peperomia (Piperaceae) novelties from Madagascar Candollea 75, 2020 Taxonomy Peperomia irrasa G. Mathieu, sp. nov. (Fig. 1). Notes. – Peperomia irrasa can easily be distinguished from other Malagasy species with small opposite or verticillate leaves by the distinct emarginate leaf apex. The short erect trichomes of the stem provide confirmation. Peperomia nicolliae G. Mathieu exhibits longer trichomes, curved in apical direc- tion, whereas P. silvicola C. DC. exhibits a villous indument. One other Malagasy Peperomia shows leaves with a distinct emarginate leaf apex: P. thomeana C. DC., but that species shows alternate leaves. Peperomia irrasa is considered as belonging to Peperomia subg. Micropiper (Miq.) Miq., as it is the case with P. nicolliae and P. silvicola (Frenzke et al., 2015). Holotypus: Madagascar. Reg. SAVA [Prov. Antsiranana]: massif de l’Anjanaharibe (pentes et sommet N), W d’Andapa, [14°43'S 49°30'E], 1700 m, 10.XII.1950 – 3.I.1951, Humbert, Capuron & Cours 24638 (P [P00106743]!; iso-: BR [BR000000651342]!). Peperomia irrasa G. Mathieu differs from P. nicolliae G. Mathieu and P. silvicola C. DC. by the pubescent stem and petiole and by the emarginate leaf apex. Perennial terrestrial herb, 10 – 15( – 20) cm tall, spreading by stolons. Stem slender, simple or 1 – 2 branched, erect or basally decumbent and there rooting at the nodes, internodes 1 – 3 cm long, pubescence moderately dense, trichomes very short, two internodal ribs faintly visible, reddish. Leaves 2 – 4( – 5)-verticil- late; petiole 2 – 4 mm long, sulcate, sulcus pubescent, reddish; Peperomia (Piperaceae) novelties from Madagascar – 77 Candollea 75, 2020 A B C 1 mm 1 mm 1 cm C 1 mm C B Fig. 1. – Peperomia irrasa G. Mathieu. A. General habit; B. Detail of node; C. Detail of leaf apex. [Humbert 24638, BR, P] [Drawing: G. Mathieu] 78 – Peperomia (Piperaceae) novelties from Madagascar Candollea 75, 2020 of “Least Concern” [LC] using the IUCN Red List Categories and Criteria (IUCN, 2012). Peperomia robusta G. Mathieu, sp. nov. (Fig. 2, 3). Holotypus: Madagascar. Reg. Alaotra-Mangoro [Prov. Toamasina]: Zahamena NP, NW border, 7 km SE of Antanandavao, along trail to camp 1 (1300 m N of camp- site), 17 °29'41"S 48°43'59"E, 1300 m, 26.IX.2001, Mathieu 446 (BR [BR000000916063]!; iso-: B [B100001403]!, G [G00341878]!, MO-5662979!, TAN!). Notes. – Peperomia robusta resembles P. portulacoides, which is essentially glabrous. However, varieties of the latter species with indument have been published: P. portulacoides var. pilosa Baker from the Seychelles and P. portulacoides var. Taxonomy hirtella Wawra from Sri Lanka. Var. pilosa shows a short, erect indu- ment. Its synonimization with P. tomentosa (Vahl) A. Dietr. (Friedmann, 2011: 78) is questionable. Peperomia tomentosa not only exhibits a villous indument but is also distinct in its (sub)sessile leaves. Var. hirtella shows much smaller leaves (1 – 1.5 cm long). It is only known from its type collection and probably belongs to P. heyneana Miq. Peperomia robusta has to be distinguished from P. pedunculata, known from La Réunion, which does show an indument, characterized however by short erect trichomes. Moreover, the leaves of that species are smaller, 2 – 4( – 5) cm long, elliptic, and usually show an obtuse apex. At last, there is some resemblance in general habit with P. mantadiana G. Mathieu. That is a glabrous species showing elliptic-lanceolate leaves. Peperomia robusta is considered as belonging to Peperomia subg. Micropiper, as it is the case with P. portulacoides and P. pedunculata (Frenzke et al., 2015). Peperomia robusta G. Mathieu differs by the pilose indument from P. portulacoides (Lam.) A. Dietr., an essentially glabrous species, and from P. pedunculata C. DC., of which the indument is characterized by short erect trichomes. Perennial terrestrial herb, occasionally epiphytic, up to 40( – 50) cm tall. Stem simple or 1 – 3 branched, erect or basally decumbent, terete, up to 8 mm diam. at the base, internodes 4 – 8 cm, pilose, dark red. Leaves opposite or 3 – 4-verticilate; petiole 8 – 10 mm long, pilose, red; lamina coriaceous to suc- culent, broad obovate, sometimes elliptic, 3 – 7 × 2 – 4 cm, small- est at the base of the stem, length/width ratio ca 1.6, apex rounded, base obtuse to acute or cuneate, 3-nerved, often only central nerve distinct, slightly impressed adaxially, both sides loosely pilose (usually more dense abaxially or at the nerves) or glabrescent, rather dark dull green adaxially, pale green abaxially, margin entire, ciliate, more dense in the apical half. Inflorescence 1 terminal spadix or 2 – 3( – 4) together; peduncle 1 – 2 cm long, basally pilose; rachis 5 – 10 cm long, glabrous, loosely to moderately densely flowered, floral bracts orbicular or slightly elliptic. Fruit globose, ca. 0.5 mm diam., subbasally attached, entirely covered with sticky papillae, individual papilla longer than wide, style wide conical, horseshoe shaped ridge around subapical stigma, highest adaxially, open abaxially, conical pseudopedicellate when mature. Paratypi. Taxonomy – Madagascar. Reg. Alaotra-Mangoro [Prov. Toamasina]: Mount Ankaroka, [17 °48'S 48°32'E], 1200 – 1400 m, X.1937, Humbert 17511 (P); Zahamena AP, massif de l’Andrangovalo, [17 °40'S 48°45'E], 1200 – 1300 m, X.1937, Humbert 17755 (P); ibid. loco, entre les rivière Sahat- avy et Sivora, 17 °36'34"S 48°47 '10"E, 709 m, 2.X.2001, Ratovoson 547 (MO). Reg. Analanjirofo [Prov. Toamasina]: Masoala NP, N ridge of Ambohitsitondroinan’Mahalevona, 25°26'16"S 49°57 '22"E, 1190 m, 23.II.2003, Lowry 6126 (MO, P). Reg. Atsinanana [Prov. Toamasina]: Rahobevava massif, [17 °59'59"S 48°46'57"E], 1250 m, 12.III.1951, Cours 4330 (P, TAN). Reg DIANA [Prov. Antsiranana]: Manongarivo, 14°02'S 48°18'E, 1050 m, 15.XI.1994, Gautier 2555 (BR, G, TAN). Reg SAVA [Prov. Antsiranana]: Marojejy massif, NE side, [14°25'S 49°43'E], 800 – 1200 m, 25.I. – 25.II.1949, Humbert 23165 (BR, P); ibid. loco, W side, 1000 – 1200 m, 9.XI. – 2.XII.1939, Humbert 31394 (P); vallée inférieure de l’Androranga, Mt Anjenabe, [14°17 'S 49°46'E], 1100 – 1130 m, 3 – 7.XII.1950, Humbert 24161bis (P); Marojejy PA, along E trail to summit, 14°26'20"S 49°44'50"E, 1150 m, 25.X.2001, Mathieu 485 (BR, MO, P, TAN); Distr. Vohemar, forêt de Binara, Daraina, 13°16'38"S 49°35'21"E, 1012 m, 20.XI.2005, Nusbaumer 1639 (BR, G, K, MO, TEF); Distr. Andapa, forêt de Betaolana, 11 km NW of Ambodiangezoka (camp 2), 14°36'22"S 49°25'30"E, 1200 m, 22.X.1999, Rakotomalaza 2025 (G); Marojejy, 14°29'S 49°38'E, 1300 – 1500 m, 21 – 22.I.1994, Rasoavimbahoaka 17 (MO, TAN); Andranomilolo, forêt située env. 13 km à l’W d’Andranopositra, 14°19'21"S 49°17 '46"E, 1380 m, 7.XI.2006, Ravelonarivo 1955 (BR, MO, P, TAN). Etymology. – The specific epithet refers to the general habit of the plant. It is one of the largest Peperomia species of Madagascar. Distribution and habitat. – The new species is endemic to North-East Madagascar from the National Park of Zahamena to the Loky-Manambato Protected Area (Daraina) where it occurs in medium altitude moist evergreen forests at an elevation of 700 – 1400 m. Because of its distribution and the number of currently known collections it is not considered as a rare species. Peperomia variilimba G. Mathieu, sp. nov. (Fig. 4, 5). Peperomia variilimba G. Mathieu differs from P. villilimba C. DC. in the alternate leaf position. Peperomia variilimba G. Mathieu, sp. nov. (Fig. 4, 5). Holotypus: Madagascar. Reg. Alaotra-Mangoro [Prov. Toamasina]: Zahamena, 17 °30'01"S 48°44'00"E, 1250 m, 26.IX.2001, Mathieu 447 (BR [BR000000916064, BR000000916124]!; iso-: B [B100001404]!, BM!, G [G00341879]!, K!, MO-5662978!, P [P00547548]!, TAN!). Peperomia variilimba G. Mathieu differs from P. villilimba C. DC. in the alternate leaf position. Conservation status. – Peperomia robusta is known from 15 locations, all but two are encompassed in the protected area network (Anjanaharibe Sud, COMATSA Nord, Corridor Ankeniheny Zahamena, Loky Manambato, Manongarivo, Marojejy, Masoala and Zahamena). With an Extent of Occur- rence (EOO) of c. 59,000 km2, an Area of Occupancy (AOO) of 60 km2 and no current threats, P. robusta is assigned a status Holotypus: Madagascar. Reg. Alaotra-Mangoro [Prov. Toamasina]: Zahamena, 17 °30'01"S 48°44'00"E, 1250 m, 26.IX.2001, Mathieu 447 (BR [BR000000916064, BR000000916124]!; iso-: B [B100001404]!, BM!, G [G00341879]!, K!, MO-5662978!, P [P00547548]!, TAN!). Peperomia (Piperaceae) novelties from Madagascar – 79 Candollea 75, 2020 A B C 0.1 mm 1 cm 1 cm C B 1 cm Fig. 2. – Peperomia robusta G. Mathieu. A. General habit; B. Detail of node; C. Fruit (lateral view). [A, B: Mathieu 446, BR; C: Mathieu 485, BR] [Drawing: G. Mathieu] 80 – Peperomia (Piperaceae) novelties from Madagascar Candollea 75, 2020 Fig. 3. – Peperomia robusta G. Mathieu. A. General habit; B. Apical part of flowering stem; C. Infrutescence. [Nusbaumer 1639] [Photos: L. Nusbaumer] A B C B B Fig. 3. – Peperomia robusta G. Mathieu. A. General habit; B. Apical part of flowering stem; C. Infrutescence. [Nusbaumer 1639] [Photos: L. Nusbaumer] Conservation status. – Peperomia variilimba is known from eleven locations, all of which are encompassed in the protected area network (Analamazaotra, Anjanaharibe-Sud, COMASTA Nord, Mantadia, Torotorofotsy, Tsaratanana and Zahamena). With an Extent of Occurrence (EOO) of c. 21,000 km2, an Area of Occupancy (AOO) of 36 km2 and no current threats, P. variilimba is assigned a status of “Least Concern” [LC] using the IUCN Red List Categories and Criteria (IUCN, 2012). Perennial herb, epiphytic on mossy trunks. Stem slender, creeping, scandent or pendent, flowering branches apically erect, simple or moderately branched, rooting from the nodes, pilose, internodes 1 – 4 cm. Fig. 4. – Peperomia variilimba G. Mathieu. A. General habit; B. Detail of node; C. Fruit (lateral view). [Mathieu 447, BR] [Drawing: G. Mathieu] is considered as belonging to subgenus Micropiper, as it is the case with P. villilimba (Frenzke et al., 2015). is considered as belonging to subgenus Micropiper, as it is the case with P. villilimba (Frenzke et al., 2015). Paratypi. – Madagascar. Reg. Boeny [Prov. Mahajanga]: Distr. Andapa, Anjanaharibe-Sud RS, around camp 2, 14°44'52"S 49°27 '58"E, 1350 m, 18.X.2001, Mathieu 468 (BR, G, MO, P, TAN). Reg DIANA [Prov. Antsiranana]: Tsaratanana massif, trail from Mangindrano up South ridge of the Maromokotro peak, 1800 – 2000 m, 8.V.1974, Gentry 11579 (MO, P); ibid. loco, 2000 – 2300 m, XI – XII.1937, Humbert 18352 (P); mountains N of Mangindrano, Ambatohafo valley, [14°12'00"S 49°06'30"E], 1200 – 1400 m, 19.I. – 12.II.1951, Humbert 25363 (P); Tsaratanana massif, 2000 m, I.1923, Perrier de la Bâthie 15351 (P); SAVA, Andapa, Doany, Andranomilolo, 10 km NW of Andranopositra, 14°18'34"S 49°19'05"E, 1128 m, 16.XI.2006, Rakotovao 3411 (BR [2 sheets]); ibid. loco, 13 km W of Andranopositra, 14°19'23"S 49°17 '54"E, 1462 m, 10.XI.2006, Ravelonarivo 2001 (BR). Reg. Alaotra- Mangoro [Prov. Toamasina]: Zahamena PA, Andrangovalo massif, [17 °40'S 48°45'E], 1200 – 1400 m, X.1937, Humbert & Cours 17802bis (P); Mantadia NP, PK 10, 18°49'48"S 48°25'58"E, 950 m, 17.IX.2001, Mathieu 431 (BR, TAN); Analamazaotra RS, 18°56'45"S 48°25'10"E, 900 m, 18.IX.2001, Mathieu 438 (BM, BR, MO, P, TAN); Ambatovy, 18°50'13"S 48°18'59"E, 1186 m, 5.V.2008, Rakotondrafara 737 (MO, TAN). Paratypi. – Madagascar. Reg. Boeny [Prov. Mahajanga]: Distr. Andapa, Anjanaharibe-Sud RS, around camp 2, 14°44'52"S 49°27 '58"E, 1350 m, 18.X.2001, Mathieu 468 (BR, G, MO, P, TAN). Reg DIANA [Prov. Antsiranana]: Tsaratanana massif, trail from Mangindrano up South ridge of the Maromokotro peak, 1800 – 2000 m, 8.V.1974, Gentry 11579 (MO, P); ibid. loco, 2000 – 2300 m, XI – XII.1937, Humbert 18352 (P); mountains N of Mangindrano, Ambatohafo valley, [14°12'00"S 49°06'30"E], 1200 – 1400 m, 19.I. – 12.II.1951, Humbert 25363 (P); Tsaratanana massif, 2000 m, I.1923, Perrier de la Bâthie 15351 (P); SAVA, Andapa, Doany, Andranomilolo, 10 km NW of Andranopositra, 14°18'34"S 49°19'05"E, 1128 m, 16.XI.2006, Rakotovao 3411 (BR [2 sheets]); ibid. loco, 13 km W of Andranopositra, 14°19'23"S 49°17 '54"E, 1462 m, 10.XI.2006, Ravelonarivo 2001 (BR). Reg. Alaotra- Mangoro [Prov. Toamasina]: Zahamena PA, Andrangovalo massif, [17 °40'S 48°45'E], 1200 – 1400 m, X.1937, Humbert & Cours 17802bis (P); Mantadia NP, PK 10, 18°49'48"S 48°25'58"E, 950 m, 17.IX.2001, Mathieu 431 (BR, TAN); Analamazaotra RS, 18°56'45"S 48°25'10"E, 900 m, 18.IX.2001, Mathieu 438 (BM, BR, MO, P, TAN); Ambatovy, 18°50'13"S 48°18'59"E, 1186 m, 5.V.2008, Rakotondrafara 737 (MO, TAN). Humbert, H. (1955). Peperomia variilimba G. Mathieu, sp. nov. (Fig. 4, 5). Peperomia (Piperaceae) novelties from Madagascar – 81 Candollea 75, 2020 A B C 1 cm 0.1 mm 1 cm B B C A 82 – Peperomia (Piperaceae) novelties from Madagascar Candollea 75, 2020 Fig. 5. - General habit of Peperomia variilimba G. Mathieu. [Photo: J.K. Spooner] Garden headquarters in Antananarivo, Madagascar. Working visits to BM, K, P, S were granted by Synthesis (European Commission-funded IHP Program). The author thanks cura- tors and staff of all mentioned herbaria for their assistance; Martin Callmander for providing the conservation status assessments; an anonymous reviewer and Martin Callmander are thanked for their valuable suggestions; finally, Louis Nus- baumer and Joanna K. Spooner are thanked for the photo- graphs of Fig. 3 and 5. Garden headquarters in Antananarivo, Madagascar. Working visits to BM, K, P, S were granted by Synthesis (European Commission-funded IHP Program). The author thanks cura- tors and staff of all mentioned herbaria for their assistance; Martin Callmander for providing the conservation status assessments; an anonymous reviewer and Martin Callmander are thanked for their valuable suggestions; finally, Louis Nus- baumer and Joanna K. Spooner are thanked for the photo- graphs of Fig. 3 and 5. is considered as belonging to subgenus Micropiper, as it is the case with P. villilimba (Frenzke et al., 2015). Les territoires phytogéographiques de Mada- gascar. Année Biol. ser. 3, 31: 439 – 448. IUCN (2012). IUCN Red List Categories and Criteria: Version 3.1. Ed. 2. IUCN Species Survival Commission, IUCN, Gland & Cambridge. Madagascar Catalogue (2020). Catalogue of the Plants of Madagascar. Missouri Botanical Garden, St. Louis & Antanana- rivo. [http://www.tropicos.org/Project/Madagascar] Mathieu, G. (2001 – 2020). TRGP (Taxonomic Repertory of the Genus Peperomia). [http://www.peperomia.net/repertory.asp] Mathieu, G. (2003a). New endemic Peperomia species (Piperaceae) from Madagascar. Syst. & Geogr. Pl. 73: 71 – 81. Mathieu, G. (2003b). Notes on tropical African plants – Peperomia nicolliae, a new endemic species (Piperaceae) from Madagascar. Syst. & Geogr. Pl. 73: 288 – 290. Peperomia variilimba G. Mathieu, sp. nov. (Fig. 4, 5). Leaves alternate, exceptionally subopposite; petiole pilose, up to 11 mm long; lamina thin to succulent, orbicular, elliptic, obovate or oblanceolate, 0.9 – 3.0 × 0.9 – 1.9 cm, length-width ratio 1 – 2.7, pilose, usually more densely around petiole insertion, sometimes glabrescent, apex rounded or obtuse, exceptionally retuse, the very tip sometimes slightly emarginate, base rounded, obtuse or acute, margin entirely ciliate. Inflorescence a solitary (sometimes two) terminal spadix; peduncle pilose, ca 1.5 cm long, somewhat widening near the rachis; rachis 3 – 7 cm long, glabrous; floral bract orbicular to elliptic, centrally peltate, ovary ellipsoid. Fruit globose, ca 0.5 mm diam., partially sunken in rachis, pericarp entirely covered with sticky papillae, individual papilla as long as wide, style flat conical, stigma apical, pseudopedicel- late when mature. Notes. – In its general habit P. variilimba resembles P. villilimba. However, the latter species is characterized by an opposite leaf position. The taxonomic concept of P. villilimba has been rather confusing for almost a century due to an inaccurate description of its leaf position in the protologue. The holotype of P. villilimba (Baron 2606, P) was considered by Casimir de Candolle as exhibiting alternate leaves (Candolle, 1911: 48). The specimen concerned is showing only 6 leaves, which were pressed and mounted with considerable superposition. After remounting (straightening of the 2 superposed spadices and turning the specimen so that its former back is in front now) the opposite leaf position became evident. This position is also obvious in the isotype at K, apparently never seen by Candolle. In P. villilimba there is a marked length difference between the indument of the leaf surfaces (long) and of the margin (short). That difference is lacking in P. variilimba. The general habit of P. variilimba also resembles P. rotundilimba C. DC., which shows an opposite (or 3-verticillate) leaf position. P. variilimba Etymology. – The specific epithet refers to the quite variable leaves, as well in shape as in succulence and indument density. Distribution and habitat. – The new species is endemic to the medium altitude moist evergreen forests at an elevation range from 900 to 2300 m. Peperomia variilimba is known from the Tsaratanana massif in the North-East to Anal- amazaotra in the Centre East. It is apparently common. References Bachman, S. & J. Moat (2012). GeoCAT – an open source tool for rapid Red List assessments. Bot. Gard. Conservation Int. J. 9. [http://geocat.kew.org] Candolle, C. de (1911). Pipéracées de Madagascar, espèces et locali- tés nouvelles. Not. Syst. (Paris) 2: 46 – 50. Frenzke, L., E. Scheiris, G. Pino, L. Symmank, P. Goetghebeur, C. Neinhuis, S. Wanke & M.-S. Samain (2015). A revised infrageneric classification of the genus Peperomia (Piperaceae). Taxon 64: 424 – 444. DOI: https://doi.org/10.12705/643.4 Friedmann, F. (2011). Peperomia. Fl. Seychelles: 75 – 78. Muséum national d’Histoire naturelle, Paris & Institut de Recherche pour le Développement, Marseille. Fig. 5. - General habit of Peperomia variilimba G. Mathieu. [Photo: J.K. Spooner] Gautier, L., J.A. Tahinarivony, P. Ranirison & S. Wohlhauser (2018). Vegetation. In: Goodman, S.M., M.J. Raherilalao & S. Wohlhauser (ed.), The terrestrial protected areas of Madagascar: Their history, description and biota: 207 – 242. Association Vahatra, Antananarivo. is considered as belonging to subgenus Micropiper, as it is the case with P. villilimba (Frenzke et al., 2015). Acknowledgements Mathieu, G. (2006). More new Peperomia species (Piperaceae) from Madagascar. Syst. & Geogr. Pl. 76: 221 – 228. DOI: https://doi. org/10.2307/20649714 Field work in Madagascar was supported financially by the Belgian King Leopold III fund for Nature Exploration & Conservation and logistically by the Missouri Botanical
https://openalex.org/W2969074820	https://zenodo.org/record/3353445/files/33441.pdf	English	null	Ethnobotany of Weeds; Weed Flora of Ramat Polytechnic Teaching and Research Farm, Maiduguri Borno State of Nigeria	International journal of advances in scientific research and engineering	2,019	cc-by	3,503	ABSTRACT This study was carried out to ascertain the species of weeds at Ramat Polytechnic Teaching and Research Farm, Maiduguri Borno State Nigeria. The objective of the study is to identify the weeds flora and ethnobotanical benefits of these weeds. Weed flora shows that 38 weed species were identified belonging to 18 different families out of which mostly, twenty-six (26) are annuals the remaining (8) are perennials. The distribution of these weeds further shows that Cleome gynandra L., Commelina benghalens is Schult. F., Euphorbia hirta linn., Crotalaria mucronata L., Senna occidentalis L., Boerhavia erecta L., Eragrotis tenella (A. Rich) Hoschst. Ex Steud., Cenchrus biflorus (Roxb.)., Corchorus olitorius L. and Tribulus terrestris L. have high (70%) occurrence at the farm. The dominant plant families are the Fabaceae, Poaceae and Malvaceae. Most of the samples weeds have uses ranging from medicinal, soup, fodder and mulch cover. Key words: Weed Flora, Ethnobotany, Families, Uses, Maiduguri. Key words: Weed Flora, Ethnobotany, Families, Uses, Maiduguri. Key words: Weed Flora, Ethnobotany, Families, Uses, Maiduguri. Ethnobotany of Weeds; Weed Flora of Ramat Polytechnic Teaching and Research Farm, Maiduguri Borno State of Nigeria Y. B. Kajidu1., Adam B. K1., R. A. Saleh2., I. B. Shehu3 and Y. M. Kundili3 1Department of Crop Production, Faculty of Agriculture, University of Maiduguri 2Desert Research Monitoring and Control Centre, Yobe State University, Nigeria 3Department of Agricultural Technology Ramat Polytechnic Maiduguri Borno State. Nigeria Ethnobotany of Weeds; Weed Flora of Ramat Polytechnic Teaching and Research Farm, Maiduguri Borno State of Nigeria Y. B. Kajidu1., Adam B. K1., R. A. Saleh2., I. B. Shehu3 and Y. M. Kundili3 1Department of Crop Production, Faculty of Agriculture, University of Maiduguri 2Desert Research Monitoring and Control Centre, Yobe State University, Nigeria 3Department of Agricultural Technology Ramat Polytechnic Maiduguri Borno State. Nigeria 1. INTRODUCTION Ethnobotany is the study of the relationship between plants and people Dhanam and Elayaraj (2014). Quite a number of plants considered as weeds in modern sciences have significant value. In ethnobotany, knowledge about local weeds is more important than local knowledge of insectsor plant diseases; some weeds are used as fodder,medicine, even toys, but others are just weeds Bentley et al., 2005.Weeds have genetic and phenotypic ability Dhanam and Elayaraj (2014); such characters enable them to pass through successfully in adverse habitats. They easily invade crop fields which are favourite habitat for their fast growth (Randall 1996; Frohlich et al., 2000; Hassan and Marwat 2001). The presence of weeds in the fields and their impact on the crop production and environment has been well documented. Sher et al., (2011) stated that the unwanted weeds are responsible for the competitive and allelopathic behavior and rendering habitats to harmful organisms. Ethnobotany can help elicit demand for research in aculturally and ecologically sensitive way.Understanding the importance of weeds is useful for taxonomists, agriculturists and scientists involved in the management of weeds.weeds easily invade crop fields which are favourite grounds for their quick growth. The presence of weeds in the fields and their impact on the crop production and environment has been well documented (Randall 1996; Frohlich et al., 2000; Hassan and Marwat 2001). There are numerous benefits of weeds as reported by Soladoye et al., (2010) such as economic and medicinal values, they protect the soil against erosion and add organic matter to the soil. Weeds also provide shelter for beneficial insects and provide nectar for bees, Some weeds serve as raw material for conventional medicine, while others have been used locally for decades for several traditional medicinal purposes ranging from simple laxative to cure dysentery (Hill and Ramsay, 1977; Soladoye et al., 2006). This study was carried out to ascertain the species of weeds at the experimental farm as this will enable researchers to be aware of This study was carried out to ascertain the species of weeds at the experimental farm as this will enable researchers to be aware of the species of plants, their nomenclature and some vital information about the weeds.  This paper study is to ascertain the species of weeds at Ramat Polytechnic Teaching and Research Farm, Maiduguri Borno State Nigeria.  This paper is to identify the weeds flora and ethnobotanic benefits of these weeds. 1. INTRODUCTION  This paper is to identify the weeds flora and ethnobotanic benefits of these weeds. Page 205 www.ijasre.net Licensed Under Creative Commons Attribution CC BY-NC 3. RESULTS AND DISCUSSIONS Table 1. Weed Flora of Ramat Polytechnic Teaching and Research Farm, Maiduguri Borno State S/N Weed Scientific name Family Common name Lifecycle 1 2 Amaranthus retroflexus L. Amaranthus spinosis L. Amaranthaceae Smooth pig weed Thorn pig weed Annual 3 4 Leptadenia hastata (Pers) Calotropis procera (Aiton) W. T. Apocynaceae Akamongot Sodom apple Annual 5 Cleome gynandra L. Cleomaceae Stink weed Annual 6 7 Commelina benghalensis Schult. F. Commelina erecta linn. Commilanaceae Climbing day flower Wandering jew Annual/perennial 8 Ipomoea eriocarpa R. Br. Convulvulaceae Tiny morning glory Annual 9 Momordica balsamina L. Curcubitaceae Balsam apple Annual 10 Cyperus rotundus L. Cyperaceae Nutsedge Perennial 11 Euphorbia hirta linn. Euphorbiaceae Asthma plant Annual 12 13 14 15 16 17 18 19 Crotalaria mucronata L. Crotalaria medicaginea L. Senna tora L. Senna occidentalis L. Tephrosia purpurea (L.) Pers.) Swartzia madagascariensisL. Faidherbia albida A. Cahev. Acacia nilotica Fabaceae Rattle pod Rattle pod Sickle senna Coffee senna Wild indigo Snake bean Acacia Gum Arabic Anuual/perennial Perennial 20 Ocimum gratissimum L. Lamiaceae African basil Annual 21 Englerina gabonensis (Engl.) Balle Loranthaceae Mistletoe Perennial 22 23 24 Waltheria americana L. Hibiscus asper L. Sida cordifolia L. Malvacea Sleepy morning wild sorrel Flannel weed Annual annual annual 35 Azadirachta indicaL. Meliaceae Neem Perennial 26 Boerhavia erecta L. Nyctaginaceae Spiderling Annual/perennial 27 28 29 30 Eragrotis tenella (A. Rich) Hoschst. Ex Steud. Eragrotis tremula Hochst. Cynodon dactylon (L.) Pers. Cenchrus biflorus (Roxb.) Poaceae Japanese love grass Cane grass Bermuda grass Indian sandbur Annual Annual Perennial Annual 31 32 Mitracarpus hirtus (L.) DC Borreria stadchydea (DC) Hutch & Dalziel Rubiaceae Girdle pod - 33 Corchorus olitorius L. Tiliaceae White jute Annual 34 Tribulus terrestris L. Zygophyllaceae Devils thorn Annual Table 1. Weed Flora of Ramat Polytechnic Teaching and Research Farm, Maiduguri Borno S Weed flora collected and identified at the experimental farm is presented on Table 1. The weeds were identified with the help of local farmers around and later translated to its various common name, scientific names and families with help of the manual Hausa plant names by Roger Blench (2007). Further clarification and confirmation was done through Google search . Twenty six (26) of the weeds were annuals while the remaining eight (8) are perennials giving a total of thirty four (34) weed species belonging to eighteen (18) families. 2. METHODOLOGY The study is conducted at the Ramat Polytechnic Teaching and Research farm (40000 m2), in Maiduguri Borno state, between June and July 2019 (at the establishment of rainfall when almost all weeds must have emerged). The ecological information (in the two months of June and July) from the area average tempereature is 36.40C-33.20C, average rainfall of 73.8mm-147.1mmin the Sudan savanna regionThe area being a research farm is characterized by numerous annual and perennial vegetation; among the tree crops are Acacia species, neem Azadriratcha indica. Wet and dry season crops such as maize Zea mays, wheat Triticum aestivum, Amaranthus Amaranthus spp, Lettuce Lactuca sativa, Carrot Daucus carota, Onion Allium cepa, Cabbage Brassica oleracea, Sorrel Hibiscus sabdariffa, Okro Hibiscus esculentusTomato Lycopersicon esculentus. Weed samples were collected from I m2 quadrant around the farm including climbing weeds on wire fence and trees. These weed sample were identified with the help of indigenous farmers, weed identification manual and elders around the community who have the knowledge about traditional medicine and are so familiar with these weeds. 3. RESULTS AND DISCUSSIONS 3. RESULTS AND DISCUSSIONS 3. RESULTS AND DISCUSSIONS Two tree species (Acacia and Neem) were included, this is because the seeds of these trees were scattered around and germinate to constitute as the weeds due to the presence of their seedlings. Page 206 DOI: 10.31695/IJASRE.2019.33441 International Journal of Advances in Scientific Research and Engineering (ijasre),Vol 5 (7), July-2019 Table 2. Weed Flora of Ramat Polytechnic Teaching and Research Farm Maiduguri, Borno State, on Frequency of Occurrence. S/N Weed Scientific name Frequency of occurrence 1 2 Amaranthus retroflexus L. Amaranthus spinosis L. + + 3 4 Leptadenia hastata (Pers) Calotropis procera (Aiton) W. T. ++ ++ 5 Cleome gynandra L. +++ 6 7 Commelina benghalensis Schult. F. Commelina erecta linn. +++ + 8 Ipomoea eriocarpa R. Br. ++ 9 Momordica balsamina L. ++ 10 Cyperus rotundus L. ++ 11 Euphorbia hirta linn. +++ 12 13 14 15 16 17 18 19 Crotalaria mucronata L. Crotalaria medicaginea L. Senna tora L. Senna occidentalis L. Tephrosia purpurea (L.) Pers.) Swartzia madagascariensis L. Faidherbia albida A. Cahev. Acacia nilotica +++ + + +++ + + ++ ++ 20 Ocimum gratissimum L. + 21 Englerina gabonensis (Engl.) Balle + 22 23 24 Waltheria americana L. Hibiscus asper L. Sida cordifolia L. + + + 35 Azadirachta indica L. ++ 26 Boerhavia erecta L. +++ 27 28 29 30 Eragrotis tenella (A. Rich) Hoschst. Ex Steud. Eragrotis tremula Hochst. Cynodon dactylon (L.) Pers. Cenchrus biflorus (Roxb.) +++ + ++ +++ 31 32 Mitracarpus hirtus (L.) DC Borreria stadchydea (DC) Hutch &Dalziel ++ ++ 33 Corchorus olitorius L. +++ 34 Tribulus terrestris L. +++ International Journal of Advances in Scientific Research and Engineering (ijasre),Vol 5 (7), July-2019 ournal of Advances in Scientific Research and Engineering (ijasre),Vol 5 (7), July-2019 International Journal of Advances in Scientific Research and Engineering (ijasre),Vol 5 (7) KEY + 50% and below low ++ 51% -70% moderate +++ 70%-100% high Table 2 shows the frequency of occurrence on the weed flora at the research farm. The frequency is categorized into three groups viz; 50% and below shows the occurrence of the weed in 20 quadrants and less. 51%-70% shows weed occurrence in 21 quadrants to 30 quadrants and 71%-100% shows the occurrence of the weed in all the sampled quadrants (40). In some special cases where parasitic weeds were identified, total number of trees in the whole farm were counted and parasitic weeds identified to give the frequency. This finding is in line with the work of Dhanam and Elayaraj (2014) who reported similar observation on the frequency occurrence of weed. www.ijasre.net Page 207 DOI: 10.31695/IJASRE.2019.33441 Table 3. DOI: 10.31695/IJASRE.2019.33441 Weed Flora of Ramat Polytechnic Teaching and Research Farm Maiduguri, Borno State, on Percentage of Weed Families S/N Weed Scientific name Family Percentage Total 100% 1 2 Amaranthus retroflexus L. Amaranthus spinosis L. Amaranthaceae 5.88 3 4 Leptadenia hastata (Pers) Calotropis procera (Aiton) W. T. Apocynaceae 5.88 www.ijasre.net Page 207 DOI: 10.31695/IJASRE.2019.33441 Table 3. Weed Flora of Ramat Polytechnic Teaching and Research Farm Maiduguri, Borno State, on Percentage of Weed Families S/N Weed Scientific name Family Percentage Total 100% 1 2 Amaranthus retroflexus L. Amaranthus spinosis L. Amaranthaceae 5.88 3 4 Leptadenia hastata (Pers) Calotropis procera (Aiton) W. T. Apocynaceae 5.88 Ramat Polytechnic Teaching and Research Farm Maiduguri, Borno State, on Percentage of Weed Families Table 3. Weed Flora of Ramat Polytechnic Teaching and Research Farm Maiduguri, Borno State, o Families Page 207 ional Journal of Advances in Scientific Research and Engineering (ijasre),Vol 5 (7), July-2019 5 Cleome gynandra L. Cleomaceae 2.94 6 7 Commelina benghalensis Schult. F. Commelina erecta linn. Commilanaceae 5.88 8 Ipomoea eriocarpa R. Br. Convulvulaceae 2.94 9 Momordica balsamina L. Curcubitaceae 2.94 10 CyperusrotundusL. Cyperaceae 2.94 11 Euphorbia hirta linn. Euphorbiaceae 2.94 12 13 14 15 16 17 18 19 Crotalaria mucronata L. Crotalaria medicaginea L. Senna tora L. Senna occidentalis L. Tephrosia purpurea (L.) Pers.) Swartzia madagascariensis L. Faidherbia albida A. Cahev. Acacia nilotica Fabaceae 23.53 20 Ocimum gratissimum L. Lamiaceae 2.94 21 Englerina gabonensis (Engl.) Balle Loranthaceae 2.94 22 23 24 Waltheria americana L. Hibiscus asper L. Sida cordifolia L. Malvacea 8.82 35 Azadirachta indica L. Meliaceae 2.94 26 Boerhavia erecta L. Nyctaginaceae 2.94 27 28 29 30 Eragrotis tenella (A. Rich) Hoschst. Ex Steud. Eragrotis tremula Hochst. Cynodon dactylon (L.) Pers. Cenchrus biflorus (Roxb.) Poaceae 11.76 31 32 Mitracarpus hirtus (L.) DC Borreria stadchydea (DC) Hutch & Dalziel Rubiaceae 5.88 33 Corchorus olitorius L. Tiliaceae 2.94 34 Tribulus terrestris L. Zygophyllaceae 2.94 l Journal of Advances in Scientific Research and Engineering (ijasre),Vol 5 (7), July-2019 International Journal of Advances in Scientific Research and Engineering (ijasre),Vol 5 A total of thirty four (34) weed species belonging to eighteen (18) families were identified as constituting the weed flora of the research site. Fabaceae has the largest family with a number of eight (8) weed species followed by the Poaceaefamily with a number of four (4) weed species.Malvaceae family having (3) weed species. All other families constituted the remaining species. A total of thirty four (34) weed species belonging to eighteen (18) families were identified as constituting the weed flora of the research site. Fabaceae has the largest family with a number of eight (8) weed species followed by the Poaceaefamily with a number of four (4) weed species.Malvaceae family having (3) weed species. All other families constituted the remaining species. This is better illustrated on chart as presented on Fig 1. The prevalence of the (3) three major families was in tandem with the families of the major weeds of the world (Akobundu, 1987). DOI: 10.31695/IJASRE.2019.33441 This is better illustrated on chart as presented on Fig 1. The prevalence of the (3) three major families was in tandem with the families of the major weeds of the world (Akobundu, 1987). Amaranthaceae 6% Apocynaceae 6% Cleomaceae 3% Commilanaceae 6% Convulvulaceae 3% Curcubitaceae 3% Cyperaceae 3% Euphorbiaceae 3% Fabaceae 24% Lamiaceae 3% Loranthaceae 3% Malvacea 9% Meliaceae 3% Nyctaginaceae 3% Poaceae 12% Rubiaceae 6% Tiliaceae 3% Zygophyllaceae 3% Fig 1. Weed Flora of Ramat Polytechnic Teaching and Research Farm Maiduguri, Borno State, on Percentage of Weed Families. Amaranthaceae 6% Apocynaceae 6% Cleomaceae 3% Commilanaceae 6% Convulvulaceae 3% Curcubitaceae 3% Cyperaceae 3% Euphorbiaceae 3% Fabaceae 24% Lamiaceae 3% Loranthaceae 3% Malvacea 9% Meliaceae 3% Nyctaginaceae 3% Poaceae 12% Rubiaceae 6% Tiliaceae 3% Zygophyllaceae 3% www.ijasre.net Page 208 DOI: 10.31695/IJASRE.2019.33441 Fig 1. Weed Flora of Ramat Polytechnic Teaching and Research Farm Maiduguri, Borno State, on Percentage of Weed Families. Fig 1. Weed Flora of Ramat Polytechnic Teaching and Research Farm Maiduguri, Borno State, on Percentage of Weed Families. Page 208 Page 208 DOI: 10.31695/IJASRE.2019.33441 Many out of the sampled weeds are used as pot herbs or soups according to different traditions. Other uses include fodder, mulching material and soil cover this is in line with the findings of many researchers on ethnobotany of weeds (Solodoye et al., 2010; Muhammad et al., 2018; ) Table 4 presents the weed flora of the research farm with their medicinal and other uses, most of the species are medicinal through their different parts (sap, leaves, stem, seeds, roots) fresh or dried, prepared with some other ingredients. Among these are the Crotalaria species and Senna occidentalis which are highly medicinal (as anti fever) especially in small children; as stated by Ogunkunle and Ladejobi (2006)local usage of the plant for these purposes and the presence of anthraquinones in its leaf extracts. Many out of the sampled weeds are used as pot herbs or soups according to different traditions. Other uses include fodder, mulching material and soil cover this is in line with the findings of many researchers on ethnobotany of weeds (Solodoye et al., 2010; Muhammad et al., 2018; ) www.ijasre.net Page 209 4. CONCLUSION In conclusion, weed flora of Ramat Polytechnic Teaching and Research Farm, Maiduguri Borno state shows that 38 weed species were identified belonging to 18 different families out of which mostly, twenty six (26) are annuals the remaining (8) are perennials. The distribution of these weeds further shows that Cleome gynandra L., Commelina benghalensis Schult. F., Euphorbia hirta linn., Crotalaria mucronata L., Senna occidentalis L., Boerhavia erecta L., Eragrotis tenella (A. Rich) Hoschst. Ex Steud.,Cenchru sbiflorus (Roxb.)., Corchorus olitorius L. and Tribulus terrestris L. have high (70%) occurrence at the farm. The dominant plant families are the Fabaceae, Poaceae and Malvaceae. Most of the samples weeds have uses ranging from medicinal, soup, fodder and mulch cover. DOI: 10.31695/IJASRE.2019.33441 International Journal of Advances in Scientific Research and Engineering (ijasre),Vol 5 (7), July-2019 Table 4. Weed Flora of Ramat Polytechnic Teaching and Research Farm Maiduguri, Borno State, with Medicinal and other Uses S/N Weed Scientificname Uses; Medicinal/others * 1 2 Amaranthus retroflexus L. Amaranthus spinosis L. Fodder - 3 4 Leptadenia hastata (Pers) Calotropis procera (Aiton) W. T. Pot herb, Medicinal ; Leaves and sap 5 Cleome gynandra L. Soup 6 7 Commelina benghalensis Schult. F. Commelina erecta linn. Fodder Medicinal 8 Ipomoea eriocarpa R. Br. Fodder 9 Momordica balsamina L. Soup, seeds are medicinal 10 Cyperus rotundus L. - 11 Euphorbia hirta linn. Medicinal 12 13 14 15 16 17 18 19 Crotalaria mucronata L. Crotalaria medicaginea L. Senna tora L. Senna occidentalis L. Tephrosia purpurea (L.) Pers.) Swartzia madagascariensis L. Faidherbia albida A. Cahev. Acacia nilotica L. Highly medicinal Highly medicinal Soup Soup Highly medicinal Medicinal Fodder Fodder, gum 20 Ocimum gratissimum L. Medicinal 21 Englerina gabonensis (Engl.) Balle - 22 23 24 Waltheria americana L. Hibiscus asper L. Sida cordifolia L. Medicinal Soup Medicinal 35 Azadirachta indica L. Medicinal 26 Boerhavia erecta L. - 27 28 29 30 Eragrotis tenella (A. Rich) Hoschst. Ex Steud. Eragrotis tremula Hochst. Cynodon dactylon (L.) Pers. Cenchrus biflorus (Roxb.) Mulch Mulch Fodder - 31 32 Mitracarpus hirtus (L.) DC Borreria stadchydea (DC) Hutch &Dalziel Sap medicinal Medicinal 33 Corchorus olitorius L. Soup 34 Tribulus terrestris L. - information given not on scientific findings but through oral interaction with the indigenous people around International Journal of Advances in Scientific Research and Engineering (ijasre),Vol 5 (7), July-2019 al Journal of Advances in Scientific Research and Engineering (ijasre),Vol 5 (7), July-2019 International Journal of Advances in Scientific Research and Engineering (ijasre),Vol 5 (7) information given not on scientific findings but through oral interaction with the indigenous people around Table 4 presents the weed flora of the research farm with their medicinal and other uses, most of the species are medicinal through their different parts (sap, leaves, stem, seeds, roots) fresh or dried, prepared with some other ingredients. Among these are the Crotalaria species and Senna occidentalis which are highly medicinal (as anti fever) especially in small children; as stated by Ogunkunle and Ladejobi (2006)local usage of the plant for these purposes and the presence of anthraquinones in its leaf extracts. RECOMMENDATION Further studies should be conducted about the allelopathic effects of these weeds, their benefit as all plants are important in one way or the other. Further studies should be conducted about the allelopathic effects of these weeds, their benefit as all plants are important in one way or the other. REFERENCES 1. Akobundu, I.O. (1987). Weed Science in the Tropics: Principles and practices. John Wiley & Sons, Chichester,pp. 522. 2. Dhanam S. and Elayara B. (2014) Ethnomedicinal aspects of some weeds from paddy fields of Villupuram district in Tamil Nadu, India International Letters of Natural Sciences 14 (2014) 1-10 ISSN 2300-9675 3. Frohlich J., S.V. Fower, A. Gianotti, R. Hill, E. Kilgore, L. Morin, C. Winks, (2000) Xth International Symp. On Biological Control of Weeds, 4-14 July 1999, Bozeman, Montana USA, P. 51-57. 4. Hassan G., K. B. Marwat, (2001) Integrated weed management in agricultural crop. National Workshop on Technologies for Sustainable,. 5. Hill, B.S. and Ramsay, J. (1977). Weeds as Indicators of Soil Conditions. Ecological Agricultural Projects Publication 67. 5. Hill, B.S. and Ramsay, J. (1977). Weeds as Indicators of Soil Conditions. Ecological Agricultural Pro 6. Jeffery W. B, Morag W., Silvio N. and Salomo P. (2005). Even Useful Weeds are Pests: Ethnobotany in the Bolivian Andes International Journal of Pest Management, 51(3): 189 – 207 7. Khan M. N., Razzaq A., Hadi F., Khan N., Basit A., Jan F., Khan N. (2018). Ethnobotanical profile of weed flora of district Charsadda, Khyber Pakhtunkhwa. RADS J. Biol. Res. Appl. Sci.; 9(1): 14-23 8. Ogunkunle A.T.J. and Ladejobi T. A. (2006) Ethnobotanical and phytochemical studies on some species of Sennain Nigeria African Journal of Biotechnology Vol. 5 (21), pp. 2020-2023 9. Randall, J. M. (1996).Weed Tech., 10 370-381. 9. Randall, J. M. (1996).Weed Tech., 10 370-381. 10. Roger B., (2007). Hausa Names for Plants and Trees. 2nd edition. http://www.rogerblench.info/RBOP.htm 11. Sher Z. F., Hussain L., Badshah M., Wahab M. (2011) Floristic composition, communities and ecological characteristics of weeds of wheat fields of Lahore, District Swabi, Pakistan. Pak J Bot.; 43(6): 2817-20. 12. Soladoye M.O., Osipitan A.A., Sonibare M.A., ,Chukwuma E.C. (2010) From ‘Vagabonds’ to Ethnobotanical Relevance: Weeds of the Campus Sites of OlabisiOnabanjo University, Ago-Iwoye, Nigeria. Ethnobotanical Leaflets 14: 546-58. 13. Soladoye, M.O., Yakubu, F.A., Kola-Oladiji, K., Alabi,, D.A. and Agbomeji, Y.O. (2006). The Collection, Conservation and Cultivation of Local Medicinal Plants for Natural Medicine Production. The Seminar/Workshop and Exhibition of natural Medicine products. Organized by Nigeria Traditional Medical Association, Ijebu-North in collaboration with Faculty of Science, OlabisiOnabanjo University, Ago-Iwoye held 19th-23rd September, 2006. Page 210 Page 210 www.ijasre.net
https://openalex.org/W3194198159	https://www.frontiersin.org/articles/10.3389/fcimb.2021.702676/pdf	English	null	Thermoresponsive Copolymer Nanovectors Improve the Bioavailability of Retrograde Inhibitors in the Treatment of Leishmania Infections	Frontiers in cellular and infection microbiology	2,021	cc-by	12,178	Keywords: Leishmania, Retro-2, retrograde inhibitors, thermoresponsive polymers, nanoaggregates, encapsulation, bioavailability, drug release ORIGINAL RESEARCH published: 19 August 2021 doi: 10.3389/fcimb.2021.702676 Evan Craig1, Anna Calarco2, Raffaele Conte2, Veronica Ambrogi3, Giovanna Gomez d’Ayala4, Philip Alabi5, Jason K. Sello5, Pierfrancesco Cerruti4* and Peter E. Kima1* 1 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, United States, 2 Research Institute on Terrestrial Ecosystems (IRET-CNR), Napoli, Italy, 3 Department of Chemical, Materials and Production Engineering (DICMaPI) – University of Naples Federico II, Napoli, Italy, 4 Institute for Polymers, Composites and Biomaterials (IPCB-CNR), Pozzuoli, Italy, 5 Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, United States Reviewed by: Reviewed by: Danilo Ciccone Miguel, State University of Campinas, Brazil Mauro Javier Cortez Veliz, University of São Paulo, Brazil Reviewed by: Danilo Ciccone Miguel, State University of Campinas, Brazil Mauro Javier Cortez Veliz, University of São Paulo, Brazil Reviewed by: Danilo Ciccone Miguel, State University of Campinas, Brazil Mauro Javier Cortez Veliz, University of São Paulo, Brazil Correspondence: Peter E. Kima pkima@uﬂ.edu Pierfrancesco Cerruti cerruti@ipcb.cnr.it Specialty section: This article was submitted to Parasite and Host, a section of the journal Frontiers in Cellular and Infection Microbiology Received: 29 April 2021 Accepted: 09 July 2021 Published: 19 August 2021 Citation: Craig E, Calarco A, Conte R, Ambrogi V, d’Ayala GG, Alabi P, Sello JK, Cerruti P and Kima PE (2021) Thermoresponsive Copolymer Nanovectors Improve the Bioavailability of Retrograde Inhibitors in the Treatment of Leishmania Infections. Front. Cell. Infect. Microbiol. 11:702676. doi: 10.3389/fcimb.2021.702676 Specialty section: This article was submitted to Parasite and Host, a section of the journal Frontiers in Cellular and Infection Microbiology Specialty section: This article was submitted to Parasite and Host, a section of the journal Frontiers in Cellular and Infection Microbiology Received: 29 April 2021 Accepted: 09 July 2021 Published: 19 August 2021 Edited by: Izabel Galhardo Demarchi, Federal University of Santa Catarina, Brazil Clinical manifestations of leishmaniasis range from self-healing, cutaneous lesions to fatal infections of the viscera. With no preventative Leishmania vaccine available, the frontline option against leishmaniasis is chemotherapy. Unfortunately, currently available anti- Leishmania drugs face several obstacles, including toxicity that limits dosing and emergent drug resistant strains in endemic regions. It is, therefore, imperative that more effective drug formulations with decreased toxicity proﬁles are developed. Previous studies had shown that 2-(((5-Methyl-2-thienyl)methylene)amino)-N-phenylbenzamide (also called Retro-2) has efﬁcacy against Leishmania infections. Structure–activity relationship (SAR) analogs of Retro-2, using the dihydroquinazolinone (DHQZ) base structure, were subsequently described that are more efﬁcacious than Retro-2. However, considering the hydrophobic nature of these compounds that limits their solubility and uptake, the current studies were initiated to determine whether the solubility of Retro-2 and its SAR analogs could be enhanced through encapsulation in amphiphilic polymer nanoparticles. We evaluated encapsulation of these compounds in the amphiphilic, thermoresponsive oligo (ethylene glycol) methacrylate-co-pentaﬂuorostyrene (PFG30) copolymer that forms nanoparticle aggregates upon heating past temperatures of 30°C. The hydrophobic tracer, coumarin 6, was used to evaluate uptake of a hydrophobic molecule into PFG30 aggregates. Mass spectrometry analysis showed considerably greater delivery of encapsulated DHQZ analogs into infected cells and more rapid shrinkage of L. amazonensis communal vacuoles. Moreover, encapsulation in PFG30 augmented the efﬁcacy of Retro-2 and its SAR analogs to clear both L. amazonensis and L. donovani infections. These studies demonstrate that encapsulation of compounds in PFG30 is a viable approach to dramatically increase bioavailability and efﬁcacy of anti-Leishmania compounds. Thermoresponsive Copolymer Nanovectors Improve the Bioavailability of Retrograde Inhibitors in the Treatment of Leishmania Infections Evan Craig1, Anna Calarco2, Raffaele Conte2, Veronica Ambrogi3, Giovanna Gomez d’Ayala4, Philip Alabi5, Jason K. Sello5, Pierfrancesco Cerruti4 and Peter E. Kima1* Evan Craig1, Anna Calarco2, Raffaele Conte2, Veronica Ambrogi3, Giovanna Gomez d’Ayala4, Philip Alabi5, Jason K. Sello5, Pierfrancesco Cerruti4* and Peter E. Kima1* INTRODUCTION treatment with Retro-2 resulted in decreased localization of Stx5 to the LPV membrane, as well as a signiﬁcant decrease in vacuole size and parasite burden (Ndjamen et al., 2010; Canton and Kima, 2012; Canton et al., 2012). Unlike Retro-2, the SAR DHQZ analogs, DHQZ 36 and DHQZ 36.1, are directly Leishmania- cidal (Craig et al., 2017). Leishmania are ﬂagellated, obligate, protozoan parasites in the Trypanosomatidae family. They are the causative agents of leishmaniases that manifest as three major clinical entities: cutaneous, mucocutaneous, and visceral infection. The prevalence of leishmaniasis is estimated at 12 to 15 million cases with 350 million people at risk worldwide. Annually, 1.5 to 2 million new leishmaniasis cases occur with approximately 70,000 deaths. In 2016, the World Health Organization (WHO) reported that of 200 countries, 87 (44%) and 75 (38%) countries were classiﬁed as endemic for cutaneous leishmaniasis (CL) or visceral leishmaniasis (VL), respectively (Arenas et al., 2017; World Health Organization, 2018; Inceboz, 2019). While CL cases are often self-limiting, VL cases are lethal requiring curative treatment with anti-leishmanial drugs. The WHO has classiﬁed leishmaniasis as a neglected disease. The few available drugs have important shortcomings, including high cost, toxicity, and the emergence of resistance strains. Control of VL has been further complicated in regions, including sub-Saharan Africa, that have high rates of human immunodeﬁciency virus (HIV) infection. HIV-VL-coinfection signiﬁcantly increases the risk of development of VL as well as decreasing the efﬁcacy of therapeutic responses (Alvar et al., 2008). As there is currently no clinically approved Leishmania vaccine, the discovery of new anti-Leishmania compounds or strategies to improve current treatments is sorely needed. While improvements in efﬁcacy through SAR analog development are encouraging, Retro-2 and the SAR analogs are limited by their hydrophobic nature. Retro-2 solubility is estimated at <0.4 mg/ml in H2O (Carney et al., 2014; Gandhi et al., 2019). It, therefore, requires the use of an organic solvent, such as dimethyl sulfoxide (DMSO), for solubilization and uptake by mammalian cells. One method to signiﬁcantly improve solubility of hydrophobic compounds while also ensuring protection against degradation is the encapsulation of the drug within nanoparticles. The latter may be made of inorganic or organic, inert, biodegradable materials that form a capsule-like structure around the compound. INTRODUCTION The beneﬁts of encapsulation include increased solubility and bioavailability, protection against degradation, and the potential for modiﬁed release kinetics or targeting to speciﬁc sites in the body, thereby reducing toxicity of the compound of interest (Banik et al., 2016; Barouti et al., 2017). This increase in bioavailability while limiting toxicity has resulted in improved safety proﬁles and efﬁcacy of a multitude of anti-Leishmania compounds including liposomal AmB that is currently approved for clinical use (Croft and Coombs, 2003; Nafari et al., 2020). Thus, it is likely that Retro-2 and SAR DHQZ analogs would greatly beneﬁt from nanoparticle encapsulation to improve efﬁcacy of these compounds through improved solubility and delivery. y A few studies have reported on the antileishmanial efﬁcacy of Retro-2, a small, organic, retrograde pathway inhibitor. Retro-2 was initially identiﬁed using high-throughput screens of a chemical library to identify compounds that could limit the toxicity of Shiga toxin [Shiga-like toxin 1 (SLT1) and 2 (SLT2)], a toxin from enterohemorrhagic Escherichia coli or Shigella dysenteriae, as well as the plant toxin, ricin. Retro-2 appeared to function through prevention of retrograde transport of these toxins (Stechmann et al., 2010). The efﬁcacy of Retro-2 on Leishmania infections was subsequently demonstrated (Canton and Kima, 2012). Craig et al. and other reports later showed that the structural–activity relationship (SAR) analogs, DHQZ 36 and DHQZ 36.1, were more efﬁcacious against both L. amazonensis and L. donovani infections (Canton and Kima, 2012; Craig et al., 2017; Gupta et al., 2017). While the precise mechanism of action of Retro-2 and its SAR analogs in Leishmania infections is not known, its application was shown to result in the shrinkage of the communal Leishmania parasitophorous vacuoles (LPVs) that harbor L. pifanoi or L. amazonensis amastigotes. LPVs had been demonstrated to be hybrid compartments that in addition to their extensive interactions with the endocytic pathway, they also displayed secretory pathway molecules (Canton et al., 2012). Acquisition of secretory pathway molecules was shown to be dependent on the actions of syntaxin 5 (Stx5), a SNARE that participates in vesicle transport and fusion at several points in the secretory pathway (Canton and Kima, 2012; Canton et al., 2012; Arango Duque et al., 2019). Loss of Stx5 function through the expression of a dominant-negative mutant or by siRNA silencing of Stx5 or g y y In this regard, amphiphilic, thermoresponsive polymers are suitable as encapsulation vectors for hydrophobic SAR analogs. Citation: August 2021 \| Volume 11 \| Article 702676 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Encapsulation to Augment Drug Bio-Availability Craig et al. Parasites To determine if a signiﬁcant proportion of C6 was not encapsulated, centrifugation experiments were performed alongside C6-loaded PFG30 aggregate samples in ﬂuorescence experiments. Samples for centrifugation were made as described above with aggregates prepared at a 1:10 w/w ratio in ﬁlter- sterilized diH2O at 37°C. Prior to serial dilution, the stock PFG30:C6 solution made at 50 mM was centrifuged at 10,000g in a microcentrifuge for 3 min at 37°C to produce a pellet. Supernatant was then collected from the pellet to remove any free C6 unassociated with the PFG30 pellet. The pellet was then resolubilized in an equal part of diH2O and chilled with gentle mixing. This solution was then reheated at 37°C for 5 min to encapsulate the remaining C6, and this solution and the supernatant were diluted in warm diH2O for a ﬁnal nominal concentration of 50 mM. Both sample pellets and the respective supernatants were then diluted to a nominal concentration of 5 mM for ﬂuorescence measurements and compared with the non-centrifuged 5 mM PFG30-C6 sample to estimate the efﬁciency of the PFG30 encapsulation procedure. ™ Promastigotes of L. amazonensis strain RAT/BA/74/LV78 (LV78) obtained from Dr. Lynn Soong (UTMB, TX) were maintained at 26°C in Schneider’s Drosophila medium supplemented with 10% heat-inactivated FBS and 10 mg/ml Gentamycin. Promastigotes of L. donovani [MHOM/SD/62/1S- C12D (SD)] obtained from Dr. Hira L. Nakhasi laboratory (FDA, MD) was maintained at 26°C in M199 medium supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin. Materials Preparation and Stock Solutions Retro-2, DHQZ 36, and DHQZ 36.1 were synthesized by Dr. Jason Sello at Brown University as described previously (Carney et al., 2014; Craig et al., 2017). Stock solutions were prepared in DMSO at concentrations of 20 or 30 mM. Retro-2 was also purchased from Sigma-Aldrich (CAS: 1429192-00-6) and prepared in DMSO, as well as at a concentration of 20 mM. Miltefosine was purchased from Sigma-Aldridge (catalog m5571), and a stock solution was prepared in ﬁltered NanoPure diH2O at a concentration of 10 mM. Coumarin 6 (C6) was purchased from Sigma-Aldrich (catalog 546283) and a stock solution was made in DMSO at a 5.3-mM solution. The tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] Cell Viability Assay Kit was purchased from Biotium (Fremont, CA). Both sets of samples were then read on a BioTek Cytation™ 5 microplate reader with excitation and emission wavelengths set to 420 and 500 nm, respectively. INTRODUCTION They are soluble in organic solvents, while their solubility in water dramatically depends on the temperature. In particular, below a critical temperature (deﬁned as low critical solution temperature, LCST), they form water-soluble nanosized micelles, which are able to encapsulate hydrophobic guest molecules in their hydrophobic core (Almeida et al., 2012). When the temperature exceeds the LCST, hydrogen-bonding interactions between hydrophilic micelle shell and water molecules weaken, and those between hydrophobic moieties become dominant leading to a collapse of polymer chains. The resulting intermicellar aggregates can aptly be used as intracellular drug delivery vectors (Sponchioni et al., 2019). Recently, it was shown that an amphiphilic statistical copolymer based on oligo(ethylene glycol) methacrylate (OEGMA, Mn = 300 g/mol) and 30%mol pentaﬂuorostyrene (PFS) (known as PFG30) forms micelles of about 10 nm at room temperature, which self-assemble into 200-nm-sized intermicellar nanoaggregates at temperatures greater than 30°C (Zuppardi et al., 2017). Another study showed that PFG copolymers of different molecular weights were able to efﬁciently encapsulate and remove a hydrophobic dye, Nile Red, from aqueous solutions by up to 90% removal (Zuppardi et al., 2020). Based on these results, this study evaluates the potential application of PFG30 for the delivery of Retro-2 and its August 2021 \| Volume 11 \| Article 702676 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 2 Encapsulation to Augment Drug Bio-Availability Craig et al. SAR analogs, DHQZ 36 and DHQZ 36.1, to Leishmania- infected cells. SAR analogs, DHQZ 36 and DHQZ 36.1, to Leishmania- infected cells. of any PFG30 precipitate due to DMSO addition, and then heated at 37°C for 5 min to induce polymer self-assembly and C6 encapsulation. Additional warm (37°C) diH2O or incomplete DMEM was added for a ﬁnal volume of 1 ml at a C6 concentration of 20 or 50 mM, respectively. This solution was then serially diluted in warm diH2O or incomplete DMEM to produce lower C6-loaded PFG30 aggregate concentrations as described in the text. Cell Lines For ﬂuorescence or NanoSight experiments, additional C6- free vehicle controls were prepared with DMSO with or without PFG30 encapsulation procedures to account for any signal due to clouding of the solution by polymer or DMSO alone. Both C6 and PFG30-C6 samples had their respective vehicle control ﬂuorescence readings subtracted out to control for any vehicle signal. Parasites Vehicle controls with equal portions of DMSO and PFG30 were used to correct for background signal caused through clouding of PFG30 polymers. These values were subtracted out of their respective C6 or PFG30-C6 measurements. Statistically signiﬁcant changes in ﬂuorescence were calculated through way ANOVA with multiple comparisons using the Holm-Šı́dák two method. PFG30 was prepared via radical polymerization under nitrogen in DMF as described previously (Zuppardi et al., 2017). PFG30 was dried via nitrogen until weight remained constant into a gel-like solid and stored at 4°C. Stock solutions were prepared in sterile-ﬁltered, NanoPure diH2O at concentrations of 10 mg/ml via gentle mixing in an Eppendorf tube at room temperature overnight. The 10-mg/ml stocks were then stored at 4°C prior to mixing with drugs or C6. MATERIALS AND METHODS Cell Lines RAW264.7 murine macrophages were obtained from ATCC (ATCC TIB-71, ATCC, USA) and maintained in Dulbecco’s Modiﬁed Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 mg/ml streptomycin- penicillin. They were cultured in a humidiﬁed atmosphere at 37°C with 5% CO2. Centrifugation Assay for the Estimation of Drug Uptake by PFG30 Aggregates As described above, PFG30 encapsulated Retro-2 or miltefosine were prepared at 100 mM or 10 mM concentrations, respectively, in warm, complete DMEM. A second set of encapsulated Retro-2 or miltefosine was subjected to centrifugation to form a pellet prior to dilution in DMEM, and the supernatant was discarded. This mixture was re-encapsulated as described above and diluted in warm, complete DMEM prior to treatment of L. amazonensis- infected macrophages in tandem with non-centrifuged samples for comparison. To determine if C6 was taken up into the intermicellar aggregates of PFG30, we used NanoSight to visualize and measure PFG30 particles with or without C6. DMSO vehicle or C6 were encapsulated in PFG30 aggregates as described above and diluted in incomplete DMEM without supplementation of 10% FBS because of the high amounts of particles from FBS. After preparation of samples, a dilution was prepared at 1:50 and then run for Nanoparticle Tracking Analysis (NTA, Malvern NanoSight NS300) at 37°C for particle size and concentration at 60 s per replicate. A second 1:50 dilution was then prepared and re-measured with a 500 nm green ﬁlter to measure the fraction of particles positive for containing C6. At least two replicates were run in these experiments. RAW264.7 macrophages were plated and infected with L. amazonensis at a 20:1 MOI as described previously. Coverslips were then treated in duplicate with Retro-2 or Miltefosine alone, PFG30-encapsulated Retro-2 or Miltefosine, or centrifugation products of PFG30-encapsulated Retro-2 or Miltefosine for 48 h at 34°C with 5% CO2. Cells were then ﬁxed with 2% PFA in PBS and IFA stained for LAMP-1 and DAPI for estimation of infection rates as described above. Statistical signiﬁcance was measured by one-way ANOVA and the Holm-Šı́dák method for multiple comparisons. PFG30 Encapsulation Procedure These centrifuged samples were run in tandem as described below with non-centrifuged samples for comparison of PFG30 loading efﬁciency between the hydrophobic compound, Retro-2, and the hydrophilic compound, miltefosine. The pellet was then resolubilized in an equivalent volume of complete DMEM as described above to make a nominal 50 or 10 µM working solution of Retro-2 or miltefosine, respectively. These centrifuged samples were run in tandem as described below with non-centrifuged samples for comparison of PFG30 loading efﬁciency between the hydrophobic compound, Retro-2, and the hydrophilic compound, miltefosine. Nanoparticle Size Characterization and Tracking Analysis The self-assembly behavior of PFG30 was investigated by dynamic light scattering (DLS), performed with a Malvern Zetasizer Nano ZS instrument (Cambridge, UK) equipped with a 4-mV HeNe laser operating at l = 633 nm, with a measurement angle of 173°. The measurements were carried out at 20°C and 37°C on a solution made up of 3.4 µl of a 30-mM Retro-2 stock in DMSO, 32 µl of a 10-mg/ml PFG30 stock, and 964.6 ml incomplete DMEM. PFG30 Encapsulation Procedure p DHQZ analog-loaded PFG30 nanoparticles were prepared by mixing 5 µl of a 20-mM Retro-2, DHQZ 36 or DHQZ 36.1 stock with 32 to 45 µl of a 10-mg/ml PFG30 stock (at a 1:10 w/w drug: polymer ratio). This mixture was cooled at 4°C to remove any PFG30 precipitate and then heated at 37°C for 5 min to induce polymer self-assembly and drug encapsulation. This solution was then raised to a 1-ml volume with warm, complete DMEM to produce a 100-mM DHQZ drug solution loaded into PFG30 aggregates. Modeling Drug Uptake Into PFG30 With C6 C6 solutions were prepared at concentrations between 0.5 and 50 mM in ﬁlter-sterilized, NanoPure diH2O or incomplete DMEM. C6-loaded PFG30 aggregates were prepared by mixing volumes of a 5.3-mM C6 in DMSO stock solution with a 10-mg/ml PFG30 stock (at a 1:10 w/w C6:polymer ratio) to produce a 20- or 50-mM working PFG30 encapsulated C6 stock solution. This mixture was cooled at 4°C to allow solubilization When noted, additional PFG30 aggregates housing Retro-2 or miltefosine were sedimented down as described above to pellet PFG30 capsules, and the supernatant was removed by pipette. August 2021 \| Volume 11 \| Article 702676 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 3 Encapsulation to Augment Drug Bio-Availability Craig et al. previously (Craig et al., 2017). IFAs were visualized and captured using a QImaging Retiga 1300C cooled CCD camera mounted on an Olympus BX50 microscope equipped with automated ﬁlters with 100× NA 1.30 oil-immersion objective. Scored LPVs were delimited by LAMP-1 reactivity and contained at least one parasite nucleus visualized by 4′,6-diamidino-2-phenylindole (DAPI) stain. The percentage of infected macrophages and the average number of parasites was determined by counting at least 100 macrophages per coverslip. Counts were done in duplicate over at least three experiments and EC50 values were determined in GraphPad Prism 8 using a four-parameter dose-response best-ﬁt curve line. Statistical signiﬁcance between treated infected cells, and the control was measured using a one-way ANOVA in GraphPad Prism 8, and multiple comparisons were measured for statistical signiﬁcance using the Holm-Šı́dák method. The pellet was then resolubilized in an equivalent volume of complete DMEM as described above to make a nominal 50 or 10 µM working solution of Retro-2 or miltefosine, respectively. Immunoﬂuorescence Assay for Drug Susceptibility RAW264.7 macrophages were plated in 100-mm dishes over glass coverslips at 1 × 106 cells/dish. After 24 h incubation, cells were infected with 96 h stationary phase L. amazonensis or 96 h PNA-selected, metacyclic L. donovani promastigotes at a 20:1 MOI for 4 h prior to two phosphate-buffered saline (PBS) washes to remove external parasites. Infected macrophages were incubated at 34°C or 37°C, respectively, with 5% CO2. Quantitative Uptake of C6-Loaded PFG30 Aggregates Into RAW264.7 Macrophages To assess the cellular uptake of PFG30 aggregates, RAW264.7 cells were seeded on round, glass coverslips at a density of 4.5 × 104 cells in 24-well plates. On the second day, each well was incubated at 37°C for 0, 30, 60, and 180 min, with 500 ml of a solution made up of 1.7 µl of a 30-mM C6 stock in DMSO, 16 µL of a 10-mg/ml PFG30 stock, and 482.3 ml incomplete DMEM. Post-treatment at the desired time points, cells were washed twice with PBS and ﬁxed (3.7% formaldehyde in PBS) for 15 min at room temperature. Coverslips were mounted onto microscope slides using ProLong Gold Antifade reagent with DAPI (Thermo Fisher Scientiﬁc, Milan, Italy). Estimation of Retro-2 Incorporation Into RAW264.7 Macrophages by Mass Spectrometry Estimation of PFG30 Cytotoxicity RAW264.7 macrophages were seeded at 1 × 104 cells/well in 96- well plates in complete DMEM at 37°C with 5% CO2 for 2 to 4 h to allow adherence, but not replication. Cells were then treated with ﬁlter-sterilized, NanoPure diH2O vehicle or PFG30 at concentrations ranging from 450 to 1 mg/ml for 48 h prior to MTT assay. After treatment, cells were incubated an additional 4 h with MTT before addition of 10% SDS in PBS with 5 N HCl to dissolve formazan crystals overnight. Absorbances were read at 570 nm with a 630 nm background subtracted out. Cell numbers were estimated based on a standard curve and statistical signiﬁcance was measured via one-way ANOVA with multiple comparisons using the Holm-Šı́dák method. RAW264.7 macrophages were plated in six-well plates at 1 × 106 cells/well overnight and infected with stationary stage L. amazonensis promastigotes as described previously. Cells were then treated 24 h-post infection with 5 or 50 mM free Retro-2 or PFG30-encapuslated Retro-2 in warm, complete DMEM. Treatments were performed at 34°C from 4 to 12 h before preparations of cell lysates with RIPA Buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% NP-40, 0.5% deoxycholate) supplemented with 1 mM PMSF and protease cocktail inhibitor, purchased from Sigma Aldrich, prior to use. Lysates were sonicated for 15 s prior to centrifugation to remove cellular debris. Supernatants and cell lysates were frozen at −80°C until ready for analysis. PFG30 Enhances C6 Solubility Through Efﬁcient Uptake Into Nanoaggregates Efﬁcient Uptake Into Nanoaggregates Previous studies had shown that Retro-2 and two SAR analogs, DHQZ 36 and DHQZ 36.1 are effective compounds against L. amazonensis and L. donovani infections (Canton and Kima, 2012; Craig et al., 2017; Gupta et al., 2017). However, these compounds are hydrophobic with low solubility in aqueous solutions. It is, therefore, likely that the efﬁcacy of these compounds could be further enhanced by increasing their solubility through polymer-based encapsulation. Polymer encapsulation has been shown to increase hydrophobic drug solubility, drug delivery, and protection from degradation in vivo (Huh and Kwon, 2011; Costa Lima et al., 2012; Nafari et al., 2020). Previously, amphiphilic co-polymers made from OEGMA and 30% PFS were found to be responsive to temperatures greater than 30°C, leading to stable nanoaggregates with a hydrophobic interior (Lutz, 2011; Kwan and Marić, 2016; Welsch and Lyon, 2017). Therefore, it is expected that these nanoparticles may enhance solubility and bioavailability of hydrophobic Retro-2 and SAR analogs. Quantitative Uptake of C6-Loaded PFG30 Aggregates Into RAW264.7 Macrophages gg g p g To assess the cellular uptake of PFG30 aggregates, RAW264.7 cells were seeded on round, glass coverslips at a density of 4.5 × 104 cells in 24-well plates. On the second day, each well was incubated at 37°C for 0, 30, 60, and 180 min, with 500 ml of a solution made up of 1.7 µl of a 30-mM C6 stock in DMSO, 16 µL of a 10-mg/ml PFG30 stock, and 482.3 ml incomplete DMEM. Infected cells were treated with free DHQZ analogs or with PFG30 encapsulated Retro-2, DHQZ 36, or DHQZ 36.1 at a 1:10 w/w (drug:PFG30) ratio as described above. When noted, some samples were centrifuged and supernatant removed as described above prior to treatment. Treatments were performed for 48 h at concentrations ranging from 100 nM to 100 mM. Miltefosine, with or without PFG30 encapsulation, was used as a positive control for parasite clearance from concentrations ranging from 100 nM to 15 mM. Cells were incubated with free drugs or PFG30 encapsulated drugs at 34°C or 37°C for L. amazonensis or L. donovani infections, respectively, for 48 h. Coverslips were ﬁxed in 4% paraformaldehyde (PFA) in PBS for 1 h prior to storage at 4°C in PBS. Coverslips were then processed in immunoﬂuorescence assays (IFAs) to visualize the distribution of LAMP-1 as described Post-treatment at the desired time points, cells were washed twice with PBS and ﬁxed (3.7% formaldehyde in PBS) for 15 min at room temperature. Coverslips were mounted onto microscope slides using ProLong Gold Antifade reagent with DAPI (Thermo Fisher Scientiﬁc, Milan, Italy). Cell-associated ﬂuorescence was qualitatively and quantitatively detected by a Cytation 3 Cell Imaging Multi- Mode Reader ﬂuorescence microscope (Biotek) at 480 nm as previously reported (Calarco et al., 2013). August 2021 \| Volume 11 \| Article 702676 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Encapsulation to Augment Drug Bio-Availability Craig et al. RESULTS Mass spectrometry analysis of the cell lysates was performed on a Shimadzu Ultra-High-Performance Liquid Chromatography (Nexera XR LC 40), coupled to an MS/MS detector (LCMS 8060, Shimadzu), and controlled by Lab Solution software. The MS/MS was operated in positive electrospray ionization (ESI+), setting the nebulizing gas ﬂow at 3 L/min, heating gas ﬂow 10 L/min, interface temperature 370°C, DL temperature 250°C, heat block temperature 450°C, and drying gas ﬂow 10 L/min. Analyzed fragments of Retro- 2 (343.00 > 39.05; 343.00 > 23.15; 343.00 > 284.3) were selected by ﬂow injection analysis mass identiﬁcation, while quantiﬁcation was operated after chromatographic separation of acetonitrile treated lysates (1:1 v/v) on a Phenomenex Kinetex polar C18 column (3 × 100 mm, 2.6 mm, Phenomenex, USA) with mobile phase consisting of acetonitrile: water + 0.01% formic acid (70:30, v/v; isocratic). A bar graph with estimated internal Retro-2 concentrations was generated in GraphPad Prism 8 and statistical signiﬁcance measured via two-way ANOVA and the Tukey method for multiple comparisons. Measurement of Vacuole Size LPV size measurements were performed as described previously (Craig et al., 2017). Brieﬂy, RAW264.7 macrophages on coverslips were infected with stationary L. amazonensis promastigotes at 10:1 MOI for 4 h after which parasites were washed off and DHQZ analogs, PFG30 encapsulated DHQZ analogs or miltefosine were added for an additional 24 h. Infected macrophages were ﬁxed with 2% PFA in PBS then processed in IFAs to visualize the distribution of LAMP-1. IFAs were visualized and captured using a QImaging Retiga 1300C cooled CCD camera mounted on an Olympus BX50 microscope equipped with automated ﬁlters with 100× NA 1.30 oil-immersion objective. Scored LPVs were delimited by LAMP-1 reactivity and contained at least one parasite nucleus visualized by DAPI stain. Vacuole size was determined by measuring vacuole area via ImageJ. At least 30 vacuoles were measured per coverslip of which there were three per concentration per biological repeat. Statistical signiﬁcance between treated infected cells and the control was measured using a one-way ANOVA in GraphPad Prism 8 with the Dunnett test for multiple comparisons. In the ﬁrst series of experiments, the uptake of poorly soluble, hydrophobic molecules into PFG30 thermoresponsive aggregates was assessed. As Retro-2 is not ﬂuorescent and its UV absorption spectrum overlaps with that of PFG30, we evaluated PFG30’s ability to encapsulate and deliver the green laser dye, coumarin 6 (C6), to obtain further insight into its efﬁciency of encapsulating hydrophobic compounds (Zuppardi et al., 2020). The ﬂuorescence of C6 is low in polar solvents bcause of its poor solubility; however, its ﬂuorescence can be restored by using amphiphilic hosts that have hydrophobic cavities (Banerjee et al., 2016). The solubility of C6 in water with or without PFG30 encapsulation was measured through changes in the ﬂuorescent proﬁle. Fluorescence of 5 and 10 µM C6 in the presence of PFG30 was signiﬁcantly greater at RFU values of 24,381 and 76,430, respectively, than water-dispersed C6, which barely ﬂuoresced at 810 and 1,319 RFU, respectively (Figure 1A). The experiment was stopped at 10 µM because of the values reaching overﬂow in PFG30 encapsulated C6 samples. August 2021 \| Volume 11 \| Article 702676 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Encapsulation to Augment Drug Bio-Availability Craig et al. A B FIGURE 1 \| Encapsulation of hydrophobic molecules in PFG30 increases their solubility. (A) Increasing concentrations of C6 alone or PFG30 + C6 was measured at 420 nm excitation and 500 nm emission. Measurement of Vacuole Size (B) 50 µM PFG30-C6 preparations were centrifuged after which the supernatant ﬂuid and pellets were diluted to 5 µM for measurement of ﬂuorescence at 420/500 nm. Experiments were run in triplicate with at least two technical replicates each. Statistical signiﬁcance was determined by two-way ANOVA in GraphPad Prism 8 with the Holm-Šídák posthoc test for multiple comparisons (*p-value < 0.001). A FIGURE 1 \| Encapsulation of hydrophobic molecules in PFG30 increases their solubility. (A) Increasing concentrations of C6 alone or PFG30 + C6 was measured at 420 nm excitation and 500 nm emission. (B) 50 µM PFG30-C6 preparations were centrifuged after which the supernatant ﬂuid and pellets were diluted to 5 µM for measurement of ﬂuorescence at 420/500 nm. Experiments were run in triplicate with at least two technical replicates each. Statistical signiﬁcance was determined by two-way ANOVA in GraphPad Prism 8 with the Holm-Šídák posthoc test for multiple comparisons (p-value < 0.001). To determine if increased ﬂuorescence of C6 was due to PFG30 encapsulation, we elected to separate free C6 in solution from encapsulated C6 via centrifugation. After removal of the supernatant and resuspension of the pellet, there was no signiﬁcant drop in the ﬂuorescence of the pellet of the 5 µM sample at 31,888 RFU compared to the control 5 µM sample (26,160 RFU) without centrifugation. In addition, the supernatant contained very little C6 with a ﬂuorescent value of only 189 RFU (Figure 1B). the number of green ﬂuorescent particles in PFG30 encapsulated C6 samples as compared with PFG30 or C6 alone. These results suggests that C6 is encapsulated within PFG30 aggregates. Considering the observations on the encapsulation of C6, the encapsulation of Retro-2 and its SAR derivatives, DHQZ 36 and DHQZ 36.1 were evaluated. 50 mM Retro-2, DHQZ 36 or DHQZ 36.1 mixed with C6 were encapsulated by PFG30 at a 1:10 w/w ratio. A 1:50 dilution in incomplete DMEM was prepared for evaluation by NanoSight. Mixing of C6, DHQZ analogs, and PFG30 resulted in an increase in the size of the particles that were formed (Figure 2A). There was also an increase in the number of PFG30 aggregates that were formed (Figure 2B). Interestingly, PFG30 particles formed upon addition of DHQZ 36 and DHQZ 36.1, were smaller but more numerous (Figures 2A, B). Measurement of Vacuole Size Retro-2 and the two DHQZ analogs mixed with C6 had a similar increase in the concentrations of green-positive particles as compared to PFG30 encapsulation of C6 alone, suggesting that the addition of drug did not affect C6 loading into PFG30 aggregates (Figure 2C). Because of the increase in C6 solubility and association with the PFG30 pellet in centrifugation experiments, DLS was used to assess whether PFG30 was able to retain its thermally triggered self-assembly properties in DMEM solution, as well as in the presence of the hydrophobic compound Retro-2. To that end, the size distribution of a PFG30 solution containing Retro-2 in DMEM was measured by DLS at 20°C and 37°C. At 20°C, polymer micelles having average hydrodynamic diameter of 10 nm were detected, with monomodal distribution and low polydispersity. When the temperature was raised to 37°C, the particle size increased up to about 200 nm, due to formation of intermicellar aggregates, demonstrating that Retro-2 did not affect the self-assembly behavior of PFG30 (Supplemental Figure 1). Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org PFG30 Enhances Efﬁcacy of SAR DHQZ Analogs of Retro-2 Against L. donovani and L. amazonensis Infections of RAW264.7 Macrophages To further afﬁrm that the increased solubility of C6 was indeed due to encapsulation of C6 into PFG30, the sizes of PFG30 intermicellar aggregates, with or without C6, were assessed by NanoSight. PFG30 aggregates without C6 were about 200 nm in size. Encapsulation of C6 with PFG30 resulted in increased particle sizes ranging from 200 to 400 nm in size. The incomplete DMEM vehicle included a small population of particles. However, they were distinctly different from PFG30 nanoparticles with sizes less than 100 nm (Figure 2A). Furthermore, the addition of PFG30 signiﬁcantly increased the magnitude of particles compared to incomplete DMEM or incomplete DMEM with free C6 (Figure 2B). Using a 500 nm wavelength ﬁlter for green ﬂuorescence in the NanoSight, we found that there was a signiﬁcant increase in We commenced these studies by assessing whether Retro-2 efﬁcacy against intracellular Leishmania amastigotes would be improved by encapsulation in PFG30 polymeric nanoparticles. RAW264.7 macrophages plated on coverslips in six-well plates were infected with L. donovani parasites for 24 h. Thereafter, several concentrations of Retro-2, DHQZ 36, or DHQZ 36.1 encapsulated in PFG30 were added to infections. After an additional 48 h, the infections were ﬁxed by adding 2% PFA. Coverslips with infected cells were then processed by immunoﬂuorescence labeling to detect the contours of the LPV and to visualize parasite nuclei. After enumerating the number of cells harboring intracellular Leishmania, the data were August 2021 \| Volume 11 \| Article 702676 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 6 Craig et al. Encapsulation to Augment Drug Bio-Availability A B C FIGURE 2 \| NanoSight measurements of PFG30 encapsulated compounds. The size and number of particles produced after encapsulation in PFG30 were analyzed as follows: C6 alone (20 mM); PFG30 alone; PFG30 + C6 (20 mM); PFG30 + C6 + Retro-2 (50 mM R2); PFG30 + C6 + DHQZ 36 (50 mM); PFG30 + C6 + DHQZ 36.1 (50 mM). Encapsulation mixtures were prepared at 1:50 dilution for NanoSight analysis. (A) The size measurements of polymer aggregates with or without the use of a 500 nm green ﬁlter for ﬂuorescence. (B) The number of particles within the samples with or without the use of a 500 nm green ﬁlter for ﬂuorescence. PFG30 Enhances Efﬁcacy of SAR DHQZ Analogs of Retro-2 Against L. donovani and L. amazonensis Infections of RAW264.7 Macrophages (C) Particles observed in samples using a 500-nm green ﬁlter were compared with the total particles in the sample observed without the ﬁlter to generate a percentage estimation of C6 uptake within PFG30 aggregates. Data were obtained from at least two biological replicates. A B C FIGURE 2 \| NanoSight measurements of PFG30 encapsulated compounds. The size and number of particles produced after encapsulation in PFG30 were analyzed as follows: C6 alone (20 mM); PFG30 alone; PFG30 + C6 (20 mM); PFG30 + C6 + Retro-2 (50 mM R2); PFG30 + C6 + DHQZ 36 (50 mM); PFG30 + C6 + DHQZ 36.1 (50 mM). Encapsulation mixtures were prepared at 1:50 dilution for NanoSight analysis. (A) The size measurements of polymer aggregates with or without the use of a 500 nm green ﬁlter for ﬂuorescence. (B) The number of particles within the samples with or without the use of a 500 nm green ﬁlter for ﬂuorescence. (C) Particles observed in samples using a 500-nm green ﬁlter were compared with the total particles in the sample observed without the ﬁlter to generate a percentage estimation of C6 uptake within PFG30 aggregates. Data were obtained from at least two biological replicates. A B C FIGURE 3 \| Evaluation of polymer-encapsulated compounds on L. donovani-infected RAW264.7 macrophages. L. donovani-infected macrophages were treated for 48 h with the following compounds: (A) free Retro-2 or PFG30 + Retro-2 (PFG30-R2); (B) free DHQZ 36 or PFG30 + DHQZ 36 (PFG30-D36); (C) free DHQZ 36.1 or PFG30 + DHQZ 36.1 (PFG30-D36.1) at concentrations ranging from 100 nM to 100 mM. Miltefosine (100 nM to 15 mM) treatment was used as a positive control for parasite clearance. Cells were ﬁxed in 2% PFA in PBS. IFAs were performed for detection of LAMP-1 and cell and parasite nuclei with DAPI. At least 200 cells were scored per coverslip and infection rates were standardized to a vehicle control before EC50 estimation. Data were compiled from at least three biological repeats. A B B C C FIGURE 3 \| Evaluation of polymer-encapsulated compounds on L. donovani-infected RAW264.7 macrophages. L. donovani-infected macrophages were treated for 48 h with the following compounds: (A) free Retro-2 or PFG30 + Retro-2 (PFG30-R2); (B) free DHQZ 36 or PFG30 + DHQZ 36 (PFG30-D36); (C) free DHQZ 36.1 or PFG30 + DHQZ 36.1 (PFG30-D36.1) at concentrations ranging from 100 nM to 100 mM. PFG30 Enhances Efﬁcacy of SAR DHQZ Analogs of Retro-2 Against L. donovani and L. amazonensis Infections of RAW264.7 Macrophages Miltefosine (100 nM to 15 mM) treatment was used as a positive control for parasite clearance. Cells were ﬁxed in 2% PFA in PBS. IFAs were performed for detection of LAMP-1 and cell and parasite nuclei with DAPI. At least 200 cells were scored per coverslip and infection rates were standardized to a vehicle control before EC50 estimation. Data were compiled from at least three biological repeats. August 2021 \| Volume 11 \| Article 702676 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Encapsulation to Augment Drug Bio-Availability Craig et al. plotted (Figure 3). PFG30 encapsulation of Retro-2 signiﬁcantly augmented the EC50 value calculated at 4.46 ± 0.94 mM compared to unencapsulated Retro-2 at 22.73 ± 3.44 mM. Due to the increased efﬁcacy observed of Retro-2 encapsulated in PFG30 to clear L. donovani infections, we proceeded to test the efﬁcacy of DHQZ 36 and DHQZ 36.1 encapsulated in PFG30. After processing in immunoﬂuorescence assays and enumeration of infected macrophages, we determined that DHQZ 36 encapsulated in PFG30 had an EC50 value of 1.07 ± 0.98 mM (Figure 3B). Encapsulated DHQZ 36.1 had an EC50 value of 0.27 ± 0.09 mM, (Figure 3C). Table 1 shows the EC50 of DHQZ 36 and DHQZ 36.1 without encapsulation. The efﬁcacy of DHQZ compounds and encapsulated formulations were run alongside miltefosine. The EC50 of miltefosine in these experiments was determined to be 1.96 ± 0.38 mM. As shown in Table 1, the EC50 values of the PFG30 encapsulated compounds on L. donovani infections were much improved and were effective at lower concentrations than miltefosine. We then proceeded to test the efﬁcacy of the encapsulated compounds just described, on L. amazonensis infections. We wanted to determine if using the same methodology for PFG30 encapsulation would improve the efﬁcacy Retro-2 and the DHQZ analogs on L. amazonensis infections. The drug killing curves that were generated for the studies on L. amazonensis infections are shown in Figure 4. From these curves we calculated the EC50 for PFG30 encapsulated Retro-2 to be 26.15 ± 2.46 mM; the EC50 of encapsulated DHQZ 36 was 2.42 ± 2.12 mM; the EC50 of PFG30 encapsulated DHQZ 36.1 was 0.36 ± 0.13 mM. These results are presented in Table 2 alongside the EC50 values of these compounds without encapsulation. PFG30 Enhances Efﬁcacy of SAR DHQZ Analogs of Retro-2 Against L. donovani and L. amazonensis Infections of RAW264.7 Macrophages Statistical signiﬁcance was measured between LogEC50 values between DHQZ compounds and encapsulated DHQZ compounds though the Extra Sum-of-Squares F test (p-value < 0.001). TABLE 1 \| Estimation of the efﬁcacy of PFG30 encapsulated DHQZ analogs on L. donovani infections. We proceeded to rule out the possibility that the increase in efﬁcacy of the encapsulated compounds was not due to toxicity of PFG30 on RAW264.7 macrophages, which may lead to stress of the parasites during infection. This was determined by using a MTT assay to measure RAW264.7 cell viability with PFG30 treatment over 48 h. In this experiment, we included much higher concentrations of PFG30 along with a ﬁlter-sterilized, NanoPure diH2O vehicle control. No signiﬁcant loss in cell viability was observed at 48 h treatments of RAW264.7 EC50 values were calculated for the plots in Figure 3 generated in GraphPad Prism 8. Statistical signiﬁcance was measured between LogEC50 values between DHQZ compounds and encapsulated DHQZ compounds though the Extra Sum-of-Squares F test (p-value < 0.001). EC50 values were calculated for the plots in Figure 3 generated in GraphPad Prism 8. Statistical signiﬁcance was measured between LogEC50 values between DHQZ compounds and encapsulated DHQZ compounds though the Extra Sum-of-Squares F test (p-value < 0.001). B A B C FIGURE 4 \| Evaluation of polymer-encapsulated compounds on L. amazonensis-infected RAW264.7 macrophages. To L. amazonensis-infected RAW264.7 macrophages, the following drug/polymer mixtures were added then incubated for an additional 48 h. (A) Retro-2 in DMSO or Retro-2 + PFG30; (B) DHQZ 36 in DMSO or DHQZ 36 + PFG30; (C) DHQZ 36.1 in DMSO or DHQZ 36.1 + PFG30 at concentrations of 100 nM to 100 mM. Miltefosine (100 nM to 15 mM) treatment served as a positive control for parasite clearance. Cells were ﬁxed in 2% PFA in PBS. IFAs were performed for detection of LAMP-1 and cell and parasite nuclei with DAPI. At least 200 cells were scored per coverslip and infection rates were standardized to a vehicle control before EC50 estimation. Data were compiled from at least three biological repeats. A B B C FIGURE 4 \| Evaluation of polymer-encapsulated compounds on L. amazonensis-infected RAW264.7 macrophages. To L. amazonensis-infected RAW264.7 macrophages, the following drug/polymer mixtures were added then incubated for an additional 48 h. PFG30 Enhances Efﬁcacy of SAR DHQZ Analogs of Retro-2 Against L. donovani and L. amazonensis Infections of RAW264.7 Macrophages Supplemental Figure 2 and Table 2 show that encapsulation of miltefosine, using the encapsulation protocol described above, did not result in a signiﬁcant change in the EC50 of miltefosine. The EC50 of miltefosine on L. amazonensis infections is 2.50 ± 0.37 mM. TABLE 1 \| Estimation of the efﬁcacy of PFG30 encapsulated DHQZ analogs on L. donovani infections. Treatment EC50 (mM) PFG30 (−) PFG30 (+) Retro-2 22.73 ± 3.44 4.46 ± 0.94* DHQZ 36 11.70 ± 0.33 1.07 ± 0.98* DHQZ 36.1 1.80 ± 0.20 0.27 ± 0.09* Miltefosine 1.96 ± 0.38 R2, Retro-2; D36, DHQZ 36; D36.1, DHQZ 36.1. EC50 values were calculated for the plots in Figure 3 generated in GraphPad Prism 8. Statistical signiﬁcance was measured between LogEC50 values between DHQZ compounds and encapsulated DHQZ compounds though the Extra Sum-of-Squares F test (*p-value < 0.001). A C FIGURE 4 \| Evaluation of polymer-encapsulated compounds on L. amazonensis macrophages, the following drug/polymer mixtures were added then incubated fo DMSO or DHQZ 36 + PFG30; (C) DHQZ 36.1 in DMSO or DHQZ 36.1 + PFG30 served as a positive control for parasite clearance. Cells were ﬁxed in 2% PFA in DAPI. At least 200 cells were scored per coverslip and infection rates were stand least three biological repeats. TABLE 1 \| Estimation of the efﬁcacy of PFG30 encapsulated DHQZ analogs on L. donovani infections. Treatment EC50 (mM) PFG30 (−) PFG30 (+) Retro-2 22.73 ± 3.44 4.46 ± 0.94* DHQZ 36 11.70 ± 0.33 1.07 ± 0.98* DHQZ 36.1 1.80 ± 0.20 0.27 ± 0.09* Miltefosine 1.96 ± 0.38 R2, Retro-2; D36, DHQZ 36; D36.1, DHQZ 36.1. EC50 values were calculated for the plots in Figure 3 generated in GraphPad Prism 8. Statistical signiﬁcance was measured between LogEC50 values between DHQZ compounds and encapsulated DHQZ compounds though the Extra Sum-of-Squares F test (*p-value < 0.001). TABLE 1 \| Estimation of the efﬁcacy of PFG30 encapsulated DHQZ analogs on L. donovani infections. Treatment EC50 (mM) PFG30 (−) PFG30 (+) Retro-2 22.73 ± 3.44 4.46 ± 0.94* DHQZ 36 11.70 ± 0.33 1.07 ± 0.98* DHQZ 36.1 1.80 ± 0.20 0.27 ± 0.09* Miltefosine 1.96 ± 0.38 R2, Retro-2; D36, DHQZ 36; D36.1, DHQZ 36.1. EC50 values were calculated for the plots in Figure 3 generated in GraphPad Prism 8. PFG30 Enhances Efﬁcacy of SAR DHQZ Analogs of Retro-2 Against L. donovani and L. amazonensis Infections of RAW264.7 Macrophages (A) Retro-2 in DMSO or Retro-2 + PFG30; (B) DHQZ 36 in DMSO or DHQZ 36 + PFG30; (C) DHQZ 36.1 in DMSO or DHQZ 36.1 + PFG30 at concentrations of 100 nM to 100 mM. Miltefosine (100 nM to 15 mM) treatment served as a positive control for parasite clearance. Cells were ﬁxed in 2% PFA in PBS. IFAs were performed for detection of LAMP-1 and cell and parasite nuclei with DAPI. At least 200 cells were scored per coverslip and infection rates were standardized to a vehicle control before EC50 estimation. Data were compiled from at least three biological repeats. FIGURE 4 \| Evaluation of polymer-encapsulated compounds on L. amazonensis-infected RAW264.7 macrophages. To L. amazonensis-infected RAW264.7 macrophages, the following drug/polymer mixtures were added then incubated for an additional 48 h. (A) Retro-2 in DMSO or Retro-2 + PFG30; (B) DHQZ 36 in DMSO or DHQZ 36 + PFG30; (C) DHQZ 36.1 in DMSO or DHQZ 36.1 + PFG30 at concentrations of 100 nM to 100 mM. Miltefosine (100 nM to 15 mM) treatment served as a positive control for parasite clearance. Cells were ﬁxed in 2% PFA in PBS. IFAs were performed for detection of LAMP-1 and cell and parasite nuclei with DAPI. At least 200 cells were scored per coverslip and infection rates were standardized to a vehicle control before EC50 estimation. Data were compiled from at least three biological repeats. August 2021 \| Volume 11 \| Article 702676 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Encapsulation to Augment Drug Bio-Availability Craig et al. TABLE 2 \| Estimated efﬁcacy on L. amazonensis infections of Retro-2 and DHQZ analogs with or without PFG30. Treatment EC50 (mM) PFG30 (−) PFG30 (+) Retro-2 50.29 ± 5.91 26.15 ± 2.46* DHQZ 36 18.00 ± 2.54 2.42 ± 2.12* DHQZ 36.1 13.05 ± 0.49 0.36 ± 0.13* Miltefosine 2.23 ± 0.27 2.11 ± 0.26 EC50 values were calculated from ﬁtted-line dose-response curves in Figure 4 of R2, Retro-2; D36, DHQZ 36; D36.1, DHQZ 36.1 Plots and analysis were performed in GraphPad Prism7. Statistical signiﬁcance was measured between LogEC50 values between Retro-2 and encapsulated Retro-2 though the Extra Sum-of-Squares F test (p-value < 0.001). TABLE 2 \| Estimated efﬁcacy on L. amazonensis infections of Retro-2 and DHQZ analogs with or without PFG30. PFG30 Enhances Efﬁcacy of SAR DHQZ Analogs of Retro-2 Against L. donovani and L. amazonensis Infections of RAW264.7 Macrophages of supernatant from the PFG30 pellet resulted in signiﬁcant loss of efﬁcacy even at 10 mM (Figure 5B), likely because of the hydrophilicity of the compound. Because of the results with C6 solubility and centrifugation experiments, it is likely that Retro-2 SAR analogs are also solubilized and packaged into PFG30 nanoaggregates. Therefore, PFG30 encapsulation and uptake could facilitate Retro-2 entry into infected cells leading to the observed increase in efﬁcacy. To ﬁrst estimate PFG30 uptake into cells, RAW264.7 macrophages were treated with PFG30 encapsulated C6 up to 180 min prior to ﬁxation. These cells were then stained with DAPI and visualized via ﬂuorescence microscopy to assess PFG30 uptake by macrophages. The intracellular C6-associated ﬂuorescence was visualized as early as 30 min post-treatment and increased gradually until 180 min (Figures 6A–I). The increasing amount of C6 uptake into cells was conﬁrmed by ﬂuorescence quantiﬁcation (Figure 6J). Additional experiments related to C6-loaded PFG30 uptake were performed on mouse ﬁbroblast cell line (L929). The result clearly demonstrated that PFG30 nanoparticles efﬁciently enter the cells, suggesting that the PFG30 cellular uptake is not related to the phagocytic nature of the cell (Supplemental Figure S4). EC50 values were calculated from ﬁtted-line dose-response curves in Figure 4 of R2, Retro-2; D36, DHQZ 36; D36.1, DHQZ 36.1 Plots and analysis were performed in GraphPad Prism7. Statistical signiﬁcance was measured between LogEC50 values between Retro-2 and encapsulated Retro-2 though the Extra Sum-of-Squares F test (p-value < 0.001). macrophages, even at 1 mg/ml concentrations of PFG30 (Supplemental Figure 3). macrophages, even at 1 mg/ml concentrations of PFG30 (Supplemental Figure 3). Retro-2 SAR Analogs Are Efﬁciently Packaged in PFG30 Aggregates and Delivered Into Macrophages In Vitro While PFG30 encapsulation experiments against L. donovani and L. amazonensis infections indicate improved efﬁcacy, it is uncertain how much DHQZ analogs are retained in the PFG30 polymer capsules and how much drug is excluded. Therefore, we reasoned that due to the physical aggregation of the capsules, these could be centrifuged into a pellet allowing for the removal of the supernatant ﬂuid containing any free drug unassociated with the polymers. Once free drug was removed, the remaining drug associated with the polymer could be tested against L. amazonensis infections and potential loss of efﬁcacy determined. We observed that with 10,000g centrifugation at 37°C, pelleted Retro-2 encapsulated in PFG30 had no signiﬁcant loss in efﬁcacy after supernatant was discarded (Figure 5A). In contrast, centrifugation of PFG30 encapsulated miltefosine and removal Based on these results, the concentration of Retro-2 uptaken by cells after PFG30 encapsulation, was then evaluated by LC/MS (Figure 6K). Infected cells were treated with various amounts of PFG30 encapsulated Retro-2 for 4 and 12 h before lysis by the addition of RIPA buffer and sonication. Early timepoints were used to limit potential drug loss from drug turnover within cellular compartments, as this compound is relatively stable in acidic conditions (Gandhi et al., 2019). Mass spectrometry analysis of the cell lysates found that levels of Retro-2 treatments of 5 and 50 µM in macrophage lysates were detectable at 4 h post-treatment with cellular uptake at 0.14 ± 0.04 µM and 3.83 ± 1.15 µM, respectively. These concentrations A B FIGURE 5 \| Evaluation of PFG30 encapsulation efﬁciency of Retro-2 and miltefosine through centrifugation separation of free and encapsulated drug. L. amazonensis- infected RAW264.7 macrophages were treated for 48 h with the following preparations: (A) Retro-2, PFG30 + Retro-2 or PFG30 + Retro-2 post-centrifugation; (B) miltefosine, PFG30 + miltefosine or PFG30 + miltefosine post-centrifugation. Cells were ﬁxed in 2% PFA in PBS. IFA’s were performed for detection of LAMP-1 and cell and parasite nuclei with DAPI. Statistical signiﬁcance was determined by one-way ANOVA in GraphPad Prism 8 with the Holm-Šídák posthoc test for multiple comparisons (n.s., not statistically signiﬁcant, p-value < 0.001). Data were compiled from three biological experiments with at least two technical replicates. B B A B FIGURE 5 \| Evaluation of PFG30 encapsulation efﬁciency of Retro-2 and miltefosine through centrifugation separation of free and encapsulated drug. L. Retro-2 SAR Analogs Are Efﬁciently Packaged in PFG30 Aggregates and Delivered Into Macrophages In Vitro amazonensis- infected RAW264.7 macrophages were treated for 48 h with the following preparations: (A) Retro-2, PFG30 + Retro-2 or PFG30 + Retro-2 post-centrifugation; (B) miltefosine, PFG30 + miltefosine or PFG30 + miltefosine post-centrifugation. Cells were ﬁxed in 2% PFA in PBS. IFA’s were performed for detection of LAMP-1 and cell and parasite nuclei with DAPI. Statistical signiﬁcance was determined by one-way ANOVA in GraphPad Prism 8 with the Holm-Šídák posthoc test for multiple comparisons (n.s., not statistically signiﬁcant, p-value < 0.001). Data were compiled from three biological experiments with at least two technical replicates. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org August 2021 \| Volume 11 \| Article 702676 Encapsulation to Augment Drug Bio-Availability Craig et al. FIGURE 6 \| PFG30 encapsulation of Retro-2 (R2) increases uptake of the drug into RAW264.7 macrophages. (A–I) Representative microscope images (magniﬁcation 10X) of PFG30 + C6 nanoparticle uptake by RAW264.7 macrophages at different incubation time. RAW264.7 macrophages were incubated at 37°C with PFG30 encapsulated C6 at the following time points: (A–C) 30 min, (D–F) 60 min, and (G–I) 180 min. (J) Cell-associated ﬂuorescence quantiﬁcation calculated as a function of time. The curve was generated by performing image analysis (ImageJ software). Results are expressed as the mean of three biological replicates ± standard deviation (n = 3). (K) LC/MS quantiﬁcation of R2 concentration in L. amazonensis-infected RAW264.7 macrophages. Cells were treated with R2 or PFG30 encapsulated R2 (PFG30-R2) for 4 or 12 h. At the indicated times, cells were recovered and supernatant was collected. The cell pellets were lysated in RIPA buffer and then sonicated for 15 s twice prior to storage at –80°C. Statistical signiﬁcance was determined by two-way ANOVA in GraphPad Prism 8 with the Tukey posthoc test for multiple comparisons (ns, not statistically signiﬁcant where p-value > 0.05, p-value < 0.01, *p-value < 0.001). Data were compiled from two biological replicates. FIGURE 6 \| PFG30 encapsulation of Retro-2 (R2) increases uptake of the drug into RAW264.7 macrophages. (A–I) Representative microscope images (magniﬁcation 10X) of PFG30 + C6 nanoparticle uptake by RAW264.7 macrophages at different incubation time. RAW264.7 macrophages were incubated at 37°C with PFG30 encapsulated C6 at the following time points: (A–C) 30 min, (D–F) 60 min, and (G–I) 180 min. (J) Cell-associated ﬂuorescence quantiﬁcation calculated as a function of time. The curve was generated by performing image analysis (ImageJ software). Retro-2 SAR Analogs Are Efﬁciently Packaged in PFG30 Aggregates and Delivered Into Macrophages In Vitro Results are expressed as the mean of three biological replicates ± standard deviation (n = 3). (K) LC/MS quantiﬁcation of R2 concentration in L. amazonensis-infected RAW264.7 macrophages. Cells were treated with R2 or PFG30 encapsulated R2 (PFG30-R2) for 4 or 12 h. At the indicated times, cells were recovered and supernatant was collected. The cell pellets were lysated in RIPA buffer and then sonicated for 15 s twice prior to storage at –80°C. Statistical signiﬁcance was determined by two-way ANOVA in GraphPad Prism 8 with the Tukey posthoc test for multiple comparisons (ns, not statistically signiﬁcant where p-value > 0.05, p-value < 0.01, **p-value < 0.001). Data were compiled from two biological replicates. size in L. amazonensis-infected RAW264.7 macrophages at 25 and 50 µM by 16.6% and 41.2%, respectively. PFG30 encapsulated Retro-2 increased this effect with signiﬁcant loss of vacuole size by 21.2% at 10 µM (Figure 7A). DHQZ 36 also showed signiﬁcant vacuole size reductions at 25 and 50 µM by 28.3% and 42.7%, respectively. PFG30 encapsulation of DHQZ 36 improved this effect signiﬁcantly at concentrations as low as 1 µM with a size reduction of 33.1% compared with the PFG30- treated control (Figure 7B). Free DHQZ 36.1 only showed signiﬁcant size reduction at 50 µM by 41.5%. However, PFG30 encapsulation of DHQZ 36.1 showed similar results to DHQZ 36 with 1 µM signiﬁcantly reducing vacuole size by 35.0% compared to the control (Figure 7C). increased over time at 12 h post-treatment to 1.18 ± 0.59 µM in the 5-µM treated sample and 20.62 ± 1.11 µM in the 50-µM treated sample. Drug levels in cells were found to be elevated with PFG30 encapsulation at all time points. At 4 h, PFG30- Retro-2 treatments of 5 and 50 µM resulted in intracellular Retro-2 levels at 1.55 ± 0.28 µM and 5.1 ± 1.13 µM, respectively. This effect was more pronounced at 12 h post-treatment with intracellular Retro-2 concentrations of 4.43 ± 1.59 µM and 41.45 ± 7.42 µM in the 5 and 50 µM PFG30-Retro-2 treated samples, respectively (Figure 6K). PFG30 Aggregates Improve Effects of DHQZ Analogs on LPV Maturation Through Prevention of Vacuole Size Increases During L. amazonensis Infection Previously, we showed that treatment with Retro-2 and the DHQZ analogs, DHQZ 36 and DHQZ 36.1, resulted in reduced sizes of maturing L. amazonensis LPVs (Craig et al., 2017). Retro-2 SAR Analogs Are Efﬁciently Packaged in PFG30 Aggregates and Delivered Into Macrophages In Vitro Considering those observations, we wanted to determine if the effects on maturing LPVs DHQZ analogs could be enhanced with PFG30 encapsulation. RAW264.7 cells were infected with L. amazonensis promastigotes for 4 h prior to treatment with DHQZ analogs, alone or PFG30 encapsulated, for an additional 24 h. We found that PFG30 enhanced the vacuole size reduction results observed previously with drug alone with all three compounds. Free Retro-2 signiﬁcantly reduced vacuole CONCLUSIONS/DISCUSSION With no preventative vaccine available for leishmaniasis, chemotherapy is the front-line option for treatment of this potentially deadly disease. However, the pool of clinically- available anti-Leishmania compounds remains relatively small. One major issue in drug development of new potentially effective compounds is the problem of solubility. It is estimated that approximately 40% of market compounds and up to 90% of pipeline candidate drugs are poorly soluble in water (Kalepu and Nekkanti, 2015). Although repurposing drugs is a viable strategy August 2021 \| Volume 11 \| Article 702676 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 10 Craig et al. Encapsulation to Augment Drug Bio-Availability A B C FIGURE 7 \| Vacuole size measurements of drug treated L. amazonensis LPVs. RAW264.7 macrophages were infected for 24 h after which they were treated Retro- 2 or DHQZ analogs, with or without encapsulation in PFG30. (A) Treatment with Retro-2 (0–50 µM) with or without PFG30 encapsulation. (B) Treatment with DHQZ 36 (0–50 µM) with or without encapsulation. (C) Treatment DHQZ 36.1 (0–50 µM) with or without PFG30 encapsulation. Data were compiled from three experiments. At least 30 vacuoles were measured per treatment via one-way ANOVA with the Šı́dák correction for multiple comparisons. (p-value < 0.05, p-value < 0.01, p- value < 0.001). A B A C FIGURE 7 \| Vacuole size measurements of drug treated L. amazonensis LPVs. RAW264.7 macrophages were infected for 24 h after which they were treated Retro- 2 or DHQZ analogs, with or without encapsulation in PFG30. (A) Treatment with Retro-2 (0–50 µM) with or without PFG30 encapsulation. (B) Treatment with DHQZ 36 (0–50 µM) with or without encapsulation. (C) Treatment DHQZ 36.1 (0–50 µM) with or without PFG30 encapsulation. Data were compiled from three experiments. At least 30 vacuoles were measured per treatment via one-way ANOVA with the Šı́dák correction for multiple comparisons. (p-value < 0.05, p-value < 0.01, *p- value < 0.001). be due to C6 encapsulation into PFG30 aggregates as tested through centrifugation and NanoSight experiments. Despite removal of the supernatant, C6 ﬂuorescence did not signiﬁcantly change compared to PFG30 encapsulated C6 without centrifugation. While PFG30 did cause some background ﬂuorescent particles likely through light refraction, the magnitude of green ﬂuorescent particles measured on NanoSight were higher in PFG30 encapsulated C6 samples compared to C6 or PFG30 alone. CONCLUSIONS/DISCUSSION DLS experiments with PFG30 encapsulation of Retro-2 at 20°C and 37°C show that thermosensitive polymer self-assembly was not impeded upon uptake of hydrophobic cargo. for discovery of anti-Leishmania candidates because of the preceding studies of their bioavailability and toxicity in vivo, it would nonetheless be beneﬁcial to utilize the large pool of hydrophobic compounds in the discovery pipeline. One way to overcome the hurdle of water solubility is the encapsulation of drugs in water-soluble, inert, biodegradable nanoparticle capsules to enhance drug solubility (Zhang et al., 2008; Rao and Geckeler, 2011). In this manuscript, we evaluated PFG30 for their capacity to encapsulate and enhance the efﬁcacy of Retro-2 and its DHQZ SAR analogs to clear L. donovani and L. amazonensis infections of macrophages in vitro. We ﬁrst decided to explore the efﬁciency of PFG30 to encapsulate hydrophobic compounds. C6 was selected due to its ﬂuorescent properties and low solubility in aqueous solution. In addition, Zuppardi et al. recently used PFG preparations of different polyﬂuorostyrene concentrations with a similar dye, Nile Red, to demonstrate efﬁcient removal of the dye from aqueous solution (Zuppardi et al., 2020). When encapsulated by PFG30, C6 solubility was signiﬁcantly increased in water compared with C6 alone as measured by 420/500 nm excitation and emission ﬂuorescence. This increase in solubility appears to Because of the improved solubility of the hydrophobic molecule, C6, after encapsulation by PFG30, we tested the improved efﬁcacy of encapsulated Retro-2, DHQZ 36, and DHQZ 36.1 against L. donovani and L. amazonensis in vitro infections of RAW264.7 macrophages. PFG30 encapsulation was found to enhance the efﬁcacy of all three DHQZ compounds with DHQZ 36.1 EC50 values in the nanomolar range, surpassing that of miltefosine, which is used clinically. It is likely that these compounds are encapsulated within PFG30 aggregates similar to August 2021 \| Volume 11 \| Article 702676 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 11 Encapsulation to Augment Drug Bio-Availability Craig et al. improve_the_bioavailability_of_retrograde_inhibitors_LC-MS_ dataset_xlsx/14553783. encapsulation of C6 as centrifugation experiments and sampling of the pellet did not affect the EC50 of DHQZ analogs encapsulated in PFG30. This suggests that these compounds are likely enclosed in the hydrophobic portion of the PFG30 aggregate. Conversely, the EC50 of miltefosine against L. amazonensis was not signiﬁcantly affected by PFG30 encapsulation, and efﬁcacy was lost upon centrifugation and sampling of the pellet. These results suggest that miltefosine was not efﬁciently encapsulated within PFG30 aggregates. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb.2021. 702676/full#supplementary-material Supplementary Figure 1 \| Size distribution of PFG30 + Retro-2 particles measured by DLS at 20°C and 37°C. The self-assembly behavior of PFG30 was investigated by DLS performed at 20°C and 37°C on a solution containing Retro-2, PFG30, and incomplete DMEM. At 20°C, 10 nm polymer micelles were detected, whereas at 37°C intermicellar aggregates of about 200 nm in size formed, demonstrating that encapsulation of Retro-2 did not affect the self-assembly behavior of PFG30. The reported size distribution curves are averaged from 3 experiments. Supplementary Figure 1 \| Size distribution of PFG30 + Retro-2 particles measured by DLS at 20°C and 37°C. The self-assembly behavior of PFG30 was investigated by DLS performed at 20°C and 37°C on a solution containing Retro-2, PFG30, and incomplete DMEM. At 20°C, 10 nm polymer micelles were detected, whereas at 37°C intermicellar aggregates of about 200 nm in size formed, demonstrating that encapsulation of Retro-2 did not affect the self-assembly behavior of PFG30. The reported size distribution curves are averaged from 3 experiments. This study presents PFG30 as a potential candidate copolymer for biological application as a drug carrier. Further studies should elucidate its uptake and release mechanisms within cells as well as its viability for use in in vivo studies. In addition, this study highlights the signiﬁcant improvement of DHQZ analog efﬁcacy with polymer encapsulation. It is likely that other nanocarrier options would provide similar improvements. As an organic polymer, PFG30 may also be further improved through the addition of targeting mechanisms to increase uptake and cell speciﬁcity. These methods may further enhance drug delivery and bioavailability within an in vivo model. Supplementary Figure 2 \| Evaluation of polymer-encapsulated miltefosine on L. amazonensis-infected RAW264.7 macrophages. To L. amazonensis-infected RAW264.7 macrophages, free or PFG30 encapsulated miltefosine was added. After 48 h treatment, cells were ﬁxed in 2% PFA in PBS. IFA’s were performed for detection of LAMP-1 and cell and parasite nuclei with DAPI. At least 200 cells were scored per coverslip and infection rates were standardized to a vehicle control before EC50 estimation. Data were compiled from at least three biological repeats. Supplementary Figure 3 \| MTT assay of RAW264.7 macrophages treated with vehicle or PFG30 aggregates. Macrophages were treated with PFG30 at concentrations ranging from 450 ng/ml to 1 mg/ml for 48 h at 37°C with 5% CO2. CONCLUSIONS/DISCUSSION Likely, because of its hydrophilic nature, miltefosine remained in the aqueous supernatant, which was removed post-centrifugation. SUPPLEMENTARY MATERIAL Cells were then incubated with MTT for 4 h before addition of 10% SDS in PBS with 5 N HCl to dissolve formazan crystals overnight. Absorbances were read at 570 nm with a 630 nm background. Cell numbers were estimated based on a standard curve from at least 3 technical replicates each over triplicate experiments. ACKNOWLEDGMENTS The authors acknowledge the H2020 - MSCA-RISE-Marie Sklodowska-Curie Actions (MSCA) Research and Innovation Staff Exchange (RISE) for funding this work in the framework of the Project VAHVISTUS - Project Number: 734759. AUTHOR CONTRIBUTIONS EC performed experiments and wrote manuscript: RC, GG performed experiments. VA and AC performed experiments and contributed analysis and writing. PA and JS synthesized most drugs used in study. PC made intellectual contributions to the study and wrote and edited manuscript. PK made intellectual contributions to the study and wrote and edited manuscript and was responsible for the overall execution of the study. All authors contributed to the article and approved the submitted version. Because of the increase in solubility of C6 and the increased efﬁcacy of Retro-2 DHQZ SAR analogs, we decided to test if this effect was due to improved delivery of Retro-2 by uptake of PFG30 aggregates using LC/MS. Small, but not signiﬁcant, increases were observed as early as 4 h post-treatment with PFG30 encapsulated Retro-2 compared to free Retro-2. However, this increase was much more pronounced by 12 h post-treatment especially in the 50-µM sample where 82.9% of the encapsulated Retro-2 was associated with the cellular lysate. This result suggests that PFG30 aggregates are efﬁciently taken up by RAW264.7 macrophages and that encapsulation of Retro-2 is a viable strategy for improving its delivery to infected cells. However, the dynamics of uptake and release of Retro-2 encapsulated in PFG30 aggregates remains unknown. The dramatically increased reduction of LPV size by PFG30 encapsulated DHQZ 36 and DHQZ 36.1 at 24 h post-treatment suggests that a large proportion of the compounds are able to target the host secretory pathway machinery. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Gupta, N., Noël, R., Goudet, A., Hinsinger, K., Michau, A., Pons, V., et al. (2017). Inhibitors of Retrograde Trafﬁcking Active Against Ricin and Shiga Toxins Also Protect Cells From Several Viruses, Leishmania and Chlamydiales. Chem. Biol. Interact. 267, 96–103. doi: 10.1016/j.cbi.2016.10.005 Huh, A. J., and Kwon, Y. J. (2011). DATA AVAILABILITY STATEMENT Supplementary Figure 4 \| L929 cellular uptake of C6-loaded PFG30 nanoparticles. 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https://openalex.org/W3199665678	https://research.monash.edu/files/370812554/369389408_oa.pdf	English	null	Evidence of disease severity, cognitive and physical outcomes of dance interventions for persons with Parkinson’s Disease: a systematic review and meta-analysis	BMC geriatrics	2,021	cc-by	8,500	RESEARCH Open Access Abstract Background: Patients with Parkinson’s Disease (PD) usually experience worsening of both motor and non-motor symptoms. Dancing has been postulated to help patients with Parkinson’s via several mechanisms that lead to improved physical, cognitive and social functions. Methods: This systematic review was conducted following Cochrane methodology and reported following the PRISMA guideline. Four databases (up to June 2021) were searched for RCTs comparing dance to standard or other physical therapy for improvements in disease severity, quality of life, cognitive and physical outcomes as well as adverse events in patients with PD. We synthesised data using RevMan and included certainty-of-evidence rating (GRADE) for major outcomes. Results: A total of 20 RCTs (N = 723) articles that evaluated Tango, Ballroom, Irish, Waltz-Foxtrot, Folk, Turo, mixed dances and a PD-tailored dance were included. Dancers (versus non-dancers) had better motor experience (MDS- UPDRS 3) (MD -6.01, 95 % CI -9.97 to -3.84; n = 148; 5 RCTs) and improved balance (MiniBest Test) (MD 4.47, 95 % CI 2.29 to 6.66; n = 95; 3 RCTs), with no consistent differences on gait, agility and cognitive outcomes. Small samples and methodological limitations resulted in low-certainty-evidence across outcomes. Conclusions: Apart from a suggestion that dance intervention modestly reduced motor disease severity and improved certain aspects of balance, there is insufficient evidence on all other outcomes, such as agility and motor function, cognitive, mood and social outcomes, quality of life as well as adverse events including the risk of fall. As evidence is insufficient to inform practice, evidence of benefits on motor disease severity and balance needs to be considered in the context of user-perception of benefit versus harm and acceptability in the development of practice guideline recommendations. Co espo de ce: sop a. as eeqa@ o .go . y 1Institute for Medical Research, National Institutes of Health, Ministry o Health, Shah Alam, Malaysia Full list of author information is available at the end of the article Correspondence: sophia.rasheeqa@moh.gov.my 1Institute for Medical Research, National Institutes of Health, Ministry of Health, Shah Alam, Malaysia Full list of author information is available at the end of the article Evidence of disease severity, cognitive and physical outcomes of dance interventions for persons with Parkinson’s Disease: a systematic review and meta-analysis Evidence of disease severity, cognitive and physical outcomes of dance interventions for persons with Parkinson’s Disease: a systematic review and meta-analysis Sophia Rasheeqa Ismail1*, Shaun Wen Huey Lee2, Dafna Merom3, Puteri Sofia Nadira Megat Kamaruddin1, Min San Chong4, Terence Ong4 and Nai Ming Lai2,5 Ismail et al. BMC Geriatrics (2021) 21:503 https://doi.org/10.1186/s12877-021-02446-w Ismail et al. BMC Geriatrics (2021) 21:503 https://doi.org/10.1186/s12877-021-02446-w © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 11 Page 2 of 11 Ismail et al. BMC Geriatrics (2021) 21:503 Ismail et al. BMC Geriatrics (2021) 21:503 Ismail et al. BMC Geriatrics Introduction incorporation of evidence into guidelines. In this review, we aimed at evaluating the effectiveness and safety of dance as compared to standard therapy or to other phys- ical intervention in improving disease severity, cognitive and physical outcomes as well as quality of life. We ad- dressed the issues of previous reviews via an up-to-date systematic review of randomised controlled trials, with robust methodology that follows the Cochrane methods and with certainty of evidence rating using an estab- lished and widely used Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology [23]. In this review, we hypothesised that dancing interventions improve motor, cognitive and so- cial outcomes, as well as quality of life for people with Parkinson’s disease. Parkinson’s disease (PD) is a progressive neurodegenera- tive condition characterised by loss of dopaminergic neurons in the substantia nigra leading to decreased levels of dopamine in the striatum [1]. With increased prevalence in older adults, PD affects 2 per 1000 people [2]. The loss of dopaminergic neurons in substantia nigra precipitates both motor and non-motor symptoms. People with PD typically have bradykinesia, rigidity, tremor and postural instability, while non-motor features includes cognitive impairment, mood disorders, fatigue, urinary and bowel symptoms [3]. Management is aimed at slowing the decline of motor and non-motor func- tions to improve quality of life mainly by pharmaco- logical modalities with or without physical therapy. However, pharmacological treatment is accompanied by significant adverse effects and pill-burden [4]. Types of studies We included randomised-controlled trials and controlled trials with unclear allocation methods. We excluded tri- als that are clearly stated as non-randomised or quasi- experimental. Participants All adults with Parkinson’s disease (PD), diagnosed via established clinical diagnostic criteria and/or neuroimag- ing and/or biomarker testing. We included studies that examine patients of all severities, stages and durations, and whether or not they are on stable therapy. Interventions Any form of dancing, either recreational or therapeutic, that involves rhythmic movement of body in response to verbal, musical or other form of cues. Examples of well- recognised types of dance include Line-dancing, Ballet, Jazz, Hip-hop, Country, Western, Flamenco, Swing, Latin, Folk dance, Waltz, Tango, Cha-cha, Rhumba, Samba, Mambo, Quickstep, Jive and Zumba. Current trends suggest that the prevalence and burden of PD are expected to continuously increase [16] thereby prompting the search for stronger evidence for the man- agement of people with PD. There are at least five re- cently published systematic reviews [17–21] and one narrative review [22] that evaluated the role of various forms dancing on various outcomes in PD. However, limitations of the aforementioned reviews included the comprehensiveness of the search, inclusion of non- randomised studies with inherent high risk-of-bias, rigour of methodology and the absence of rating of cer- tainty of evidence which has become essential in the Methods g Dance is characterised by the synchronization of movement to musical rhythms that integrates multiple physical, cognitive, emotional and social elements. The complex sensorimotor activities has been shown to acti- vate the medial geniculate nucleus thereby potentially improving motor and non-motor features of the disease [5]. Mirroring, a technique used in dancing, activates the mirror neurons in the brain which would then lead to hormonal changes, positive effects on cognitive function, engagement of body memories for reminiscence therapy, and improvement in social cognitive theory constructs [6–11]. Consequently, positive group dynamics, mutual support and trust, corrective emotional experiences, em- powerment, probing social roles, and enactive interper- sonal learning experiences would be enhanced. Dance intervention focuses on maintaining posture, balance, movement and strength leading to improvements in gait, balance and reduce risk of fall as seen in healthy older adults [12]. Physical, psychological and social outcomes improvements were also seen in elderly people with de- mentia, depression and cancer [13–15]. Although the role of exercise in PD has been recognised in treatment guidelines, dance is less often highlighted therefore re- quires further exploration as compared to standard of care and other physical treatments. The systematic review is registered with PROSPERO (CRD42018081017). The registered protocol is available at https://www.crd.york.ac.uk/prospero/display_record. php?RecordID=81017. Comparisons We compared dance interventions with standard therapy but no dancing, or with other intervention in the form of physical or non-physical activities. We accepted stud- ies that compare two types of dancing interventions. Results The search yielded 4,506 records after removal of dupli- cates. We shortlisted 199 articles and included 20 re- ports, with eight studies awaiting full-texts and five on- going studies. There were 20 studies described in these 23 reports, as three pairs of reports described the same studies [32–37]. The PRISMA flow diagram of the study identification process is shown in Fig. 1. Among the 20 included studies, 16 were parallel- group, individually randomised, two-arm RCTs. One study was a randomised, crossover trial [38], one study was a three-arm controlled trial with alternate allocation [37], and two more studies [39, 40] had unclear methods of allocation. Eleven studies were conducted in the United States of America (USA) [36, 37, 40–48], two each in the United Kingdom (UK) [34, 35] and Italy [49, 50], with one each in Ireland [51], Canada [52], Germany Secondary outcomes Cognitive outcomes (memory, processing time, execu- tion and reaction time at so on), quality of life measured using the disease-tailored Quality of Life Scale (e.g. PDQ-39). We used the I2 statistic to quantify the degree of hetero- geneity in the results [29], with a cut-off of 50 % and above considered as substantial heterogeneity. We pooled data and performed meta-analysis where appro- priate, using a random-effect model in the RevMan 5.4 software [27]. We reported our results using mean dif- ference (MD) and risk ratio (RR) for continuous and di- chotomous outcomes respectively with their 95 % confidence intervals (CI). We performed certainty of evi- dence using the GRADE approach for all the outcomes and highlighted some major outcomes using one ‘Sum- mary of findings’ table for each comparison (Additional File 5) [23]. Further details on our methodology, includ- ing how we addressed heterogeneity, undertook meta- analysis and performed GRADE certainty-of-evidence rating are available in Additional File 3. Key references to tools used in the evaluation of major outcomes in this review are reported in Add- itional File 1. Outcome measures Disease severity measured using established instruments such as MDS-UPDRS (total score of part I-IV) (total Page 3 of 11 Ismail et al. BMC Geriatrics (2021) 21:503 Ismail et al. BMC Geriatrics outcome assessors, incomplete outcome data and select- ive outcome reporting. Each domain was judged either low, high or unclear risk of bias (Additional File 4). score 0-209, higher worse) [24], Webster Rating Scale (0–30, higher worse) [25] or other scale evaluating motor disturbance, and specific physical outcomes such as gait balance and range of motion. Selection of studies, data extraction and management Selection of studies, data extraction and management We followed the Cochrane methods, as described in the Cochrane Handbook for Systematic Reviews of Interven- tions [26]. A detailed description of our methods is avail- able in Additional File 3 and following is a summary. and with additional incorporation of the random effect model using DerSimonian and Laird method [30], which includes the following additional formula to calculate the prediction interval that takes into account of the variance between studies: M tk2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Tau2 þ SE M ð Þ2 q [31]. Two authors independently screened the titles and/or abstracts for potentially eligible studies, and two authors then independently evaluated the abstract and full texts of the shortlisted articles to determine eligibility. We de- lineated the study selection process in a PRISMA dia- gram. Two review authors independently extracted and coded all data from each included study using a data col- lection form, including design, population, intervention, comparison and outcomes. Disagreements along these steps were resolved via discussion, with the input of a third author if necessary. One review author transferred the data to Review Manager 5.4 software [27], and a sec- ond review author checked the accuracy of data entry. ween studies: M tk2 Tau2 þ SE M ð Þ2 q [31]. Search strategies We searched MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials (CENTRAL) for published studies, and trial registries including the WHO Inter- national Trial Registry Platform and ClinicalTrials.gov for on-going studies, from inception up to 18 June 2021, without language restriction. We reviewed the references lists of retrieved articles for further trials for inclusion and contacted the authors of relevant trials to request details of any additional relevant published, unpublished or ongoing studies. Our search strategy for Medline is listed in Additional File 2. We adapted the same search strategy for other databases. We performed our meta-analysis by entering all data into the Review Manager software version 5.4, where the effect size calculations were automatically performed, using the inverse variance approach with the following core formula in calculating weighted average: Genericinverse varianceweightedaverage ¼ P Y ið1=SEi2Þ P ð1=SEi2Þ , Effects estimates In total, 18 studies with 723 participants contributed data, while the outcome data of two studies [34, 47] were not reported sufficiently for meta-analysis. Four major comparisons were evaluated, namely, (1) dancing in various forms versus no dancing; (2) two different forms of dance intervention: (i) specifically-tailored Dance for Parkinson’s Disease (D4PD) versus Tango [39], (ii) Tango versus mixed dances [54], and (iii) part- nered versus non-partnered dance [45]; 3) Dance versus different exercises, as follows: i).Tango versus treadmill [37], ii).Tango versus stretching and flexibility exercises [37, 46] and iii) Tango versus Tai-Chi [53]; and 4) Dance versus physiotherapy [49]. Assessment of risk-of-bias The Cochrane risk-of-bias tool for randomised trials was used for risk of bias assessment of all included studies [28]. Two review authors assessed each included trial for risk-of-bias independently according to the following six major criteria: sequence generation, allocation conceal- ment, blinding of patient and personnel, blinding of Page 4 of 11 Ismail et al. BMC Geriatrics Ismail et al. BMC Geriatrics (2021) 21:503 Fig. 1 The PRISMA flow diagram of the study identification process from screening to analysis Fig. 1 The PRISMA flow diagram of the study identification process from screening to analysis [53], Australia [54] and Republic of Korea [38]. The number of participants ranged from 10 [42] to 119 [37]. [53], Australia [54] and Republic of Korea [38]. The number of participants ranged from 10 [42] to 119 [37]. A list of the essential characteristics of each included study is included in Additional Table 1. A more detailed description of the characteristics of the included studies and their detailed risk-of-bias evaluation, as well as the characteristics of excluded and on-going studies are available in Additional File 4. participants and personnel and incomplete outcome data. Meanwhile, a large proportion of included studies did not provide sufficient information to enable a mean- ingful risk-of-bias assessment. A detailed description of the risk-of-bias of each study is provided in Additional File 4. A list of the essential characteristics of each included study is included in Additional Table 1. A more detailed description of the characteristics of the included studies and their detailed risk-of-bias evaluation, as well as the characteristics of excluded and on-going studies are available in Additional File 4. Risk-of-bias in included studies The proportions of studies with low, high, and unclear risks-of-bias in each domain is illustrated in Fig. 2, and the risk-of-bias judgement of each included study in each domain is depicted in Fig. 3. Overall, there was a wide variation in the risks-of-bias of the studies across domains, with the majority judged to have low risk in blinding of outcome assessment, incomplete outcome data and selective reporting, while a minority having low risk in random sequence generation, allocation conceal- ment and least of all, blinding of participants and personnel. A small but significant proportion of studies were judged to have high risk-of-bias in random se- quence generation, allocation concealment, blinding of Data from 4 studies were extrapolated from the graphs [37, 42, 43, 48]. The data from one study were reported Page 5 of 11 Ismail et al. BMC Geriatrics (2021) 21:503 Ismail et al. BMC Geriatrics Fig. 2 The risk-of-bias graph: proportions of studies with low, high, and unclear risks of bias in each domain At 3 months, our studies (n = 148) revealed. At 3 months, our studies (n = 148) revealed. in median and interquartile ranges [51], so they were un- suitable to be included in meta-analysis. The data were displayed in a separate table (Additional File 6). Based on five studies, dancers appeared to have lower scores in MDS-UPDRS subscale 3 at 3 months (MD -6.91, 95 % CI -9.97 to -3.84; n = 148; I2 = 0 %). Based on three studies, dancers also had lower MDS-UPDRS sub- scale 3 scores at both 6 months (MD -7.26, 95 % CI -11.68 to -2.85; n = 131; I2 = 0 %) and 12 months (MD -14.91, 95 % CI -19.77 to -10.05; n = 62; I2 = 0 %)(low- certainty evidence, downgraded due to concerns on risk- of-bias and imprecision) (Fig. 4). See Summary of findings Tables 1, 2, 3, 4, 5, 6, 7 and 8 (Additional File 5) for certainty-of-evidence ratings for all outcomes along with reasons for downgrading. A list of all outcome estimates per comparison and their cor- responding Forest plots are available in Additional File 7 and Additional File 8 respectively. Below is a summary of the major outcome estimates. g c. MDS-UPDRS 4 (dyskinesia, 0–13, fluctuation, 0–10) (higher score indicates more severe disease). Comparison 1: Dancing versus no dancing Based on a single study (n = 33), there were no signifi- cant differences in the score in MDS-UPDRS 4 subscale between dancers and non-dancers (dyskinesia subscale: MD -0.10, 95 % CI -0.79 to 0.59; fluctuation subscale: MD 0.60, 95 % CI -0.92 to 2.12;) (low-certainty evidence, downgraded due to concerns on risk-of-bias and imprecision). There were ten studies that compared dance interven- tions with no dancing. Nine studies compared with usual care and one study compared with education [40]. The types of dances included Tango, Waltz, Foxtrot, ball- room, Latin American dances and Sardinian folk dances adapted to persons with PD. Usual care was mainly de- fined as the continuation of medications, medical ap- pointments, and any existing usual exercise programs. p 2. Balance. a. Mini-BEST Test (0–28, higher score indicates better balance). a. Mini-BEST Test (0–28, higher score indicates better balance). Primary outcomes Based on three studies (n = 95), dancers may have bet- ter balance, assessed using Mini-BEST Test compared to non-dancers (MD 4.47, 95 % CI 2.29 to 6.66; I2 = 0 %) (moderate-certainty evidence, downgraded due to con- cern on risk-of-bias). 1. Disease severity. a MDS-UPDRS subscales 1 (non-motor experiences of daily living, 0–16) and 2 (motor experiences of daily living, 0–56) (higher score indicates more severe disease). b. Berg Balance Scale (0–56, higher score indicates better balance). Due to large degree of heterogeneity when the results of the five included studies were pooled, we separated the studies into subgroups based on the timing of out- come measurement (at 2, 3, 4 and 6 months), and found a difference in the scores of Berg’s Balance Scale at 3 months favouring dance (MD 8.42, 95 % CI 3.68 to 13.17; n = 32), but no significant differences at 2 months (MD 1.10, 95 %CI -0.81 to 3.01; n = 41), at 4 months (MD 5.10, 95 % CI -0.00 to 10.20; n = 48) and 6 months (MD -1.60, 95 % CI -4.54 to 1.34; n = 46) (low-certainty Based on the same two studies (n = 23), dancers ap- peared to have lower but insignificant scores in both MDS-UPDRS subscale 1(MD -3.50, 95 % CI -18.68 to 11.67) and MDS-UPDRS subscale 2 (MD -2.09, 95 % CI -7.57 to 3.40) (low-certainty evidence, downgraded due to concerns on risk-of-bias and imprecision). b. MDS-UPDRS 3 (Motor examination, 0-108) (higher score indicates a more severe disease). b. MDS-UPDRS 3 (Motor examination, 0-108) (higher score indicates a more severe disease). Page 6 of 11 Ismail et al. BMC Geriatrics (2021) 21:503 Ismail et al. BMC Geriatrics evidence, downgraded due to concerns on risk-of-bias and imprecision). Fig. 3 The risk-of-bias summary: the risk-of-bias judgement of each c. Activity-specific balance confidence (0-100, higher score indicates better balance confidence). Based on one study (n = 46), it was unclear whether dancers had better balance confidence compared to non-dancers, as assessed using Activity-specific balance confidence scale (MD 0.20, 95 % CI -12.72 to 13.12) (Low-certainty evidence, downgraded due to concern on risk-of-bias and imprecision). p ) 3. Gait. 3. Gait. 3. Gait. a. Freezing of gait questionnaire (0–24, higher score indicates more severe condition). Based on three studies (n = 89), dancers appeared to have lower questionnaire score (MD -0.39, 95 % CI -2.99 to 2.24; I2 = 0 %) (low-certainty evidence, downgraded due to concern on risk-of-bias and imprecision). p 4. Range or motion, speed, flexibility or agility. 4. Range or motion, speed, flexibility or agility. a. Timed Up and Go Test (TUG). a. Timed Up and Go Test (TUG). 1. Disease severity. Seven studies measured agility using TUG (second). Dancers appeared to perform slightly better in TUG Test (MD -1.16, 95 % CI -2.17 to -0.15; participants = 200; studies = 7; I2 = 47 %) (low-certainty evidence, downgraded due to concern on risk-of-bias, inconsist- ency and imprecision). There was substantial heterogeneity in the pooled esti- mate for this outcome, as shown by an I2 statistic of 47 %. We found that removing Kunkel 2017 [35] re- duced I2 to 0 %, while removing one or more others did not reduce I2 substantially. We explored possible factors that could have accounted for the difference between Kunkel 2017 and other included studies, from study conduct (risk-of-bias) to characteristics of the popula- tion, intervention/comparison, outcome measurement, and risk-of-bias profile. We found a major difference be- tween Kunkel 2017 [35] and other included studies, as the final outcome measurement took place at 6 months, well after the completion of the intervention which lasted for 10 weeks, while all other studies had their final outcome measured at or soon after the completion of the intervention. However, we still considered appropri- ate to pool the outcome estimates of the six studies in a meta-analysis, rather than placing Kunkel 2017 in a dif- ferent subgroup. b. Five Times Sit-to-Stand Test (FTSTT) (seconds, shorter better). b. Five Times Sit-to-Stand Test (FTSTT) (seconds, shorter better). Based on a single study, dancers appeared to perform better in the FTSTT test (MD -4.90, 95 % CI -6.51 to -3.29; participants = 19) (low-certainty evidence, down- graded due to concerns on risk-of-bias and imprecision). c. Sit-and-Reach Test (SRT) (cm, shorter distance better). Based on a single study, there was no clear differences between groups in the performance in SRT (MD 4.60, Ismail et al. BMC Geriatrics (2021) 21:503 Page 7 of 11 Ismail et al. BMC Geriatrics Fig. 4 The Forest plot for one of the major outcomes: MDS-UPDRS 3 subscale (motor examination, 1-108, higher score indicates a more severe disease) Fig. 4 The Forest plot for one of the major outcomes: MDS-UPDRS 3 subscale (motor examination, 1-108, higher score indicates a more severe disease) certainty evidence, downgraded due to concern on risk- of-bias and imprecision). 95 % CI -2.78 to 11.98; participants = 19) (low-certainty evidence, downgraded due to concerns on risk-of-bias and imprecision). g. Back-Scratch test. p d. Walking distance: six-minute walk test (meters). 1. Disease severity. A single study showed no clear difference between groups in the BST (MD 5.30, 95 % CI -2.94 to 13.54; par- ticipants = 19)) (low-certainty evidence, downgraded due to concern on risk-of-bias and imprecision). Due to large degree of heterogeneity, we separated the four included studies into two subgroups based on the type of dance offered. We found that among the three studies that evaluated Tango (n = 104), there were no significant difference between groups in walking distance in six minutes (MD -1.34, 95 % CI -53.91 to 51.24; I2 = 49 %), but in the single study that evaluated Sardinian folk dance, dancers appeared to perform better in the six-minute-walk-test (MD 238.80, 95 % CI 157.99 to 319.61; participants = 19) (low-certainty evidence, down- graded due to concern on risk-of-bias and imprecision). e Forward velocity (meters/second) h. Spinal mouse inclination degree. A single study showed no clear difference between groups in the spinal mouse inclination degree (MD 0.80, 95 % CI -3.61 to 5.21; participants = 46) (low-certainty evidence, downgraded due to concern on risk-of-bias and imprecision). 4. Adverse events: falls during study A single study showed no significant differences be- tween groups in the risk of fall during the study period (RR 0.56, 95 % CI 0.11 to 2.90, participants = 33) (moder- ate-certainty evidence, downgraded due to concern on imprecision). e. Forward velocity (meters/second). Based on one study (n = 48), there were no significant differences between groups in forward velocity (meters/ second) (MD 0.10, 95 % CI -0.10 to 0.30) (low-certainty evidence, downgraded due to concern on risk-of-bias and imprecision). Tango versus Tai-Chi Based on a single study (n = 29), there were no signifi- cant differences between groups in quality of life (PDQ- 39) and Life satisfaction (Brief Multidimensional Life Satisfaction Scale (BMLSS) scores. The certainty of evi- dence is low for both outcomes due to serious concerns on the risk-of-bias and imprecision (downgraded one level) (Additional File 5). Dance for Parkinson’s disease (D4PD) versus Tango There were no significant differences between groups in all outcomes estimates under this comparison, including disease severity (MDS-UPDRS subscale 3), Timed Up and Go Test, six-minute walk test, forward velocity and quality of life (for details please refer to Additional File 7). The certainty of evidence is low for all outcomes in- cluded in this comparison, due to serious concerns on the risk-of-bias of the included study and imprecision (Additional File 5). Comparison 3: Dancing versus different exercises There were three comparisons made under this category: i).Tango versus treadmill [37], ii).Tango versus stretch- ing and flexibility exercises [37, 46] and iii) Tango versus Tai-Chi [53]. Tango versus mixed dances There were no significant differences between groups in all outcomes estimates under this comparison, including Timed Up and Go Test, Functional Gait Assessment, Freezing Gait Questionnaire, Berg Balance Scale, disease severity (MDS-UPDRS 2 & 3), and quality of life (PDQ39) (for details please refer to Additional File 7). The certainty of evidence is low for all outcomes in- cluded in this comparison, due to serious concerns on the risk-of-bias of the included study and imprecision (Additional File 5). Tango versus stretching or flexibility exercises Tango versus stretching or flexibility exercises There were no significant differences between groups in all outcomes estimates under this comparison, including disease severity (MDS-UPDRS subscale 3), balance (Mini Best Test and Berg Balance Scale), Freezing of gait ques- tionnaire score, six-minute walk test, forward velocity and quality of life (PDQ-39) (for details please refer to Additional File 7). The certainty of evidence is low to very low for all outcomes included in this comparison, due to either serious or very serious concerns on the risk-of-bias of the included study, which resulted in a downgrade of one or two levels respectively, and impre- cision (downgraded one level) (Additional File 5). Secondary outcomes f. Standing-start 180 degree turn test and spinal mouse inclination. There were no significant differences between groups in all secondary outcomes under this comparison, including cognitive function (Montreal Cognitive Assessment Scale), activity participation (Activity Card Sort), depres- sive symptoms (Beck Depression Inventory), apathy (Ap- athy Scale), fatigue (Krupp Fatigue Severity Scale) and quality of life (PDQ-39) (for detail please refer to Add- itional File 7) (low-certainty evidence for all outcome Based on one study (n = 46), there were no significant differences between groups in 180 degree turn test in the number of steps taken (MD 1.30, 95 % CI -0.38 to 2.98) and the time taken to complete the turn (MD 0.40, 95 % CI -0.18 to 0.98), as well as spinal mouse inclin- ation degree (MD 0.80, 95 % CI -3.61 to 5.21) (low- Page 8 of 11 Page 8 of 11 Ismail et al. BMC Geriatrics (2021) 21:503 Ismail et al. BMC Geriatrics estimates, downgraded due to concern on risk-of-bias and imprecision) (Additional File 5). estimates, downgraded due to concern on risk-of-bias and imprecision) (Additional File 5). Best Test), six-minute walk test, forward velocity and quality of life (PDQ-39) (for details please refer to Add- itional File 7). The certainty of evidence is very low for all outcomes included in this comparison, due to very serious concerns on the risk-of-bias of the included study (downgraded two levels) and serious concern on imprecision (downgraded one level) (Additional File 5). Comparison 2: Two different forms of dance interventions Three included studies compared two different forms of dance intervention. One study compared a specific dance for PD (D4PD) and Tango [39], another study compared tango with mixed dances [54], and one study compared partnered dancing with non-partnered dan- cing [45]. Partnered vs. non-partnered dance Based on one study in this comparison (n = 39), there were no significant differences between groups in bal- ance (Berg Balance Scale) as well as Timed Up and Go Test (for details please refer to Additional File 7). The certainty of evidence is very low for both outcomes, due to very serious concerns on risk-of-bias of the included study (downgraded two levels) and imprecision (down- graded one level) (Additional File 5). Based on one study in this comparison (n = 24), dancers appeared to have lower severity of disease in the motor examination subscale of the MDS-UPDRS subscale 3 (MD -3.60, 95 % CI -6.42 to -0.78), improved balance as measured by the Berg Balance Scale (MD 7.20, 95 % CI 0.36 to 14.04) and better ratings in the Freezing of Gait Questionnaire (MD -5.30, 95 % CI -8.11 to -2.49). How- ever, there were no significant differences in the risks of fall of any cause between groups either during the time of intervention or during the entire study period (during the time of intervention: RR 3.00, 95 % CI 0.13 to 67.06; during the entire study: RR 0.90, 95 % CI 0.60 to 1.36), as well as the quality of life (PDQ-39) (MD -5.40, 95 % CI -12.63 to 1.83) (for details please refer to Additional File 7). Discussion disease, Carapellotti 2020 [21] was recently published and included the most articles thus far. However, four RCTs were missed, strength of evidence was not in- cluded, and intervention comparisons were only grouped into dance versus no or active intervention only. The lack of certainty of evidence rating might lead to in- appropriate confidence in the findings, as possibly reflected in the reviews above in the tone of their con- clusions about the effects or lack thereof of dance therapy. Based on limited evidence, this review highlights the modest improvement of motor function, non-motor function, and balance in people with Parkinson’s disease who underwent dance intervention compared to usual care. Irish dance intervention also offered modest im- provement in motor function, balance and gait, as com- pared to physiotherapy. There were no clear differences between groups in most other outcomes evaluated in the meta-analysis. The available evidence was limited by the small samples and serious concerns on the risk of bias of the included studies. Apart from two studies, the rest of the included stud- ies were conducted in Europe or North America, thereby limiting the generalisability of the findings. Findings were also limited due to the very low to low certainty of evidence in almost all outcomes which were contributed by serious concerns of risk-of-bias, imprecision of the outcome estimates and inconsistency of the estimates. Serious concerns were made for performance bias, selec- tion bias, and attrition bias domains. As many outcomes such as disease severity, balance and quality of life were subjective outcomes, the lack of blinding of participants and care personnel in all studies could have had poten- tial impact on outcome performance. Although partici- pant blinding would be impossible, blinding of outcome assessor would have been possible and would have lim- ited this concern. Serious concerns on attrition bias were due to the high withdrawal rates in individual studies (15–54 %) and reasons for withdrawal were likely outcome-related thereby causing under- or overesti- mation of reported outcomes. Imprecision of outcome estimates, as reflected by wide confidence intervals, was due to the small sample sizes in single studies which were underpowered to detect important differences in the effect estimates. The overall findings of this review were consistent with the findings of other reviews [17, 18, 20, 21]. Tango versus treadmill The certainty of evidence is low for all outcomes in- cluded in this comparison, due to serious concerns on the risk-of-bias of the included study and imprecision (downgraded one level each) (Additional File 5). There were no significant differences between groups in all outcomes estimates under this comparison, including disease severity (MDS-UPDRS subscale 3), balance (Mini Page 9 of 11 Ismail et al. BMC Geriatrics (2021) 21:503 Page 9 of 11 Ismail et al. BMC Geriatrics (2021) 21:503 Ismail et al. BMC Geriatrics Conclusions Although certainty of evidence was low, our review suggests that dance intervention modestly reduced motor disease severity and improved certain aspects of balance, while there is insufficient evidence on all other outcomes. The wide variety of dance interven- tion types and outcome assessed diluted the strength of the evidence base on the effectiveness of dance in people with PD. Evidence of benefits on motor dis- ease severity and balance needs to be considered in the context of user-perception of benefit versus harm and acceptability in the development of practice guideline recommendations. Given the difficulties in achieving blinding of participants and personnel, it is essential that future RCTs adhere to rigorous stan- dards in random sequence generation, allocation con- cealment and blinding of outcome assessor, with clear documentation, to offer any improvement in the over- all certainty of evidence. Discussion Based on five RCTs (159 participants), Dos 2018 [17] con- cluded that dance conferred significant improvements in disease severity as measured using MDS-UPDRS III, as well as reaction time measured using TUG test, com- pared to other types of exercise. All five RCTs included in Dos 2018 [17] were included in our review. Even with the inclusion of mixture of RCTs and non-RCTs as in Lötzke 2015 [18] and Kalyani 2019 [20], motor and cog- nitive improvements were similar. Both Lötzke 2015 [18] and Kalyani 2019 [20] concluded that there were moder- ate motor and possibly cognitive benefits in favour of Argentine Tango specifically [18] and dance in general [20], although the certainty of evidence was unclear. Cognitive assessment was evaluated in two studies that compared dance with usual care or education and showed small, insignificant cognitive improvements. The review by Zhang 2019 [19] evaluated mainly cogni- tive and mood symptoms and included seven RCTs (185 participants). While only two of the seven RCTs included in Zhang 2019 [19] was included in our re- view, due possibly to different scope of search, the au- thors concluded that dance improved executive function but did not seem to improve global cognitive and mood outcomes. The lack of cognitive assessment and the resulting inconsistent results as compared to sensorimotor assessment was highlighted in a review by Bek 2020 [22]. Supplementary Information Supplementary Information Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12877-021-02446-w. Additional file 1. Key references to tools used in the evaluation of major outcomes. Additional file 2. MEDLINE (PubMed) search strategy. Additional file 3. Additional details on the methodology. Additional file 4. Characteristics of studies in greater details. Additional file 5. Summary of findings tables with certainty of evidence rating. Additional file 6. Outcome data for the study with skewed data. Additional file 7. Summary of all outcome estimates. Additional file 8. Forest plots. Additional file 9. Completed PRISMA checklist for reporting of systematic review. Additional file 10: Table S1. Characteristics of included studies Received: 12 February 2021 Accepted: 29 August 2021 Funding Thi k 17. Dos Santos Delabary M, Komeroski IG, Monteiro EP, Costa RR, Haas AN. Effects of dance practice on functional mobility, motor symptoms and quality of life in people with Parkinson’s disease: a systematic review with meta-analysis. Aging clinical and experimental research. 2018;30(7):727–35. g This work was supported by Taylor’s University Ageing Flagship Research Grant, Malaysia, with provision of salary for a research assistant who assisted in retrieving papers and extracting study-related data. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 18. Lötzke D, Ostermann T, Büssing A. Argentine tango in Parkinson disease: a systematic review and meta-analysis. BMC Neurology. 2015;15(1):226. 19. Zhang Q, Hu J, Wei L, Jia Y, Jin Y. Effects of dance therapy on cognitive and mood symptoms in people with Parkinson’s disease: A systematic review and meta-analysis. Complementary therapies in clinical practice. 2019;36:12–7. Availability of data and materials The dataset used and analysed during this review are available from the corresponding author on reasonable request. 20. Kalyani HHN, Sullivan K, Moyle G, Brauer S, Jeffrey ER, Roeder L, et al. Effects of Dance on Gait, Cognition, and Dual-Tasking in Parkinson’s Disease: A Systematic Review and Meta-Analysis. J Parkinsons Dis. 2019 9(2):335–49. Acknowledgements W ld l k h 10. Koch SC. Arts and health: Active factors and a theory framework of embodied aesthetics. The Arts in Psychotherapy. 2017;54:85–91. g We would like to thank the Director General of Health, Malaysia, for his permission to publish this article. Our deep gratitude to Dr Dorit Kunkel for generously providing the protocol and the full-text of her study. 11. Meng X, Li G, Jia Y, Liu Y, Shang B, Liu P, et al. Effects of dance intervention on global cognition, executive function and memory of older adults: a meta-analysis and systematic review. Aging Clin Exp Res. 2020;32(1):7–19. 12. Merom D, Mathieu E, Cerin E, Morton RL, Simpson JM, Rissel C, et al. Social Dancing and Incidence of Falls in Older Adults: A Cluster Randomised Controlled Trial. PLoS medicine. 2016;13(8):e1002112-e. References Atkins R, Deatrick JA, Gage GS, Earley S, Earley D, Lipman TH. Partnerships to Evaluate the Social Impact of Dance for Health: A Qualitative Inquiry. J Community Health Nurs. 2019;36(3):124–38. 10. Koch SC. Arts and health: Active factors and a theory framework of embodied aesthetics. The Arts in Psychotherapy. 2017;54:85–91. Authors’ contributions SRI: screened and selected studies, extracted data, assessed risk of bias, performed certainty of evidence rating, and wrote the review. NML: conceived the review, screened and selected studies, inspected the abstracts or full texts, extracted data, checked accuracy of data entry in RevMan, assessed risk of bias, performed certainty of evidence rating, and wrote the review. SWHL, PSNMK, MSC: screened and selected studies, extracted data, and wrote the review. TO: inspected the abstracts or full texts for selection, and wrote the review. DM: critically revised search strategy, and wrote the review. The authors read and approved the final manuscript. 13. Goodill Sharon W. Accumulating Evidence for Dance/Movement Therapy in Cancer Care. 2018;9:1778. 14. Klimova B, Valis M, Kuca K. Dancing as an Intervention Tool for People with Dementia: A Mini-Review Dancing and Dementia. Current Alzheimer research. 2017;14(12):1264–9. 15. Meekums B, Karkou V, Nelson EA. Dance movement therapy for depression. The Cochrane database of systematic reviews. 2015(2):CD009895-CD. 16. Global, regional, and national burden of Parkinson’s disease, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Neurol. 2018;17(11):939–53. Consent for publication Consent for publication Not applicable. 23. Schünemann HJ, Oxman AJ, Vist GE, Higgins JP, Deeks JJ, Glasziou P. Chapter 12: Interpreting results and drawing conclusions. In: Higgins JP, Green S, editor(s). Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0 (updated March 2011). The Cochrane Collaboration; 2011. Competing interests h h d l h Competing interests The authors declare that they have no competing interests. Declarations 21. Carapellotti AM, Stevenson R, Doumas M. The efficacy of dance for improving motor impairments, non-motor symptoms, and quality of life in Parkinson’s disease: A systematic review and meta-analysis. PLOS ONE. 2020; 15(8):e0236820. Ethical approval and consent to participate Not applicable Strengths and Limitations We believe we have captured most literature that are relevant to this review through our comprehensive search strategy from multiple databases with independ- ent screening, selection, and assessment of eligible stud- ies. However, future inclusion of the eight on-going studies may change some findings due to the small num- ber of studies and participants in most outcomes of this review. Additionally, our review has the advantage of in- corporating the certainty of evidence rating, which was not included in the aforementioned reviews. Of the five similar reviews on dance interventions and Parkinson’s Page 10 of 11 Page 10 of 11 Ismail et al. BMC Geriatrics (2021) 21:503 Ismail et al. BMC Geriatrics (2021) 21:503 Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12877-021-02446-w. Additional file 1. Key references to tools used in the evaluation of major outcomes. Additional file 2. MEDLINE (PubMed) search strategy. Additional file 3. Additional details on the methodology. Additional file 4. Characteristics of studies in greater details. Additional file 5. Summary of findings tables with certainty of evidence rating. Additional file 6. Outcome data for the study with skewed data. Additional file 7. Summary of all outcome estimates. Additional file 8. Forest plots. Additional file 9. Completed PRISMA checklist for reporting of systematic review. Additional file 10: Table S1. Characteristics of included studies Consent for publication Not applicable. 22. Bek J, Arakaki AI, Lawrence A, Sullivan M, Ganapathy G, Poliakoff E. Dance and Parkinson’s: A review and exploration of the role of cognitive representations of action. Neurosci Biobehav Rev. 2020;109:16–28. References References 1. Elbaz A, Carcaillon L, Kab S, Moisan F. Epidemiology of Parkinson’s disease. Rev Neurol. 2016;172(1):14–26. 2. Tysnes OB, Storstein A. Epidemiology of Parkinson’s disease. Journal of neural transmission. 2017;124(8):901–5. 3. Jankovic J. Parkinson’s disease: clinical features and diagnosis. Journal of neurology, neurosurgery, and psychiatry. 2008;79(4):368–76. 4. Kalia LV, Lang AE. Parkinson’s disease. The Lancet. 2015;386(9996):896–912. 5. Brown S, Martinez MJ, Parsons LM. The neural basis of human dance. Cerebral cortex. 2006;16(8):1157–67. 6. Karkou V, Meekums B. Dance movement therapy for dementia. Cochrane Database Syst Rev. 2017;2(2):Cd011022. 7. Woods B, Spector A, Jones C, Orrell M, Davies S. Reminiscence therapy for dementia. Cochrane Database Syst Rev. 2005(2):Cd001120. 8. Colcombe S, Kramer AF. Fitness effects on the cognitive function of older adults: a meta-analytic study. Psychol Sci. 2003;14(2):125–30. 9. Atkins R, Deatrick JA, Gage GS, Earley S, Earley D, Lipman TH. Partnerships to Evaluate the Social Impact of Dance for Health: A Qualitative Inquiry. J Community Health Nurs. 2019;36(3):124–38. References 1. Elbaz A, Carcaillon L, Kab S, Moisan F. Epidemiology of Parkinson’s disease. Rev Neurol. 2016;172(1):14–26. 2. Tysnes OB, Storstein A. Epidemiology of Parkinson’s disease. Journal of neural transmission. 2017;124(8):901–5. 3. Jankovic J. Parkinson’s disease: clinical features and diagnosis. Journal of neurology, neurosurgery, and psychiatry. 2008;79(4):368–76. 4. Kalia LV, Lang AE. Parkinson’s disease. The Lancet. 2015;386(9996):896–912. 5. Brown S, Martinez MJ, Parsons LM. The neural basis of human dance. Cerebral cortex. 2006;16(8):1157–67. 6. Karkou V, Meekums B. Dance movement therapy for dementia. Cochrane Database Syst Rev. 2017;2(2):Cd011022. 7. Woods B, Spector A, Jones C, Orrell M, Davies S. Reminiscence therapy for dementia. Cochrane Database Syst Rev. 2005(2):Cd001120. 8. Colcombe S, Kramer AF. Fitness effects on the cognitive function of older adults: a meta-analytic study. Psychol Sci. 2003;14(2):125–30. 9. Atkins R, Deatrick JA, Gage GS, Earley S, Earley D, Lipman TH. Partnerships t Evaluate the Social Impact of Dance for Health: A Qualitative Inquiry. J Community Health Nurs. 2019;36(3):124–38. 1. Elbaz A, Carcaillon L, Kab S, Moisan F. Epidemiology of Parkinson’s disease. Rev Neurol. 2016;172(1):14–26. 1. Elbaz A, Carcaillon L, Kab S, Moisan F. Epidemiology of Parkinson’s disease. Rev Neurol. 2016;172(1):14–26. 2. Tysnes OB, Storstein A. Epidemiology of Parkinson’s disease. Journal of neural transmission. 2017;124(8):901–5. 2. Tysnes OB, Storstein A. Epidemiology of Parkinson’s disease. Journal of neural transmission. 2017;124(8):901–5. adults: a meta-analytic study. Psychol Sci. 2003;14(2):125–30. Additional file 10: Table S1. Characteristics of included studies 9. Author details 1 Author details 1Institute for Medical Research, National Institutes of Health, Ministry of Health, Shah Alam, Malaysia. 2School of Pharmacy, Monash University Malaysia, Subang Jaya, Malaysia. 3University of Western Sydney, Perth, Australia. 4University of Malaya Medical Centre, Kuala Lumpur, Malaysia. 5School of Medicine, Faculty of Health and Medical Sciences, Taylor’s University, Subang Jaya, Malaysia. 24. Goetz CG, Tilley BC, Shaftman SR, Stebbins GT, Fahn S, Martinez-Martin P, et al. Movement Disorder Society-sponsored revision of the Unified Parkinson’s Disease Rating Scale (MDS-UPDRS): scale presentation and clinimetric testing results. Mov Disord. 2008;23(15):2129–70. 25. Kennard C, Munro AJ, Park DM. The reliability of clinical assessment of Parkinson’s disease. J Neurol Neurosurg Psychiatry. 1984;47(3):322–3. Page 11 of 11 Page 11 of 11 Page 11 of 11 Ismail et al. BMC Geriatrics (2021) 21:503 Ismail et al. BMC Geriatrics Ismail et al. 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https://openalex.org/W4362630526	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Table_S1_from_Radiofrequency_Ablation_Remodels_the_Tumor_Microenvironment_and_Promotes_Neutrophil-Mediated_Abscopal_Immunomodulation_in_Pancreatic_Cancer/22546010/1/files/40009478.pdf	English	null	Supplementary Table S1 from Radiofrequency Ablation Remodels the Tumor Microenvironment and Promotes Neutrophil-Mediated Abscopal Immunomodulation in Pancreatic Cancer	null	2,023	cc-by	110	Supplemental Table 1 List of antibodies for IMC Metal labeling Specificity Nd144 Ly6C Nd145 Ly6G Nd146 CD11b Sm147 a-SMA Nd148 Pan-CK Sm149 CD11c Sm152 CD163 Gd155 pSTAT1 Gd156 Endomucin Gd160 Foxp3 Dy162 CD8a Dy164 PD-L1 Er166 CD44 Er167 Ki-67 Tm169 PD-1 Er170 B220 Yb171 NK1.1 Yb174 CD4 Lu175 CD86 Yb176 F4/80 Supplemental Table 1 List of antibodies for IMC Metal labeling Specificity Nd144 Ly6C Nd145 Ly6G Nd146 CD11b Sm147 a-SMA Nd148 Pan-CK Sm149 CD11c Sm152 CD163 Gd155 pSTAT1 Gd156 Endomucin Gd160 Foxp3 Dy162 CD8a Dy164 PD-L1 Er166 CD44 Er167 Ki-67 Tm169 PD-1 Er170 B220 Yb171 NK1.1 Yb174 CD4 Lu175 CD86 Yb176 F4/80 Supplemental Table 1 List of antibodies for IMC.
https://openalex.org/W2984964144	https://ojs.unud.ac.id/index.php/psikologi/article/download/37124/22490	Indonesian	null	KONSEP SEHAT DAN SAKIT PADA INDIVIDU DENGAN UROLITHIASIS (KENCING BATU) DI KABUPATEN KLUNGKUNG, BALI	Jurnal Psikologi Udayana	2,018	cc-by-sa	8,572	Abstrak Pada era globalisasi ini, dengan perkembangan pengobatan medis yang semakin maju, masih banyak dari masyarakat yang tetap menggunakan pengobatan tradisional, yang dipercaya mampu mengobati berbagai penyakit. Menurut Asimo (1995) penggunaan pengobatan tradisional tidak terlepas dari ketidakpuasan terhadap pengobatan medis. Hal ini juga terlihat pada masyarakat Bali yang masih banyak menggunakan pengobatan tradisional. Gagasan tersebut didukung dengan pre-eliminary study yang menunjukan adanya ketergantungan terhadap pengobatan tradisional yang mengakibatkan keterlambatan pengobatan medis. Penelitian ini menggunakan metode penelitian kualitatif dengan desain penelitian studi kasus. Pengambilan data pada penelitian ini menggunakan teknik wawancara dan observasi. Responden yang digunakan pada penelitian ini merupakan salah seorang kepala keluarga dengan kasus keterlambatan pengobatan medis, dengan dua orang informan untuk memperkuat data penelitian. Hasil penelitian ini menjelaskan bahwa konsep sehat dan sakit pada responden penelitian dipengaruhi oleh; 1) faktor biologis yakni pemahaman terhadap kondisi fisiologisnya; 2) faktor psikologis yang mempengaruhi responden terhadap konsep sehat dan sakitnya dan cara yang dilakukan untuk menjaga kesehatannya; 3) faktor sosial yakni pengaruh masyarakat dan keluarga terhadap konsep sehat dan sakitnya. Health seeking behavior responden terbentuk, karena dipengaruhi oleh perilaku yang dibentuk berdasarkan pengetahuan dan sikap responden serta health system model yang terdiri dari karakteristik predisposisi, karakteristik pendukung, dan karakteristik kebutuhan yang juga dipengaruhi oleh faktor biologis, psikologis, serta sosialnya. Berdasarkan hasil temuan tersebut didapatkan bahwa health seeking behavior responden dalam menggunakan pengobatan usada dan juga medis dipengaruhi oleh konsep sehat dan sakit yang dimilikinya. Kata Kunci: konsep sehat dan sakit, health seeking behavior, health system model KONSEP SEHAT DAN SAKIT PADA INDIVIDU DENGAN UROLITHIASIS (KENCING BATU) DI KABUPATEN KLUNGKUNG, BALI Samuel Dwi Krisna Triyono, Yohanes K. Herdiyanto Samuel Dwi Krisna Triyono, Yohanes K. Herdiyanto Program Studi Psikologi, Fakultas Kedokteran, Universitas Udayana samuelkrisnha@gmail.com Jurnal Psikologi Udayana 2017, Vol.4, No.2, 263- 276 Jurnal Psikologi Udayana 2017, Vol.4, No.2, 263- 276 Program Studi Psikologi, Fakultas Psikologi, Universitas Udayana ISSN: 2354 5607 Kata Kunci: konsep sehat dan sakit, health seeking behavior, health system model LATAR BELAKANG atau tidak, digunakan dalam pemeliharaan kesehatan serta dalam pencegahan, diagnosa, perbaikan atau pengobatan penyakit secara fisik dan juga mental. Definisi kesehatan menurut Undang-Undang nomor 36 tahun 2009 adalah “keadaan sehat, baik secara fisik, mental, spiritual maupun sosial untuk memungkinkan setiap orang untuk hidup produktif secara sosial dan ekonomi” (Undang- undang tentang kesehatan tahun 2009). Kesehatan merupakan hal yang sangat penting dalam kehidupan manusia, sehat juga merupakan keadaan dari kondisi fisik yang baik, mental yang baik, dan juga kesejahteraan sosial, tidak hanya merupakan ketiadaan dari penyakit atau kelemahan (WHO, 1948). Pengobatan tradisional memiliki keuntungan yakni dari segi biaya yang lebih murah jika dibandingkan dengan pengobatan modern, sedangkan kelemahannya adalah pengobatan tradisional ini tidak pernah melalui uji ilmiah sehingga kelayakan dari pengobatan tradisional ini masih sering dipertanyakan. Bahkan pada beberapa kasus, penggunaan pengobatan tradisional mengakibatkan keterlambatan penanganan pengobatan medis sehingga membuat penyakit yang diderita menjadi semakin parah. Pengertian sakit adalah berasa tidak nyaman di tubuh atau bagian tubuh karena menderita sesuatu (demam, sakit perut, dan lain-lain). Sakit juga merupakan gangguan dalam fungsi normal individu sebagai totalitas, termasuk keadaan organisme sebagai sistem biologis dan penyesuaian sosialnya (Parson, 1972). Sakit juga dapat disebabkan oleh beberapa hal, baik itu yang berasal dari gaya hidup yang kurang sehat, lingkungan yang tidak bersih, ataupun karena menurunnya metabolisme tubuh. Menurut Asimo (1995), pengobatan tradisional dibagi menjadi dua bagian yaitu cara penyembuhan tradisional atau traditional healing yang terdiri dari pijatan, kompres, akupuntur dan sebagainya, serta obat tradisional atau traditional drugs yaitu menggunakan bahan-bahan yang telah tersedia dari alam sebagai obat untuk menyembuhkan penyakit. Obat tradisional terbagi menjadi tiga bagian. Obat yang pertama merupakan obat yang berasal dari sumber nabati yang diambil dari bagian-bagian tumbuhan seperti buah, daun, kulit batang dan sebagainya, yang kedua adalah yang bersumber dari hewani seperti bagian kelenjar-kelenjar, tulang-tulang maupun dagingnya dan yang ketiga adalah berasal dari sumber mineral atau garam-garam yang bisa didapatkan dari mata air yang dikeluarkan dari tanah. Saat ini, berbagai fasilitas medis sudah semakin diperhatikan terkait dengan perkembangan penyakit yang berbeda di tiap tahunnya, pelayanan kesehatan sudah banyak disediakan dengan berbagai alat modern dalam menunjang pekerjaannya. Tidak lupa juga adanya tenaga profesional yang membantu dokter dalam pekerjaannya, pada umumnya tenaga profesional ini termasuk ke dalam tenaga kesehatan. Abstract In this globalization era, with the development of medical treatment, there are still plenty of people who still use traditional medicine, which is believed to cure various diseases. According to Asimo (1995) the use of the traditional treatment is inseparable from dissatisfaction with medical treatment. It also looks at the Balinese people who still use traditional treatment. This explanation was supported by the preliminary study that shows dependence of the traditional healing that result delay on the medical treatment. This study use qualitative research method with case study research design. Collecting data in this study use interview and observation techniques. Respondent were used in this study was a householder with case of delays in medical treatment, and with two significant others to strengthen data obtained. The result of this research explains that the concept of health and sick on respondent are influenced by: 1) biological factor that the comprehension of the physiological form; 2) psychological factor that influence the concept of the health and illness, and how to maintain his health; 3) social factor that affected by family and society. Health seeking behavior of respondent is formed,as influenced by behavior that was formed by the knowledge and attitudes, and health system model that consisting of predisposing characteristic, enabling characteristic, and need characteristic and also affected by his biological, psychological, and social factors. Based on these findings, show that the health seeking behavior of respondent in using usada treatment and medical treatment is influenced by the respondent’s health and sick concept. Keyword: the concept of health and illness, health seeking behavior, health system model Keyword: the concept of health and illness, health seeking behavior, health system model 263 KONSEP SEHAT DAN SAKIT PADA INDIVIDU DENGAN UROLITHIASIS bahwa pengobatan tradisional merupakan pengobatan alternatif yang digunakan masyarakat apabila pengobatan konvensional tidak memberikan hasil yang memuaskan. ke medis atau non medis tergantung dari jenis penyakitnya, dan juga bisa menggunakan kedua jalur tersebut sekaligus” (Asmara, 2015). Seperti yang dikatakan oleh Subandi, dan Utami (1996) bahwa adanya proses evaluasi setelah penggunaan pengobatan dari professional maupun non-profesional. Hal ini mengakibatkan tidak jarang masyarakat akan beralih ke pengobatan alternatif ketika pengobatan modern memberikan hasil yang kurang memuaskan, dan begitu juga sebaliknya. Dewasa ini, walaupun ilmu dan teknologi kedokteran sudah mengalami kemajuan pesat, namun peran dan eksistensi pengobatan usada di Bali sebagai pengobatan alternatif masih cukup menonjol. Seperti yang dialami oleh WS, salah seorang masyarakat di Bali , yang berdomisili di Kabupaten Klungkung. WS merupakan salah seorang individu dengan urolitiasi (kencing batu) yang selama lima tahun terakhir menggunakan pengobatan tradisional usada, tanpa diikuti dengan penanganan secara medis, sehingga satu tahun terakhir WS sempat melakukan operasi karena hampir terjadinya keterlambatan pengobatan terhadap sakit kencing batu yang dialami oleh WS. Perilaku menentukan arah pengobatan dikenal dengan istilah health seeking behavior, Notoadmojo (2014) mengatakan bahwa ketika seseorang mengalami sakit maka akan memunculkan beberapa respon yaitu tidak bertindak, tindakan mengobati diri sendiri, mencari pengobatan tradisional, dan mencari pengobatan ke fasilitas-fasilitas pengobatan modern. Notoadmojo (2014) menambahkan bahwa setiap elemen masyarakat memiliki konsep sehat dan sakit yang berbeda-beda sehingga akan mempengaruhi health seeking behavior ketika mengalami kondisi sakit, sehingga persepsi masyarakat terhadap sehat dan juga sakit memiliki hubungan yang erat terhadap health seeking behavior. Kondisi ini terjadi menurut berbagai kalangan karena pengobatan usada, disamping masih fungsional secara sosial dan lebih murah biayanya, juga cukup efektif untuk menyembuhkan jenis atau golongan penyakit tertentu (Sukarma, 2013). Berobat ke balian sudah menjadi kebiasaan, masyarakat akan cenderung pergi ke balian ketika mengalami sakit, latar belakang melakukan pengobatan usada adalah dikarenakan tidak adanya hasil terhadap pengobatan medis yang sudah dilakukan, faktor lainnya yang mempengaruhi adalah perbedaan biaya yang dikeluarkan dengan pengobatan modern. Pada budaya di Bali pengobatan tradisional ini disebut dengan pengobatan usada sedangkan seseorang yang memiliki kemampuan untuk melakukan pengobatan usada disebut dengan balian. Secara etimologi kata usada berasal dari kata ausadhi yang berarti tumbuh-tumbuhan yang mengandung khasiat obat-obatan (Nala, 1992). Usada adalah ilmu pengobatan tradisional Bali yang dikenalkan oleh para leluhur dan merupakan ilmu pengetahuan penyembuhan yang dijiwai oleh nilai-nilai agama Hindu (Sukantara, 1992). LATAR BELAKANG Masyarakat akan menentukan arah berobat atau melakukan pengobatan, baik itu ke pengobatan tradisional maupun modern, namun pada dasarnya budaya juga mengambil peran yang penting dalam pembentukan perilaku dan kepercayaan ini, seperti penelitian yang dilakukan oleh Quah dan Bishop (1996). Quah dan Bishop (1996) melakukan penelitian terhadap warga Cina asli dengan Cina-Amerika terkait dengan persepsi terhadap kesehatan, warga asli Cina menganggap bahwa penyakit muncul akibat adanya ketidakseimbangan dalam tubuh, hal ini sama dengan budaya di Cina yang menganggap bahwa seseorang dikatakan sehat apabila memiliki keseimbangan antara Yin dan Yang, sedangkan warga Cina-Amerika mengatakan bahwa suatu penyakit muncul diakibatkan oleh virus-virus, sehingga warga Cina asli akan memilih berobat ke pengobatan tradisional Cina sedangkan warga Cina-Amerika akan lebih memilih untuk berobat ke tenaga kesehatan (Matsumoto & Juang, 2008). Semakin majunya dunia kesehatan tidak berjalan beriringan dengan perilaku sehat dari masyarakat. Perilaku sehat pada dasarnya adalah respon seseorang terhadap stimulus yang berkaitan dengan sakit dan penyakit, sistem pelayanan kesehatan, makanan, serta lingkungan (Simons – Morton et al., 1965). Dasar orang berperilaku dapat ditentukan oleh nilai, sikap, dan pendidikan atau pengetahuan (Notoadmojo, 2005). Masyarakat sering kali enggan untuk pergi ke rumah sakit yang umumnya disebabkan karena biaya pengobatan di rumah sakit yang terbilang cukup tinggi bagi masyarakat dengan tingkat perekonomian menengah kebawah. Terdapat dua jenis pengobatan yang sering digunakan oleh masyarakat antara lain pengobatan modern, dan pengobatan tradisional. Pengobatan modern adalah pengobatan yang berkembang saat ini, yakni dengan metode medis dan kedokteran, pengobatan modern dilakukan dengan cara-cara ilmiah atau telah diujicobakan dengan penelitian dan dipertanggungjawabkan hasilnya, dan pengobatan tradisional menurut WHO (2000) adalah jumlah total pengetahuan, keterampilan, dan praktek-praktek yang berdasarkan teori- teori, keyakinan, dan pengalaman masyarakat yang mempunyai adat dan budaya yang berbeda, baik dijelaskan Penelitian yang dilakukan oleh Quah dan Bishop (1996) juga memiliki kemiripan terhadap kehidupan tradisional masyarakat di Indonesia. Hal ini cukup terlihat pada masyarakat yang berdomisili di Bali yang menggunakan kedua teknik pengobatan yaitu pengobatan modern dan pengobatan tradisional, seperti yang dikatakan Asimo (1995) 264 Responden Responden didapatkan dengan cara menggunakan teknik purposive sampling. Purposive sampling adalah teknik pengambilan sampel sumber data dengan pertimbangan tertentu, sehingga memerlukan adanya kriteria tertentu yang ditujukan kepada sampel yang akan digunakan (Sugiyono, 2014). Responden yang digunakan pada penelitian ini merupakan seorang kepala rumah tangga yang berdomisili di Desa Sampalan, Kabupaten Klungkung, Bali. Responden penelitian yang digunakan pada penelitian ini memiliki kasus pengunaan pengobatan tradisional yang tidak diimbangi dengan penggunaan pengobatan medis selama mengalami penyakit kencing batu (urolithiasis), sehingga hampir terjadinya keterlambatan penanganan. Tipe Penelitian Jenis penelitian yang digunakan pada penelitian ini merupakan jenis penelitian kualitatif dengan menggunakan pendekatan studi kasus. Metode penelitian kualitatif adalah metode penelitian yang berlandaskan pada filsafat pospositivisme, digunakan untuk meneliti pada kondisi objek yang alamiah, yang memposisikan peneliti sebagai instrument kunci, pengambilan sampel sumber data dilakukan secara purposive sampling, teknik pengumpulan dengan triangunlasi atau gabungan, analasis data bersifat induktif atau kualitatif, dan hasil penelitian kualitatif menekankan makna daripada generalisasi (Sugiyono, 2014). Unit Analisis Unit analisis adalah suatu kajian kerja yang terdapat pada penelitian kualitatif. Satuan kajian penelitian kualitatif akan mempengaruhi dalam penentuan sampel, besarnya sampel dan strategi sampling yang akan digunakan (Moleong, 2000). Pada penelitian yang dilakukan, menggunakan satuan kajian perseorangan yang berasal dari suatu daerah tertentu di Bali. Daerah dari penelitian yang akan dilakukan, merupakan daerah yang masih memegang adat serta kebudayaan. Penggunaan responden yaitu individu dalam suatu daerah di Bali dengan adat serta budaya yang masih dijaga didasarkan kepada fokus penelitian bahwa health seeking behavior terbentuk akibat adanya intervensi dari lingkungan, sehingga individu akan cenderung memiliki suatu bentuk kepercayaan terhadap konsep sehat dan sakit yang dipengaruhi oleh lingkungan yang menghasilkan health seeking behavior yang berbeda-beda. Sebagian besar dari pasien yang menggunakan pengobatan usada mengatakan bahwa penyakitnya dapat sembuh. Saat ini, berbagai fasilitas kedokteran medis sudah mengalami perkembangan yang pesat, berbagai obat bahkan untuk penyakit yang awalnya tidak terdeteksi dapat diberikan intervensi dengan cepat dan akurat, namun banyak dari masyarakat yang masih mempercayakan kondisi kesehatan terkait dengan penyakit-penyakit tertentu yang diderita kepada pengobatan tradisional. Kepercayaan masyarakat dalam menggunakan pengobatan tradisional ini kemudian memunculkan keingintahuan yaitu untuk mengetahui konsep sehat dan sakit pada masyarakat yang kemudian mengarahkan health seeking beahavior terhadap pengobatan tradisional. S.D. K.TRIYONO & Y. K. HERDIYANTO atau kapha berada dalam keadaan seimbang dan berfungsi dengan baik. akan diselidiki, dan apabila fokus penelitian terletak pada fenomena kontemporer atau kejadian masa kini di dalam konteks kehidupan nyata. Yin (2014) mengatakan bahwa tujuan penggunaan penelitian studi kasus tidak hanya untuk menjelaskan seperti apa objek yang diteliti, tetapi menjelaskan bagaimana keberadaan dan mengapa kasus tersebut dapat terjadi. Sebaliknya manusia akan sakit apabila unsur-unsur panca brahma sebagai kekuatan panas, dan unsur-unsur panca tirta sebagai kekuatan dingin saat bereaksi dengan udara, ada dalam keadaan tidak seimbang (Sukarma, 2013). Jika terjadi ketidakseimbangan pada unsur-unsur tersebut maka akan menimbulkan penyakit tertentu. Hal inilah yang mempengaruhi masyarakat Bali pergi ke balian untuk berobat yang didasari dengan kepercayaan terhadap ketidakseimbangan unsur-unsur tersebut yang mengakibatkan munculnya penyakit-penyakit tertentu. Teknik dan cara pengobatan yang dilakukan oleh balian adalah dengan menggunakan pengobatan non-medis yakni melalui beberapa upacara adat, “Balian memberikan tirta dan juga mantra kepada ayahnya.” (Asmara, 2015) dan kemudian dilanjutkan dengan memberikan obat-obatan herbal. KONSEP SEHAT DAN SAKIT PADA INDIVIDU DENGAN UROLITHIASIS “Menurut responden, balian merupakan seseorang yang memiliki kemampuan melebihi manusia pada umumnya, kemampuan ini bisa didapatkan ataupun diperoleh” (Dewi, 2015), merupakan salah satu kutipan dari hasil wawancara terkait dengan balian. Responden mempercayai bahwa balian merupakan seseorang yang memiliki kemampuan untuk menyembuhkan orang lain dari penyakit yang bersifat medis maupun non–medis. Hal ini sama seperti yang disampaikan oleh responden berdasarkan hasil dari wawancara yang dilakukan, “Responden mengatakan bahwa dulu responden pernah mengalami sakit kepala yang tidak sembuh-sembuh selama beberapa hari. Responden kemudian memutuskan untuk pergi ke dokter, namun sesampainya di rumah sakit dokter hanya mengatakan bahwa ini cuma sakit kepala biasa” (Dewi, 2015). Hal seperti ini dianggap wajar apabila kebiasaan ini dilakukan disaat belum adanya kemajuan dalam dunia medis. Dalam konteks sistem medis suku Bali atau usada dan konsepsi balian tentang sehat-sakit, bahwa orang bisa disebut sebagai manusia sehat apabila semua sistem dan unsur pembentuk tubuh atau panca maha bhuta yang terdiri dari : pertiwi atau tanah yang berarti segala sesuatu yang bisa disentuh, dirasakan, kokoh dan nata, apah atau air yang berarti kebalikan dari pertiwi yakni segala sesuatu yang lentur, mengalir, fleksibel, luwes, mendinginkan, dan tidak memiliki bentuk yang kokoh, teja atau api yang membawa dua hal yaitu panas dan cahaya, bayu atau angin yang berarti segala sesuatu yang melindungi atau melingkupi, dan akasa yang diartikan sebagai eter , dan unsur dalam tubuh yang dikenal dengan isitilah tri dosha, yaitu udara atau vatta, api atau pitta, dan air Bidang medis sudah mengalami perkembangan yang sangat cepat dari segi diagnosis maupun dari sisi pengobatan, namun disaat bidang medis sudah mengalami kemajuan, banyak dari masyarakat masih cenderung menggunakan pengobatan usada. Meskipun hal ini tidak bisa dipukul rata kepada semua masyarakat di Bali, di beberapa daerah terutama perkotaan terlihat bahwa pengobatan usada merupakan pilihan alternatif ketika pengobatan secara modern atau medis tidak memberikan hasil yang baik dan memuaskan, seperti hasil wawancara yang telah dilakukan sebelumnya, “Responden biasanya menyarankan orang yang dikenalnya untuk berobat 265 Tempat Penelitian Pengambilan data penelitian di lakukan di Kabupaten Klungkung, dengan responden yaitu salah satu anggota dari keluarga yang berdomisili di Kabupaten Klungkung, hal ini dikarenakan di Klungkung adat istiadat serta penggunaan pengobatan usada masih terasa cukup kental. Responden yang digunakan berasal dari desa Sampalan, desa Sampalan merupakan salah satu desa adat di Kabupaten Klungkung. Pendekatan yang digunakan pada penelitian ini adalah pendekatan studi kasus. Studi kasus merupakan strategi penelitian yang digunakan apabila peneliti hanya memiliki sedikit peluang untuk mengontrol peristiwa-peristiwa yang 266 1. Triangulasi Sumber Triangulasi sumber untuk menguji kredibilitas data dilakukan dengan cara mengecek data yang telah diperoleh melalui beberapa sumber yang lainnya, seperti menggunakan lingkungan sekitar responden, ataupun significant others untuk memperoleh lebih banyak informasi. Pemantapan Kredibilitas Data Pemantapan kredibilitas penelitian yang dilakukan pada penelitian ini adalah dengan menggunakan teknik triangulasi. Triangulasi merupakan pengecekan data dari berbagai sumber dengan berbagai cara, dan berbagai waktu (Wiersma, 1986). Triangulasi kemudian dibagi kedalam tiga bagian yaitu triangulasi sumber, triangulasi teknik pengumpulan data, dan triangulasi waktu (Sugiyono, 2014). 1. Teknik Analisis Sebelum di Lapangan Teknik analisis data yang digunakan sebelum memasuki lapangan adalah dengan melakukan analisis seperti pengecekan dan pemahaman yang dilakukan terhadap data hasil studi pendahuluan yang akan digunakan untuk menentukan fokus penelitian, dan fokus penelitian dapat berkembang setelah masuk dan selama di lapangan (Sugiyono, 2014). Teknik Penggalian Data Teknik penggalian data yang dilakukan pada penelitian ini menggunakan tekni wawancara semi terstruktur. Esterberg (2002) mendefinisikan bahwa wawancara merupakan pertemuan dua orang untuk bertukar informasi dan ide melalui tanya jawab, sehingga dapat dikonstruksikan makna dalam suatu topik tertentu. Wawancara semiterstruktur atau semistructure interview yang termasuk dalam kategori in- depth interview yang menjelaskan di dalam pelaksanaannya lebih bebas bila dibandingkan dengan wawancara terstruktur. Tujuan dari wawancara jenis ini adalah untuk menemukan permasalahan secara lebih terbuka. Di dalam wawancara semiterstruktur responden diminta pendapat, dan ide-idenya. KONSEP SEHAT DAN SAKIT PADA INDIVIDU DENGAN UROLITHIASIS dengan mereduksi data maka akan memberikan gambaran yang lebih jelas, dan mempermudah dalam melakukan pengumpulan data selanjutnya, dan mencarinya bila diperlukan. Reduksi data dapat dilakukan dengan menggunakan theoretical coding yang terdiri dari open coding, axial coding, dan selective coding. Desa adat atau yang di Bali lebih dikenal dengan desa pakraman memiliki perbedaan dengan desa dinas. Desa adat merupakan desa yang masih menggunakan spiritual keagamaan yang paling mendasar bagi pola hubungan dan pola interaksi sosial di dalam masyarakat Bali, di dalam desa adat juga terdapat awig-awig atau aturan-aturan tertentu yang didasarkan pada adat dan budayanya, sehingga desa adat tentunya memiliki nilai kebudayaan yang lebih kental jika dibandingkan dengan desa dinas. 2. Triangulasi Teknik Analisis data dalam penelitian kualitatif dilakukan sejak sebelum memasuki lapangan, selama di lapangan, dan setelah selesai di lapangan (Sugiyono, 2014). Triangulasi teknik untuk menguji kredibilitas data dilakukan dengan cara mengecek data kepada sumber data yang sama dengan teknik yang berbeda, misalnya pada data awal pengambilan data dilakukan dengan wawancara, maka data yang kedua diambil dengan menggunakan observasi, dan dokumentasi. b. Pengaruh faktor psikologis terhadap konsep sehat dan sakit Dari faktor psikologis, kemudian juga ditemukan konsep sehat dan sakit pada responden. Menurut Sarafino dan Smith (2011) faktor psikologis dibagi menjadi tiga bagian, yaitu pengaruh dari kognisi, emosi, dan motivasi. 1. Pengaruh dari sisi kognisi Kognisi merupakan aktivitas mental yang mencakup cara menerima, belajar, mengingat, berpikir, menginterpretasi, mempercayai, dan menyelesaikan masalah (Sarafino & Smith, 2011). Dari kondisi kognisi dapat dilihat bahwa responden menganggap kesehatan merupakan hal yang sangat luar biasa dan juga sulit untuk mendapatkannya, responden mengatakan bahwa kesehatan merupakan segala-galanya, sehingga bagi responden kesehatan memiliki nilai yang sangat mahal, namun hal ini mungkin saja muncul setelah responden mengalami sakit kencing batunya, hal ini dikarenakan setelah melakukan operasi responden mengatakan lebih waspada terhadap kondisi kesehatannya saat ini. 1. Pengaruh dari sisi kognisi Kognisi merupakan aktivitas mental yang mencakup cara menerima, belajar, mengingat, berpikir, menginterpretasi, mempercayai, dan menyelesaikan masalah (Sarafino & Smith, 2011). Dari kondisi kognisi dapat dilihat bahwa responden menganggap kesehatan merupakan hal yang sangat luar biasa dan juga sulit untuk mendapatkannya, responden mengatakan bahwa kesehatan merupakan segala-galanya, sehingga bagi responden kesehatan memiliki nilai yang sangat mahal, namun hal ini mungkin saja muncul setelah responden mengalami sakit kencing batunya, hal ini dikarenakan setelah melakukan operasi responden mengatakan lebih waspada terhadap kondisi kesehatannya saat ini. 3. Triangulasi Waktu Triangulasi waktu untuk menguji kredibilitas data dilakukan dengan cara melakukan pengambilan data dengan latar belakang waktu yang berbeda, karena kondisi responden dari waktu ke waktu akan selalu berbeda. 2. Teknik Analasis Selama sampai dengan Selesai di Lapangan 2. Teknik Analasis Selama sampai dengan Selesai di Lapangan Teknik analisis data yang digunakan pada tahap ini adalah teknik yang dikemukakan oleh Miles dan Huberman (1984) yaitu data reduction atau reduksi data, data display atau penyajian data, dan conclusion drawing, verification atau penarikan kesimpulan dan verifikasi. Reduksi data adalah kegiatan merangkum, memilih hal- hal yang pokok, memfokuskan pada hal-hal yang penting, mencari tema dan pola serta membuang yang tidak perlu, 267 S.D. K.TRIYONO & Y. K. HERDIYANTO komponen dari tubuh individu saling bekerja dan berinteraksi untuk menciptakan kondisi sehat, sehingga apabila dilihat dari kondisi responden saat ini, bahwa kondisi tubuh dari responden berada dalam keadaan yang tidak sehat, karena sempat mengalami kencing batu yang berkepanjangan, ditambah lagi dengan kondisi kebugaran responden yang semakin hari semakin menurun, dan pendengarannya yang semakin memburuk. 1. Konsep sehat dan sakit Berdasarkan hasil penelitian yang telah dilakukan, ditemukan faktor-faktor yang membentuk konsep sehat dan juga sakit pada responden. Adapun faktor-faktor yang mempengaruhi konsep sehat dan juga sakit pada responden yaitu faktor biologis, psikologis, dan sosial budaya (Sarafino & Smith, 2011). Di sisi lainnya, dengan perkataan dari responden yang mengungkapkan bahwa kesehatan merupakan segalanya, responden tidak terlalu baik dalam menjaga kesehatan, seperti bekerja terlalu keras sehingga sering melupakan kondisi kesehatannya. Sarafino dan Smith (2011) mengatakan bahwa kondisi sakit tidak hanya diakibatkan oleh faktor biologis saja, namun juga bisa diakibatkan oleh perilaku dari individu. a. Pengaruh dari faktor biologis terhadap konsep sehat dan sakit a. Pengaruh dari faktor biologis terhadap konsep sehat dan sakit a. Pengaruh dari faktor biologis terhadap konsep sehat dan sakit Dari faktor biologis dapat dilihat bagaimana responden menyadari penyakitnya yaitu kencing batu, responden sudah menderita sakit kencing batu tersebut selama lebih dari lima tahun, bukanlah waktu yang sebentar terlebih lagi dengan penyakit kencing batunya tersebut, namun pada awalnya responden hanya mengetahui bahwa responden terkena kencing batu dari beberapa artikel yang dibaca, dan juga saat responden melakukan percakapan dengan teman-teman seprofesi. Hal ini kemudian ditambahkan oleh significant others yang mengatakan bahwa penyakit tersebut terlihat seperti penyakit biasa, hanya panas dibagian perut saja. Dari faktor biologis dapat dilihat bagaimana responden menyadari penyakitnya yaitu kencing batu, responden sudah menderita sakit kencing batu tersebut selama lebih dari lima tahun, bukanlah waktu yang sebentar terlebih lagi dengan penyakit kencing batunya tersebut, namun pada awalnya responden hanya mengetahui bahwa responden terkena kencing batu dari beberapa artikel yang dibaca, dan juga saat responden melakukan percakapan dengan teman-teman seprofesi. Hal ini kemudian ditambahkan oleh significant others yang mengatakan bahwa penyakit tersebut terlihat seperti penyakit biasa, hanya panas dibagian perut saja. 2. Pengaruh dari sisi emosi Hal ini juga bisa dilihat dari sisi emosi responden, significant others yaitu istri responden mengatakan bahwa selama masih bisa ditahan, maka responden akan menahan rasa sakitnya dan tetap melakukan aktivitasnya seperti biasanya, responden juga mempercayakan kondisi tubuhnya terhadap minuman herbal yang dikonsumsi oleh responden saat sedang mengalami kondisi sakit, namun kemudian permasalahan muncul, saat mengalami sakit responden seringkali tidak memeriksakan diri Menurut Sarafino dan Smith (2011) kondisi biologis tersebut berhubungan dengan bagaimana masing-masing 268 3. Pengaruh dari budaya Di sisi lainnya, faktor budaya juga mengambil peran yang sangat penting dalam pembentukan konsep sehat dan sakit dari responden. Matsumoto dan Juang (2008) juga mengatakan bahwa budaya dapat mempengaruhi kesehatan dari berbagai sisi. Dalam wawancara yang dilakukan, responden mengatakan bahwa hidup harus berdasarkan keseimbangan, sehingga apabila sudah seimbang maka tubuh akan selalu sehat. c. Pengaruh faktor sosial dan budaya terhadap konsep sehat dan sakit c. Pengaruh faktor sosial dan budaya terhadap konsep sehat dan sakit Pembentukan konsep sehat dan sakit dari responden tidak semata-mata terbentuk begitu saja, Sarafino dan Smith (2011) mengatakan bahwa faktor sosial dan budaya juga mengambil peran yang penting dalam hal tersebut. KONSEP SEHAT DAN SAKIT PADA INDIVIDU DENGAN UROLITHIASIS ke dokter, dan lebih memilih menggunakan pengobatan tradisional saja. ke dokter, dan lebih memilih menggunakan pengobatan tradisional saja. masyarakat yang seringkali memberikan referensi untuk menentukan arah pengobatan yang digunakan. Bahkan saat mengalami sakit kencing batu, responden sempat bertanya kepada masyarakat di lingkungan tempat tinggalnya, baik kepada teman- teman dekatnya maupun kepada beberapa tetangga di banjar, yang kemudian menyarankan responden untuk menggunakan pengobatan usada dengan memanfaatkan tumbuhan yang tumbuh di alam, sehingga memang benar bahwa faktor sosial terutama masyarakat seringkali mempengaruhi konsep sehat dan sekit dari responden dan menentukan arah pengobatan dari responden. Seperti yang dikatakan oleh Sarafino dan Smith (2011) bahwa emosi juga bisa mempengaruhi arah pengobatan dari seseorang. Istri responden mengatakan bahwa responden menggunakan pengobatan tradisional dikarenakan memiliki kecemasan apabila pergi ke dokter, bentuk kecemasan tersebut adalah kecemasan akan diambilnya tindakan operasi ketika penyakit dari responden telah diketahui. 3. Pengaruh dari sisi motivasi Kondisi ekonomi dari responden tersebutlah yang kemudian memotivasi responden untuk tetap menggunakan pengobatan tradisional yaitu pengobatan usada untuk mengobati penyakit kencing batunya. Selama kurang lebih lima tahun responden menggunakan pengobatan usada tersebut, menggunakan obat tradisional seperti loloh dan juga boreh. Tidak hanya saat itu, bahkan setelah operasi, responden tetap menggunakan pengobatan tradisional seperti memanfaatkan daun kecibling yang kemudian direbus dan dikonsumsi untuk mencegah munculnya penyakit kencing batu tersebut. 2. Pengaruh dari keluarga Hal ini tentunya mendapatkan dukungan dari istri responden, istri dari responden juga mempercayai dan sering menggunakan pengobatan usada apabila mengalami sakit. Muprhy dan Bennet (2004) mengatakan bahwa seseorang bertumbuh dan berkembang di dalam keluarga sejak dari masa kanak-kanak sehingga keluarga memberikan pengaruh yang paling kuat selama masa pertumbuhan dan perkembangan. Hal ini sejalan dengan yang diungkapkan oleh istri responden, penggunaan pengobatan usada sudah dilakukan sejak masih anak-anak. Hal ini dikarenakan responden mendapat informasi bahwa penyakit kencing batu tersebut sewaktu-waktu dapat muncul kembali, sehingga cara mencegahnya menurut responden adalah dengan menggunakan daun kecibling tersebut. Responden mengatakan selama mengkonsumsi rebusan daun kecibling tersebut maka responden tidak akan terkena penyakit kencing batu lagi. S.D. K.TRIYONO & Y. K. HERDIYANTO alam. Responden juga mengatakan bahwa hubungan dengan sesama manusia haruslah tetap dijaga, dalam kepercayaan di Bali terdapat suatu ajaran yang mengatur bagaimana cara untuk menjaga pikiran, perkataan, dan perbuatan yang dilakukan. dari responden untuk ke dokter karena takut akan dilakukan tindakan operasi karena kondisi perekonomian yang kurang memadai saat itu. Disamping hal tersebut responden juga mempercayai bahwa selama responden tetap mengkonsumsi obat-obat tradisional, tetap menggunakan pengobatan usada, maka responden akan terhindar dari berbagai penyakit, dan dapat mencegah timbulnya penyakit-penyakit lainnya. Responden mempercayai bahwa pengobatan usada merupakan cara yang tepat dalam menyembuhkan penyakit dari responden, dikarenakan responden mempercayai bahwa munculnya penyakit terjadi akibat adanya ketidakseimbangan, seperti konsep tri hita karana yang telah disampaikan. Dalam kepercayaan di Bali, responden juga mengatakan tentang tanggung jawab, tanggung jawab seorang kepala rumah tangga untuk memenuhi kebutuhan keluarganya, sehingga kemudian responden mengatakan hal inilah yang juga mendasari responden untuk tetap bekerja meskipun dalam keadaan sakit. karena responden memiliki sebuah keluarga, dan merupakan tanggung jawabnya untuk memberikan nafkah kepada keluarga, sehingga responden akan tetap bekerja meskipun dalam keadaan yang kurang sehat. Dalam beberapa kasus menurut responden kondisi sakit juga bisa diakibatkan oleh beberapa penyebab yang di luar medis, seperti adanya janji-janji di masa lalu, ataupun permasalahan dalam perilaku responden sehari- hari, sehingga pengobatan usada dilakukan untuk mengetahui jenis dan penyebab penyakitnya, dan juga cara penyembuhannya. Seperti yang dikatakan Nala (2006) bahwa usada merupakan tata cara yang digunakan untuk menyembuhkan penyakit, cara pengobatan atau kuratif, pencegahan atau preventif, memprakirakan jenis penyakit atau diagnosis, perjalanan penyakit atau prognosis, maupun pemulihannya, baik dengan berkonsultasi dengan praktisinya yaitu balian ataupun tidak. Responden juga mempercayai bahwa munculnya suatu penyakit dapat berasal dari atas yaitu dari Tuhan maupun dari bawah yaitu dari alam ataupun sesama manusia, berdasarkan hal tersebut maka ketika mengalami sakit, responden akan bertanya kepada praktisi pengobatan usada yaitu balian untuk mengetahui penyebab penyakitnya dan kemudian mendapatkan pengobatan dari balian tersebut. Hal ini juga kemudian yang membuat responden tidak bisa terlepas dari pengobatan etnomedisin di Bali, yaitu pengobatan usada dan praktisinya yang disebut dengan balian. d. Pengaruh ketiga faktor terhadap konsep sehat dan sakit d. Pengaruh ketiga faktor terhadap konsep sehat dan sakit 2. Kepercayaan masyarakat Bali terhadap pengobatan Usada Konsep sehat dan sakit pada responden adalah didasari dengan pemikirannya secara kognisi yang mengatakan bahwa sehat adalah ketika tubuh masih bisa beraktivitas, dan sakit adalah ketika tubuh sudah tidak bisa beraktivitas lagi, sehingga meskipun dalam kondisi tubuh yang kurang sehat, selama masih bisa melakukan aktivitas, maka responden akan tetap beraktivitas seperti bekerja. Responden juga mengatakan bahwa responden mempercayai pengobatan usada, ketika mengkonsumsi obat-obatan herbal yang berasal dari pengobatan usada maka tubuhnya akan merasa lebih baik. Kondisi secara biologis dari responden terlihat bahwa responden mengalami kondisi sakit, yaitu merasa sakit saat membuang air kecil yang mengakibatkan perasaan tidak nyaman, serta penurunan kesehatan dari responden seperti masalah pendengaran dan juga kebugaran. Berdasarkan hasil wawancara yang telah dilakukan, terlihat bahwa responden dan juga keluarga sangat mempercayakan kesehatannya terhadap pengobatan tradisional dan juga praktisi dari pengobatan usada yaitu yang bernama balian. Seperti yang terlihat selama sakit kencing batu, responden selalu menggunakan pengobatan usada untuk mengobati sakit kencing batunya. 1. Pengaruh dari masyarakat Dalam kepercayaan di Bali, keseimbangan tersebut dikenal dengan istilah Tri Hita Karana, yang berarti keseimbangan antara manusia dengan Tuhan, sesama manusia, dan juga alam. Apabila ada salah satu bagian yang tidak seimbang, hal tersebut bisa mendasari munculnya penyakit. Seperti yang dikatakan Sukarma (2013) prinsip hubungan keharmonisan dan keseimbangan dari tri hita karana dipercaya oleh masyarakat Bali sebagai konsep dasar dalam mencegah dan menanggulangi penyakit. Hal tersebut terlihat dari bagaimana responden mendapatkan informasi dari lingkungan di sekitarnya, di lingkungan sekitar tempat tinggal responden banyak masyarakat yang menggunakan pengobatan usada tersebut, dan mengaku bahwa merasa puas dan mendapat kesembuhan dari pengobatan tersebut. Hal ini kemudian memperkuat keyakinan serta kepercayaan responden terhadap pengobatan usada. Hal ini seperti yang dikatakan oleh Green (dalam Notoadmojo, 2014) yang mengatakan bahwa salah satu faktor dari individu dalam menentukan arah pengobatan adalah faktor pendorong atau reinforcement factors yaitu Hal ini kemudian yang mendasari responden bahwa kesehatan dapat diperoleh daripada Tuhan, dengan meminta kepada Tuhan, dan memanfaatkan serta menjaga 269 b. Faktor pembentuk kepercayaan pada responden Tentunya kepercayaan ini tidak dibentuk begitu saja, selain karena beberapa faktor dari kebiasaan di dalam keluarga, juga terdapat faktor lainnya, seperti faktor informan yang kemudian membuat responden bisa mempelajari pengobatan usada. Seperti yang dikatakan Sarafino dan Smith (2011) bahwa masyarakat seringkali akan menanamkan nilai- nilai serta kebudayaan terhadap masyarakat yang lainnya sehingga mempengaruhi perilakunya terhadap kondisi sehat dan juga sakit. Responden mengatakan bahwa penyakit bisa berasal dari bawah maupun dari atas. Penyakit yang berasal dari bawah dapat diakibatkan oleh sesama manusia, dalam hal ini adalah penyakit kiriman atau yang lebih dikenal dengan nama guna- guna, dan juga bisa berasal dari alam, sehingga pengobatan usada saja tidak cukup, dan kemudian juga memerlukan praktisinya yaitu balian untuk membantu menyembuhkan penyakit. Penyakit yang dari atas dapat berupa penyakit yang diberikan oleh Tuhan ataupun karena adanya kejadian di masa lalu, dalam kepercayaan masyarakat Bali dikenal dengan reinkarnasi, adanya janji dari pendahulu di masa lalu, sehingga memerlukan balian untuk membantu dalam penyembuhan penyakit tersebut. Dalam hal ini balian akan menyampaikan hal-hal yang harus dilakukan untuk menyembuhkan penyakit, seperti melakukan upacara keagamaan atau dikenal dengan istilah meluasin. Responden sangat mempercayakan kesehatannya dan kesehatan keluarga dari responden kepada praktisi dari pengobatan usada yaitu balian. Setiap kali mengalami sakit, maka responden akan pergi ke balian untuk berkonsultasi ataupun meminta bantuan penyembuhan terhadap penyakitnya. Hal ini sama seperti yang disampaikan Green (dalam Notoadmojo, 2003) yang mengatakan bahwa salah satu faktor yang mempengaruhi perilaku kesehatan adalah faktor predisposisi yang terdiri dari pengetahuan dan sikap. Pengetahuan dari responden dipengaruhi oleh berbagai informasi yang diterima selama hidupnya, yang kemudian memunculkan sikap responden untuk mempercayakan kesehatannya keapada pengobatan usada. Beberapa kebudayaan tersebut kemudian membuat responden menaruh kepercayaan yang sangat besar terhadap pengobatan etnomedisin yaitu pengobatan usada dengan praktisinya yang bernama balian, sehingga seringkali responden akan menggunakan pengobatan usada apabila mengalami suatu penyakit, baik datang langsung dan berkonsultasi kepada balian, ataupun membuat sendiri obat- obatan herbal dan kemudian mengkonsumsinya. Lingkungan masyarakat di sekitar tempat tinggal responden juga mengatakan bahwa masyarakat di lingkungan sekitar tempat tinggal responden menyukai berbagai bentuk pengobatan yang bersifat alami, hal ini kemudian juga membuat responden semakin mempercayai pengobatan tradisional dapat mengobati berbagai penyakit yang ada. a. Penggolongan pengobatan usada Masyarakat Bali mempercayai bahwa munculnya suatu penyakit diakibatkan oleh adanya ketidakseimbangan, responden mengatakan bahwa konsep keseimbangan dalam masyarakat Bali dikenal dengan istilah tri hita karana. Berdasarkan hal tersebut maka pengobatan usada yang ada di Bali dapat digolongkan ke dalam sistem medis naturalistik. Seperti yang dikatakan oleh Foster dan Anderson (2011) bahwa penyebab munculnya penyakit menurut sistem medis naturalistik diakibatkan oleh adanya ketidakseimbangan di dalam tubuh. Penurunan kondisi fisiologis bukanlah masalah bagi responden, karena responden beranggapan bahwa selama responden masih bisa beraktivitas, maka responden masih menganggap dirinya dalam keadaan sehat, namun kondisi ini juga dipengaruhi dari keadaan perekonomian responden yang memaksa responden untuk bekerja keras dalam memenuhi kebutuhan sehari-hari, juga kecemasan 270 KONSEP SEHAT DAN SAKIT PADA INDIVIDU DENGAN UROLITHIASIS bisa saja diakibatkan dari merusak alam sehingga adanya ketidakseimbangan dengan alam dan kemampuan dari balian adalah bisa mengetahui letak ketidakseimbangan tersebut, dan memberikan penanganan terhadap ketidakseimbangan tersebut. b. Faktor pembentuk kepercayaan pada responden Pengaruh agama dan juga budaya tidak kalah pentingnya, seperti konsep tri hita karana yang mengajarkan keseimbangan manusia dengan Tuhan, sesama manusia, dan juga alam, sehingga untuk mendapatkan kesehatan haruslah menjaga keseimbangan tersebut, seperti apabila sakit dapat menanyakan kepada balian apakah sakit tersebut berasal dari atas yaitu berasal dari Tuhan ataukah dari bawah yang bisa diakibatkan oleh alam ataupun manusia. Hal ini selaras dengan yang dikatakan oleh Sukarma (2013) bahwa konsep keharmonisan tri hita karana dalam masyarakat Bali dipercaya sebagai konsep dasar dalam mencegah dan menanggulangi penyakit. S.D. K.TRIYONO & Y. K. HERDIYANTO pengobatan konvensional tidak memberikan hasil yang memuaskan. Terlebih lagi responden dan istri dari responden mengatakan bahwa setelah melakukan pengobatan usada tubuh menjadi lebih sehat. Bisa dilihat bahwa adanya kepuasan yang membuat responden kembali menggunakan pengobatan usada ketika mengalami sakit. penggunaan pengobatan usada merupakan hal yang sudah dilakukan secara turun temurun, sehingga bahan-bahan yang digunakan untuk melakukan pengobatan usada sangat mudah ditemui, responden juga mengatakan baru saja mencari daun kecibling yang kemudian akan digunakannya untuk dijadikan ramuan obat herbal. Akses untuk menggunakan balian juga tidaklah sulit jika dibandingkan dengan fasilitas kesehatan yang sedikit sulit ditemui di Desa Sampalan. Karena kecenderungan masyarakat dalam menggunakan balian sehingga hampir seluruh masyrakat mengetahui akses untuk ke balian. Terlebih lagi pengobatan usada tidak memerlukan biaya sebesar pengobatan medis, pada umumnya pengobatan usada hanya menggunakan biaya seikhlasnya dari pasien yang datang, dibandingkan dengan pengobatan medis yang menurut responden jauh lebih mahal dibandingkan dengan pengobatan usada. Seperti yang dikatakan Anderson (dalam Notoadmojo, 2014) bahwa kemampuan dari penghasilan dan juga biaya dapat mempengaruhi individu dalam menentukan arah pengobatan yang akan dilakukan. Green (dalam Notoadmojo, 2014) juga mengatakan bahwa faktor pendorong juga merupakan salah satu faktor yang penting dalam memunculkan health seeking behavior. Berdasarkan hasil wawancara didapatkan bahwa responden mengatakan masyarakat sangat menyukai pengobatan usada, serta mendapatkan kesembuhan setelah melakukan pengobatan tersebut, sehingga semakin meningkatkan kepercayaan responden terhadap pengobatan usada, dan responden mempercayakan kesehatannya terhadap pengobatan usada. b. Health system model atau sistem model kesehatan Anderson (dalam Notoadmojo, 2014) mengatakan sistem model kesehatan dapat digambarkan melalui tiga kategori yaitu, karakteristik predisposisi, karakteristik pendukung, dan karakteristik kebutuhan. 3. Kaitan antara konsep sehat dan sakit terhadap health seeking behavior a. Perilaku yang dipengaruhi faktor predisposisi, pendukung, dan juga pendorong Green (dalam Notoadmojo, 2014) mengatakan bahwa faktor yang mempengaruhi perilaku kesehatan dari masyarakat adalah faktor predisposisi, pendukung, dan juga pendorong. Green (dalam Notoadmojo, 2014) mengatakan bahwa faktor predisposisi terdiri dari pengetahuan dan juga sikap yang kemudian mempengaruhi perilaku kesehatan. Pengetahuan dan sikap dari responden dapat dilihat dari konsep sehat dan sakit yang dimilikinya. b. Health system model atau sistem model kesehatan c. Ketergantungan terhadap pengobatan usada Responden mengatakan bahwa responden sangat mempercayai pengobatan usada, mempercayai balian sebagai praktisi dalam pengobatan usada. Bahkan anak dari responden mengatakan bahwa menggunakan pengobatan usada seperti pergi ke balian sudah menjadi suatu kebiasaan di dalam keluarga, sudah menjadi suatu ketergantungan, sehingga seringkali setiap mengalami suatu permasalahan, responden dan keluarganya akan menggunakan pengobatan usada sebagai pengobatan utama. Responden juga sempat mengatakan bahwa seringkali ketika melakukan pengobatan medis, rekam medis menunjukan kondisi yang sehat sedangkan terkadang pasien masih merasakan keadaan yang tidak nyaman. Jika dilihat dari pemaparan tersebut, bisa dilihat adanya bentuk ketidakpuasan terhadap pengobatan medis, karena tidak mendapat kesembuhan dari pengobatan medis, hal ini seperti yang dikatakan Asimo (1995) yaitu pengobatan tradisional merupakan pengobatan alternatif yang digunakan apabila Responden mengatakan bahwa dirinya mempercayai munculnya penyakit bisa saja diakibatkan oleh ketidakseimbangan, bisa saja karena responden kurang melakukan ibadah sehingga munculnya ketidakseimbangan kepada Tuhan, ataupun melakukan perbuatan yang buruk sehingga munculnya ketidakseimbangan dengan manusia, dan 271 1. Karakteristik predisposisi Responden memiliki konsep sehat dan sakit yaitu selama masih bisa melakukan aktivitasnya, maka responden dapat menyimpulkan bahwa masih dalam kondisi yang sehat. Hal ini bisa dilihat dari aktivitasnya sehari-hari, bahkan saat responden mengalami sakit kencing batu responden tetap bekerja dan melakukan aktivitasnya seperti biasa. Anderson (dalam Notoadmojo, 2014) mengatakan bahwa karakteristik predisposisi dipengaruhi oleh ciri individu yang digolongkan ke dalam beberapa kelompok, yang didasarkan pada struktur sosial seperti suku, agama, ras, dan pekerjaan, serta kepercayaan terhadap suatu metode pengobatan. Pada penelitian yang telah dilakukan, didapatkan bahwa responden merupakan salah satu masyarakat yang berasal dari suku Bali yang masih memegang teguh adat serta kebudayaan. Terlebih lagi responden merupakan salah seorang pemangku atau pemuka agama di Bali sehingga secara langsung mempelajari serta mengamalkan nilai-nilai dari agama Hindu. Hal ini juga mempengaruhi responden untuk mempercayai bahwa suatu penyakit timbul akibat adanya ketidakseimbangan, seperti yang dikatakan oleh Sukarma (2013) bahwa keharmonisan dari keseimbangan tri hita karana merupakan konsep dasar yang dipercayai oleh masyarakat di Bali untuk mencegah dan menyembuhkan penyaklit. Selain itu responden juga mempercayai bahwa munculnya penyakit dikarenakan adanya ketidakseimbangan, dalam kepercayaan masyarakat di Bali dikenal dengan tri hita karana, penyakit juga bisa datang baik dari atas yaitu berasal dari Tuhan maupun dari bawah yaitu berasal dari alam ataupun sesama manusia. Adanya ketidakteraturan dalam berpikir, berbicara, dan berperilaku juga dapat menyebabkan timbulnya penyakit. Pengetahuan dari responden kemudian memunculkan sikap yaitu melalui pengobatan usada maka hal- hal tersebut dapat terlihat, letak ketidakseimbangan dan ketidakteraturan, ketidakseimbangan dan ketidakteraturan dapat diobati dengan pengobatan usada, baik melalui arahan dari balian maupun dengan secara langsung mengkonsumsi obat-obatan herbal. Responden juga mempercayai bahwa salah satu teknik pengobatan yang bisa dilakukan apabila terjadi suatu kondisi sakit, yang responden percaya diakibatkan oleh adanya ketidakseimbangan tersebut adalah melalui pengobatan usada, Green (dalam Notoadmojo, 2014) mengatakan bahwa faktor pendukung dari predisposisi tersebut adalah sarana dan prasarana dari pengobatan tersebut. Pada kasus responden, 272 2. Karakteristik pendukung Adapun kepercayaannya tersebut seperti tri hita karana, dan juga kepercayaan terhadap datangnya penyakit baik dari atas maupun dari bawah, sehingga pengobatan usada bagi responden merupakan salah satu jalan untuk dapat mendiagnosis dan melakukan pengobatan terhadap sakitnya tersebut. Di lain sisi kondisi perekonomian responden saat itu memaksanya untuk tetap bekerja dan menggunakan pengobatan usada dikarenakan biaya yang lebih murah, hal ini juga dikarenakan responden sudah memiliki pemahaman yang buruk terhadap pengobatan medis. Anderson (dalam Notoadmojo, 2014) mengatakan bahwa karakteristik ini merupakan faktor yang mendukung predisposisi individu untuk menentukan arah pengobatan, bisa berupa biaya, pelayanan kesehatan, dan lainnya, namun disisi lainnya, kondisi psikologis yang terdiri dari kognisi, emosi, dan motivasi juga bisa dimasukkan ke dalam faktor pendukung, karena secara tidak langsung akan mempengaruhi individu untuk menentukan arah pengobatan. Dari hasil penelitian didapatkan bahwa responden memiliki kecemasan untuk melakukan pengobatan secara medis, yaitu pergi ke dokter, dikarenakan cemas bahwa dokter akan langsung melakukan tindakan operasi setelah responden memeriksakan dirinya, hal ini kemudian juga dipengaruhi karena kondisi perekonomian dari responden yang menurutnya hanya pas-pasan saja. Pengaruh lingkungan tempat tinggal responden juga membuatnya lebih mempercayai pengobatan usada, responden sudah melihat banyak orang yang sembuh dan juga mendapat kepuasan dari pengobatan usada, bahkan setiap kali menggunakan pengobatan usada, responden merasa lebih baik dan juga lebih segar daripada sebelumnya. Responden lebih sering menggunakan pengobatan usada dibanding pengobatan medis, meskipun dalam beberapa kasus seperti ketika anak responden mengalami sakit demam berdarah, responden membawa anaknya ke rumah sakit, namun setelah itu responden tetap menggunakan pengobatan usada dengan menanyakan ke balian agar anaknya cepat mendapatkan kesembuhan. Terlebih lagi lingkungan keluarga dan masyarakat yang mendukung perilaku responden dalam mengkonsumsi obat- obatan herbal, meskipun di lain sisi anak dari responden tetap mengajaknya ke dokter untuk memeriksakan diri, namun responden selalu menolaknya karena responden beranggapan masih bisa melakukan aktivitasnya dengan baik. 3. Karakteristik kebutuhan Anderson (dalam Notoadmojo, 2014) mengatakan bahwa faktor yang mendukung seseorang dalam menentukan arah pengobatannya ditentukan oleh adanya kebutuhan terhadap pengobatan tersebut. yang sudah secara turun temurun dilakukan pada masyarakat di Bali. yang sudah secara turun temurun dilakukan pada masyarakat di Bali. yang sudah secara turun temurun dilakukan pada masyarakat di Bali. masyarakat Bali yaitu agama Hindu. Responden juga sangat memegang teguh nilai serta kebudayaan di Bali, sehingga pengobatan usada diniliai lebih menimbulkan efek yang positif, atau dengan kata lain kesembuhan baginya. 2. Karakteristik pendukung 1. Konsep sehat dan sakit Konsep sehat dan sakit pada responden dipengaruh oleh tiga faktor yaitu faktor biologis, , faktor psikologis, dan faktor sosial dan budaya. Faktor biologis dari responden merupakan kondisi kesehatan dari responden, riwayat penyakit yang pernah diderita oleh responden. Faktor psikologis dari responden merupakan pemahaman dari responden terhadap kondisi sehat dan juga sakit, serta arah pengobatan yang dilakukan oleh responden ketika mengalami kondisi sakit, dan faktor sosial dan budaya dari responden merupakan kondisi kebudayaan yang berada di tempat tinggal dari responden, pemahaman responden tentang konsep keagamaan yang mempengaruhi kondisi sehat dan juga sakit. Berdasarkan hasil penelitian yang telah dilakukan, responden juga mengatakan bahwa kondisi perekonomiannya saat itu membuatnya lebih memilih pengobatan tradisional dibandingkan dengan pengobatan medis. Bagi responden pengobatan medis mengharuskannya mengeluarkan biaya yang cukup besar, meskipun responden belum mencoba untuk melakukan pengobatan secara medis saat mengalami kencing batu, sehingga sudah munculnya perasaan negatif dari responden terhadap pengobatan medis. Kondisi emosi tersebut membuat responden lebih memilih dan membutuhkan pengobatan usada jika dibandingkan dengan pengobatan medis. Konsep sehat dan sakit yang didapatkan dari responden banyak dipengaruhi oleh faktor kebudayaan yang ada di lingkungannya, yang membentuk kepercayaan dari responden, seperti konsep tri hita karana yang dipandang oleh responden sebagai konsep keseimbangan sehat dan juga sakit, kemudian kepercayaan tersebut juga diperkuat oleh kondisi lingkungan masyarakat dan lingkungan keluarga yang memiliki pemahaman yang sama terhadap responden, menguatkan dan memunculkan persepsi responden terhadap kondisi sehat dan juga sakit yang kemudian membuat responden mampu untuk menentukan kemana arah pengobatan yang akan dilakukan. c. Kaitan antara konsep sehat dan sakit terhadap health seeking behavior c. Kaitan antara konsep sehat dan sakit terhadap health seeking behavior Beberapa hal tersebut diatas kemudian membuat responden untuk lebih memilih mempecayakan pengobatan usada, dalam mengobati kencing batunya saat itu. Hal ini dikarenakan pengobatan usada didasari oleh ajaran kepercayaan masyarakat di Bali, yang dituliskan di dalam lontar-lontar yang mengambil budi luhur dari kepercayaan 273 3. Kaitan konsep sehat dan sakit terhadap health seeking behavior Health seeking behavior dipengaruhi oleh faktor predisposisi, pendukung, dan juga pendorong, serta adanya health system model. Faktor-faktor tersebut berupa: Saran juga diberikan kepada penelitian berikutnya yaitu dapat menggunakan pendekatan lain untuk mendapatkan hasil yang lebih baik, menggunakan responden penelitian yang lebih banyak, serta memperluas kajian penelitian dan juga menggunakan lebih banyak referensi penelitian. Menggunakan lokasi penelitian yang berbeda karena faktor demografi merupakan salah satu faktor yang mempengaruhi health seeking behavior. a. Keyakinan responden berdasarkan kepercayaan terhadap suatu pengobatan, kepercayaan responden terhadap munculnya suatu penyakit, b. kemudahan akses dalam menggunakan pengobatan usada, c. kondisi ekonomi dari responden, disamping pengobatan usada tidak membutuhkan biaya yang terlalu besar, S.D. K.TRIYONO & Y. K. HERDIYANTO Konsep sehat dan sakit yang dimiliki oleh responden berkaitan dengan munculnya health seeking behavior. Responden mempercayai bahwa kondisi sehat dan juga sakitnya dipengaruhi oleh faktor keseimbangan yang ada di lingkungannya, konsep keseimbangan tersebut dipengaruhi oleh adat serta budaya di tempat tinggal responden. Health seeking behavior pada responden juga tidak terlepas dari kebudayaan yang ada di lingkungan responden, kebudayaan tersebut kemudian terenkulturasi dengan lingkungan sekitar tempat tinggal responden, dan kondisi kehidupan responden. Enkulturasi tersebut kemudian menghasilkan proses-proses psikologis yang di dalamnya terdapat belief atau kepercayaan, terhadap kondisi sehat dan juga sakit yang kemudian mempengaruhi health seeking behavior dari responden, baik dalam penggunaan pengobatan usada maupun medis. 2. Kepercayaan terhadap pengobatan usada 2. Kepercayaan terhadap pengobatan usada Responden sangat mempercayai pengobatan usada, karena pengobatan usada didasari oleh nilai-nilai agama dan kebudayaan yang ada di Bali. Kepercayaan responden dibangun dari keluarga dan juga masyarakat sekitar tempat tinggal responden. Responden juga mempercayai bahwa datangnya penyakit diakibatkan oleh adanya ketidakseimbangan. Konsep tri hita karana yang dipercaya oleh responden sebagai konsep sehat dan juga sakit. Responden mempercayai bahwa salah satu cara untuk menyembuhkan sakit yang diakibatkan oleh ketidakseimbangan tersebut adalah dengan menggunakan pengobatan usada melalui praktisinya yang bernama balian. Manfaat dari pengobatan usada adalah mampu untuk melihat serta mengobati ketidakseimbangan tersebut, terutama dengan menggunakan praktisi dari pengobatan usada yaitu balian. Beberapa saran diberikan berdasarkan dari hasil penelitian yang telah dilakukan, saran diberikan kepada lembaga- lembaga kesehatan agar meningkatkan promosi kesehatan kepada masyarakat supaya lebih terdorong untuk melakukan pengobatan dan pemeriksaan ke layanan kesehatan. Mengkolaborasikan pengobatan medis dan juga tradisional agar masyarakat bisa mempertimbangkan penggunaan pengobatan medis, ataupun mengkombinasikan pengobatan medis dan juga tradisional. Kepercayaan responden terhadap pengobatan usada juga dipengaruhi oleh keluarga dari responden, sejak kecil responden terbiasa untuk menggunakan pengobatan usada, banyak pengalaman yang dialami oleh responden, banyak kesembuhan yang didapatkan dari pengobatan usada, sehingga memunculkan kepercayaan responden terhadap pengobatan usada. Kepada responden penelitian juga diberikan saran untuk mempertimbangkan pengobatan medis sebagai pilihan kedua setelah pengobatan tradisional, agar dapat dijadikan solusi alternatif ketika mengalami sakit. Melakukan cek rutin ketika mengalami gejala-gejala yang tidak wajar, dan menyeimbangkan antara penggunaan pengobatan tradisional dan juga pengobatan medis. 3. Kaitan konsep sehat dan sakit terhadap health seeking behavior KONSEP SEHAT DAN SAKIT PADA INDIVIDU DENGAN UROLITHIASIS adolescence: The place of internet. Social Science & Medicine , 60, 1467-1478. Ardani, I. (2013). Eksistensi dukun dalam era dokter spesialis. 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https://openalex.org/W3216062294	https://hv.diva-portal.org/smash/get/diva2:1621195/FULLTEXT01	English	null	Value Propositions in Public Collaborations: Regaining Organizational Focus Through Value Configurations	British journal of management	2,021	cc-by	12,528	Value Propositions in Public Collaborations: Regaining Organizational Focus Through Value Configurations This focus is argued to be less appropriate in contemporary society, in which an increasing outward orientation (such as col- laborating with other organizations as well as in- dividual citizens) among PSOs is called for (e.g. Mintzberg, 2015). The most common feature of such outwardness is probably the need to collabo- rate, across PSOs (Agranoff and McGuire, 2004), across sectors (Klijn and Koppenjan, 2012) with citizens (Cooper, Bryer and Meek, 2006), or with Value Propositions in Public Collaborations: Regaining Organizational Focus Through Value Configurations Erik Eriksson ,1,2 Christian Gadolin ,3 Thomas Andersson,4,5 Andreas Hellström1 and Svante Lifvergren1,6 1Department of Technology Management and Economics, Chalmers University of Technology, Gothenburg, SE-412 96, Sweden, 2Department of Work Life and Social Welfare, University of Borås, Borås, SE-503 32, Sweden, 3Department of Health Sciences, University West, Trollhättan, SE-461 32, Sweden, 4School of Business, University of Skövde, Skövde, SE-541 45, Sweden, 5VID Specialized University, Oslo, NO-0370, Norway, and 6Skaraborg Hospital Group, Skövde, SE-542 24, Sweden Corresponding author email: erik.eriksson@chalmers.se Erik Eriksson ,1,2 Christian Gadolin ,3 Thomas Andersson,4,5 Andreas Hellström1 and Svante Lifvergren1,6 1Department of Technology Management and Economics, Chalmers University of Technology, Gothenburg, SE-412 96, Sweden, 2Department of Work Life and Social Welfare, University of Borås, Borås, SE-503 32, Sweden, 3Department of Health Sciences, University West, Trollhättan, SE-461 32, Sweden, 4School of Business, University of Skövde, Skövde, SE-541 45, Sweden, 5VID Specialized University, Oslo, NO-0370, Norway, and 6Skaraborg Hospital Group, Skövde, SE-542 24, Sweden Corresponding author email: erik.eriksson@chalmers.se There is consensus that complex problems of contemporary society call for public ser- vice collaborations. So-called public service logic (PSL) focuses on joint value creation among a multiplicity of actors in service ecosystems. Despite recognizing various ac- tors, this logic is essentially user-centric, with the service user being the one realizing the value. Consequently, single and collaborating organizations cannot deliver value, only potential value, or so-called value propositions. The elusive public service logic takes a network value configuration for granted and as a starting point. Drawing from two cases in Swedish healthcare, this paper argues that two other value configurations (chain and shop) are also relevant for understanding the development of value propositions – and that these may be related to both intra- and inter-organizational processes. Theoretically, we conclude that just like public service logic, other collaborative public management theo- ries need to recognize the importance of a multiplicity of value configurations and that these are often related to both intra- and inter-organizational processes. We conclude that managers should not adopt the latest network trends without first reflecting on the relevance of existing internal processes. focus on internal production processes) public ser- vice organizations (PSOs) fit to address relatively simple challenges (Ansell, Sørensen and Torfing, 2021; Osborne, 2020). This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distri- bution and reproduction in any medium, provided the original work is properly cited. The authors wish to thank the anonymous reviewers for valuable comments on earlier versions of this pa- per, as well as comments received at the British Academy of Management Conference, 3−5 September 2019. This study was funded by grants from Regional Cancer Cen- tre West and the Swedish Research Council for Health, Working Life and Welfare [Grant No. 2018-01196]. © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management. Published by John Wiley & Sons Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK and 350 Main Street, Malden, MA, 02148, USA. Introduction It is commonly argued that both public adminis- tration and new public management (NPM) tra- jectories have helped make inward-oriented (e.g. The authors wish to thank the anonymous reviewers for valuable comments on earlier versions of this pa- per, as well as comments received at the British Academy of Management Conference, 3−5 September 2019. This study was funded by grants from Regional Cancer Cen- tre West and the Swedish Research Council for Health, Working Life and Welfare [Grant No. 2018-01196]. 2 E. Eriksson et al. all of the above actors and sectors (Eriksson and Hellström, 2021). ualized conceptualization of value with so-called public value(s), a construct that focuses on value at the collective level (Moore, 1994), such as the com- mon good or the public interest (Beck Jørgensen and Bozeman, 2007). Despite these developments, Cluley and Radnor (2020a) argued that what value is remains largely unidentified. The value concept, and the creation thereof, will be discussed further in the theoretical section below. The call for various forms of collaboration is based on the alleged complexity of the problems that PSOs are responsible for addressing (Ash- worth et al., 2013; Bryson et al., 2017). The ad- vances of modern society – such as medical and technological progress – have also brought risks (Beck, 1992): climate change, forced migration, pandemics and the like – societal and global issues paramount for the responsible PSO to solve alone (Sørensen and Torfing, 2011). These complex chal- lenges are not only difficult to solve (Christensen, 2012; Geuijen et al., 2017), but also to define be- cause of the inherent uncertainty and likely goal conflict among stakeholders (Peters and Pierre, 2017; Pollitt and Bouckaert, 2017). Consequently, since the early 1990s, many countries have imple- mented various forms of collaboration and net- works to address such complex public challenges (Turrini et al., 2010). Thus, the developments in PSL have been important for increasing our understanding of value creation, not least by focusing on the citizen/user level – often as a pivotal actor in inter- organizational collaborations in public service ecosystems (e.g. Petrescu, 2019). However, the conceptualization of value in these ecosystems is rather elusive, which has meant that the organi- zational level has been left relatively unelaborated in PSL. Introduction This level is argued to be vital in order to understand the prerequisites for value creation as in the development of potential value – to eventu- ally be realized as real value by citizens and other actors (Eriksson et al., 2020; O’Cass and Ngo, 2011; Skålén et al., 2018). Like other collaborative and network approaches, PSL places focus on the interface between organizations in the system. PSL also recognizes the individual citizen or ser- vice user to be essential in these collaborations, and in realizing value from value propositions. We argue that both these foci are important, but that the organizational focus has become lost. Just as in other collaborative theories (Span et al., 2012), it is assumed that value creation in PSL builds on a network idea. We argue that network is just one configuration for creating value, and the other configurations may influence a joint value proposition. Therefore, building on two empirical cases, this paper seeks to put the focus on three different value configurations (network, chain and shop) that are essential for the organization to develop value propositions – both directly with other public organizations and indirectly as orga- nizational conditions that influence collaborations with other actors in the ecosystem. Thus, the research question of this paper is: How do different value configurations influence the development of potential value through public service organization collaboration? Again, the word ‘potential’ is used because it is a premise in PSL that the individual user realizes value. Th d f ll Th th Similarly, a variety of collaborative or net- work theoretical approaches emerged in the early 2000s (e.g. Agranoff and McGuire, 2004; Ansell and Gash, 2008). However, the way in which value is created in these collaborations remains under-theorized in the public administration and management literature and poorly understood in practice (Jo and Nabatchi, 2016). Recently, public service logic (PSL) (Engen et al., 2021; Osborne, Nasi and Powell, 2021) has gained increased at- tention in public administration and management literature. In PSL, value creation is an essential concept that focuses on collaborations between service provider and citizen at the micro-level (e.g. Hardyman, Daunt and Kitchener, 2015) and/or between a multiplicity of actors in public service ecosystems at the macro-level (e.g. Petrescu, 2019) in their efforts to create value. However, the value concept is elusive. © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management. © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management Collaborations in the public sector There is general consensus that, in an increasingly complex world, there is a similarly increasing need for collaboration (Cristofoli, Meneguzzo and Riccucci, 2017), not only to improve a particular public service, but to solve meta-problems of public service delivery (Keast and Brown, 2002). Thus, collective impact is sought, rather than leaving the issue at hand to be solved by the re- sponsible PSO alone (Denhardt and Denhardt, 2015; Pollitt, 2003). For example, in healthcare, which is our studied case, value is seen as being created in complex constellations, which involve many different actors in the patient’s health service network, rather than just one PSO alone (Nord- gren, 2015). It is also argued that the need to coordinate fragmented services is a consequence of past trajectories (Christensen and Lægreid, 2011), such as decentralization of accountability of NPM (Andersson and Liff, 2012). Collabo- rations are proposed to be viable only when the advantages of the collaboration are clear (Åhgren, 2014) and when inter-organizational interaction entails purposive forms of service integration (Åh- gren and Axelsson, 2005; Nordgren, Planander and Leifland, 2020). Ideally, the benefit of these collaborations includes more appropriate use of common resources and improved service delivery to citizens (Koliba et al., 2017; Meier and O’Toole, 2003). Collaborations may also help participat- ing PSOs achieve their own goals (Christensen and Lægreid, 2015; Ferlie, 2017), as well as goals shared with the other actors (Koliba et al., 2017; Willem and Lucidarme, 2014). However, a benefit is that (successful) collaborations may nurture fur- ther collaborations by learning from one another, , ) Naturally, the structure of collaborations may differ. In a seminal paper, Provan and Kenis (2008) presented three types of collaboration. In the first, all participating organizations share responsibility in an informal and decentralized way. In the sec- ond, one organization takes the lead in a central- ized and formalized way, managing the network. In the third, a centralized and separate adminis- trative entity is created to govern the network in a formalized way. It has been argued that coor- dinating mechanisms become more important in decentralized collaborations, whereas the presence of a lead organization tends to entail more tradi- tional managerial activities and control in practice (Markovic, 2017). Because the ideal of informal- ity, non-hierarchy and consensus is often the point of departure of research on collaborations, the presence of top-down aspects is often neglected (Span et al., 2012). Introduction Accord- ing to seminal work in service management and marketing, which inspired the early elaborations of PSL, value is not an objective construction, but should be understood to be individually deter- mined (Grönroos, 2011; Vargo and Lusch, 2004). Moreover, value should be understood to be context-bound (Chandler and Vargo, 2011), par- ticularly to social context (Rihova et al., 2013) and, consequently, value to emerge intersubjec- tively (Helkkula, Kelleher and Pihlström, 2012). In a public management context, PSL (e.g. Osborne, 2020) has sought to balance the original individ- The paper proceeds as follows. The theo- retical background offers an overview of the Value Propositions in Public Collaborations 3 generating trust (Agranoff and McGuire, 2004; O’Leary and Vij, 2012). collaboration field, followed by a section on value creation. We then present the three ideal types of value configuration, which we use to analyse the empirical material. The methods section presents data collection and analysis, followed by the results from the two cases in the findings section that il- lustrate different value configurations. The discus- sion theorizes value creation in collaborations by analysing how the value configurations influence value propositions through collaboration. The pa- per concludes with implications for research, as well as policymakers and practitioners. Despite the potential, collaborations involve numerous challenges. For instance, collaborations may decrease PSOs’ accountability and trans- parency and may lead to increased conflict and deadlock (Sørensen and Torfing, 2009). Moreover, the presence of the authorizing environment and the bureaucratic structures of each organization are often evident, entailing differences in prior- itization, goals, legislation and culture between the participating organizations (Agranoff and McGuire, 2004; Klijn and Koppenjan, 2012; Willem and Lucidarme, 2014). Moreover, the informal ideal of collaborations entails fragility because of the dependence of individual enthusi- asts and an unaccustomedness to work informally among the participants. The informality may also entail a stronger focus on participating individuals, which means that the differences in status, power and mandate they bring to the table become an important factor (Agranoff and McGuire, 2004). However, the inter-personal dimension in collabo- rations, as part of the organizational level, remains under-theorized (Cristofoli, Meneguzzo and Riccucci, 2017). © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management. Collaborations in the public sector Moreover, the collaborative ideals may be hampered and rejected in a context in which managers still draw from traditional public administration and NPM (Hansen and Waldorff, 2020). However, it is commonly argued that traditional management may be ill fit in collaborations in which an ‘integrative leadership’approach (Crosby 4 E. Eriksson et al. E. Eriksson et al. and Bryson, 2010) may be more appropriate, fo- cusing on stimulating interaction between par- ticipants, exchange of resources and how to de- sign a service with respect to the common goal (Crosby, Hart and Torfing, 2017). In practice, collaborations may be governed by ‘distributed leadership’ (Crosby, Hart and Torfing, 2017), in which employees without formal managerial au- thority are expected to lead as coordinators, fa- cilitators, mediators and so forth (Bryson et al., 2017; Cristofoli, Meneguzzo and Riccucci, 2017; Eriksson et al., 2020). Connective capacities are es- sential for this role (Edelenbos, van Buuren and Klijn, 2013), including stimulation of interactions, building trust and commitment, solving conflicts and leading the network towards a common goal (Klijn, Steijn and Edelenbos, 2010). However, the difference between the goals of the participating organizations must be recognized as well (Vangen, 2017). Value creation in collaborations The emerging PSL stems from a critique of NPM’s alleged inherent manufacturing logic, in which PSOs have been organized as if they produce and deliver tangible goods, and has led to an inter- nal focus on processes (Grönroos, 2019; Osborne, 2018). In (public) services, the production and con- sumption processes cannot be separated (Osborne and Strokosch, 2013; Osborne, Nasi and Powell, 2021) as the service is intangible and occurs in the service meeting when provider and service user in- teract (Normann, 2001). Consequently, the user is always a co-producer in a service approach, entail- ing that the relationship and interactions between staff and user are crucial (Eriksson, 2019). Re- cently, Cluley and Radnor (2020a) challenged the dominant focus of value co-creation on provider– user interaction and proposed a framework that focuses on value co-creation as a continuous – and relational, fluid, heterogenous and changeable – process rather than an interaction or outcome, composed of a multiplicity of elements: human, environmental, cultural and material. It is argued that organizational learning may be better in bureaucratic networks than in collabora- tive ones (and other ‘post-bureaucratic’ organiza- tions), due to the absence of a stable core in the latter (Andersson, Stockhult and Tengblad, 2021; Pollitt, 2009). Collaborations in the public sector However, Ferlie et al. (2011) found that a long-term career in a network offered a sta- ble core that enabled organizational learning and memory in post-bureaucratic organizations, such as team-based leadership (rather than individual) among healthcare professionals (Martin, Currie and Finn, 2009). – process rather than an interaction or outcome, composed of a multiplicity of elements: human, environmental, cultural and material. Rather than producing and delivering value, PSOs can only offer potential value, so-called value propositions (Grönroos, 2019; Skålén et al., 2018); these value propositions sometimes need to be coordinated among several organizations (Eriksson et al., 2020). Value is then realized in the user’s life situation, which means that the PSO’s potential value is combined and integrated by the user with other actors’ potential value offerings and resources, including knowledge, skills and so on from friends and family, private enterprises and third-sector organizations (Osborne et al., 2015). Thus, value is a subjective phenomenon that will vary between different people and change over time (Cluley and Radnor, 2020a). In sum, collaborations in the public sector have often focused on structure and leadership, and more recently, on aspects such as trust and re- lationship (Cristofoli, Meneguzzo and Riccucci, 2017). Collaboration is often assumed good per se: ‘[c]ollaboration has become a hammer and nearly all problems have become nails’ (Silvia, 2018, p. 472). However, collaboration is only relevant if it has the potential to develop potential value, in- cluding better organizational performance, out- comes or lowered costs (Agranoff, 2007; Bardach, 1998; Kenis and Provan, 2009; Klijn, Steijn and Edelenbos, 2010; Sørensen and Torfing, 2009). Nylén (2007) argued that collaborations should be evaluated more on value creation than cost effec- tiveness. The value creation in collaborations has often been taken for granted, focusing research on how to collaborate instead of the more specific how to collaborate to develop potential value. Lately, PSL has increasingly focused beyond the provider–user interface to include a number of ac- tors participating in the collaboration of mutual value creation (Petrescu, 2019). Thus, value co- creation in these public service systems includes ac- tors from public, private and third sectors, as well as citizens/service users (Eriksson and Hellström, 2021; Osborne et al., 2015). The premise is that all actors engage in mutually beneficial value cre- ation in which they contribute with various knowl- edge and skills (and physical products) (Kinder et al., 2020; Petrescu, 2019). © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management. Value configurations We argue that PSL – by focusing on distancing it- self from NPM – neglects previous models of value creation that have their merits. Instead, different situations and problems require different types of value creation. For example, not all problems, even in collaborations, are to be considered complex, many intra- and inter-organizational processes de- veloped to solve particular problems (not complex) are likely to be valid. In this subsection, we present three ideal types of value configuration that are all relevant for collaborations and that we use to anal- yse the empirical material. In the 1990s, Stabell and Fjeldstad (1998) pre- sented their three ideal types of value configura- tion that are generic across sectors: value chain, value shop and value network. In a Weberian sense, ideal should not be understood as ‘better’, but rather as ‘pure’, which means that, in practice, versions and combinations of the configurations are likely to be found. The value shop is appropriate when the prob- lem is diffuse, hard to define and the focus is on problem definition by gathering competences. This is the traditional way of organizing healthcare, a legacy from an era when the causes of illness were rather unfamiliar (Stabell and Fjeldstad, 1998). In the shop, individualized solutions to the problem are needed when users seek public services with manifestations that are hard to attribute (Chris- tensen, Grossman and Hwang, 2009). Rather than sending the public service user around between various PSOs, in the value shop, competences and expertise are gathered and various exami- nations/tests are carried out more or less at the same time. Thus, value creation through a value shop stipulates ‘value is created by mobilizing resources and activities to resolve a particular customer problem’ (Stabell and Fjeldstad, 1998, p. 414). ) In sum, value creation in PSL has shifted the fo- cus from the provider–user sphere to public service ecosystems. In both cases, however, only the indi- vidual actor can realize value by combining po- tential value provided by others (the PSO, fam- ily and friends, etc.). Like NPM, PSL draws on developments in the private sector. Consequently, much of the elaboration has been about the dif- ference between private and public sector: besides individual value, collective or public value is es- sential for PSOs (Alford, 2016). Collaborations in the public sector Recently, it has also been recognized that value at the different levels 5 Value Propositions in Public Collaborations (individual users, groups and society) needs to be recognized in these public service systems (Cluley, Parker and Radnor, 2021; Dudau, Glennon and Verschuere, 2019). Consequently, public services are not only a concern for the responsible PSO, but also require the involvement of all system actors (Osborne, 2020; Radnor et al., 2014). Again, be- cause value cannot be delivered in PSL – neither as products, nor as policies – it is important to un- derstand how combinations of resources are used by the actors (Osborne, 2020). the reluctance, fear and discomfort among public service users (e.g. patients, prisoners) are likely to be higher than for private sector users (Eriksson and Nordgren, 2018). PSL has added important aspects to value creation, but the focus on the indi- vidual customer and/or ecosystems has meant that the organization of the value proposition, or de- velopment of potential value, has not received suf- ficient attention. The recognition of value at the three levels (in- dividual users, groups and society) also entails that perceptions of value between levels may be in con- flict (Eriksson and Nordgren, 2018). In addition, value perceptions may vary within each level: for instance, individual public service users (prison inmates, for instance) and citizens are likely to perceive different benefits from public prisons (Os- borne, 2020), and value perceptions may vary be- tween collaborating organizations (De Graaf and Van der Wal, 2010). Moreover, it is often assumed in PSL that public service users are rational actors, which is not a matter of course (Cluley and Rad- nor, 2020b). In addition, in a public sector context, it should not be assumed that beneficial outcomes are always the case (Engen et al., 2021; Voorberg, Bekkers and Tummers, 2015). Consequently, there has been a call for a more nuanced understanding of value and the creation of value (Dudau, Glen- non and Verschuere, 2019) in public management; for instance, to recognize that value should not be assumed to be created equally for all, but that disvalue (Cluley, Parker and Radnor, 2021), value destruction (Järvi, Kähkönen and Torvinen, 2018) or value diminution (Vafeas, Hughes and Hilton, 2016) is as likely an outcome (for one or more actors across levels) as creation of value. © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management. Setting The empirical material draws from two cases in Swedish public healthcare, in which collaborations are central. The cases – cancer care and elderly care – demonstrate the complexities in aging so- cieties in which an increasing proportion of the population is older and suffers from multiple and chronic illnesses, while at the same time the work- force decreases (Osborne, 2020). This particularly puts mainly tax-financed healthcare systems un- der strain. Hence, the changed population struc- ture and disease panorama also require healthcare services to change. Both cases are set in Västra Götaland, Sweden’s second-largest region, located in the southwest- ern part of the country. In the decentralized and mainly tax-financed Swedish healthcare system, the main responsibility of the regions is to pro- vide healthcare to their inhabitants (1.7 million in Västra Götaland and 10.4 million in Sweden in to- tal; Statistics Sweden, 2021) in public hospitals and primary care units. Within each region, the munic- ipalities’ responsibilities include providing elderly care at public institutions or their homes. At both levels, there are private and third-sector actors that provide care on behalf of, and compensated by, re- gion or municipality, respectively. At an overarch- ing level, national governments and agencies stip- ulate laws, directives and recommendations for all 21 regions and 290 municipalities in the country. The value network is particularly advantageous in long-term services, such as chronic diseases, where the patient can take great personal respon- sibility for managing their disease, but still needs support in various forms from PSO experts – often assisting with a network through which public service users can support each other and through IT solutions that report and receive feed- back from professionals (Stabell and Fjeldstad, 1998). The network configuration is increasingly mentioned as a form of care suitable for elderly people with multiple illnesses (Eriksson et al., 2020), but is also applicable to more preventive measures. The first case (hereafter referred to as Case A) is set in cancer care and covers the whole of the present region (as well as the northern parts of another region). A national cancer strategy was launched in 2009 (Statens offentliga utredningar, 2009). A central feature of the strategy of western Sweden was the appointment of clinically active physicians as so-called process-owners of their respective team, often based on cancer diagnosis. © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management. Value configurations A returning ‘cus- tomer’ is good news in the private sector, but a returning client to a social service office may be understood as a service failure (Osborne, 2018); E. Eriksson et al. 6 © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management. Methods The value chain (Porter, 1985) attracted in- creased attention in the 1990s in order to mitigate the fragmentation caused by silo-ization in many bureaucratic organizations (Christensen and Læ- greid, 2011; Pollitt, 2003). In the chain, value is added in pre-defined steps in a linear process of refinement (Stabell and Fjeldstad, 1998). The value chain has been shown to be appropriate in cases where the problem is known and standardization and best practice may be the most appropriate way to organize services. Thus, the chain requires that problems can be solved with great precision and in the same way for most users. The impact of the chain in public services has been massive and claimed to have been a positive contribution to the public sector, even by PSL proponents (Osborne et al., 2015). However, too much focus on measur- ing quantifiable output has been criticized for hav- ing reduced the trust in public employees, and in- creased the administrative burden on them (Quist and Fransson, 2014). Setting The second case (hereafter referred to as Case B) is set in elderly care and covers 15 of the re- gion’s 49 municipalities. In Case B, management and employees at municipalities, primary care and hospitals developed a care model for the elderly with chronic and multiple diseases based on collaboration, trying to bridge a fragmented and poorly coordinated system (Statens offentliga utredningar, 2020). The collaboration consisted of A few years before Stabell and Fjeldstad (1998), Porter’s value-chain model was criticized by Nor- mann and Ramírez (1993). Instead, they proposed that value was co-created in value-creating systems as ‘synchronic and interactive, not linear and tran- sitive’ (Ramírez, 1999, p. 50) and involved a multi- plicity of actors who reconfigured their roles and relations to create value in new forms (Normann and Ramírez, 1993). The systems understanding of value creation – underpinned by its technological development – entailed that the potential role of the service users in particular broadened in rela- tion to what they can do, where they can do it, when things can be done and with whom (Levin and Nor- mann, 2001; Normann, 2001). Value Propositions in Public Collaborations 7 Table 1. Respondents’ backgrounds and data collection Case Type of healthcare Data collection Number of respondents Profession A Cancer care Focus groups, semi-structured 18 Process-owners (all physicians) B Elderly care Individual interviews, semi-structured 34 Managers, coordinators, staff in teams (physicians, nurses, nurse assistants) Total 52 7 Value Propositions in Public Collaborations the final step the second-order themes were sorted into dimensions based on the three different value configurations (shop, chain and network). Some themes were omitted (for instance, themes ad- dressing problem types and solutions to address these problems, as they were not clearly found in the empirical material, but were central in the value configuration literature). Other aspects were given more room than is typical in the respective configuration; for instance, trust is a key feature in contemporary Swedish public administration and was widely discussed by the respondents. For val- idation (Lincoln and Guba, 1985), and to ensure that nothing was misunderstood or that empirical material was not ‘forced’ into a priori themes based on theory, tentative themes were presented for stakeholders (and many of the respondents) on various occasions to ensure they recognized the data and the themes. Setting Figure 1 shows the final second-order themes, primary coding categories (expressions close to the interviews/focus groups) and overarching dimensions (constructed based on the three ideal types of value configuration). three levels: the management network, the learning network in which practitioners on the floor shared experiences and mobile teams at the patients’ homes. The coordinators held collaborations together, both vertically and horizontally. Data collection and analysis In Case A, five focus group interviews with the process-owners were conducted. Between three and five process-owners participated in each fo- cus group. In total, 18 process-owners participated in the semi-structured focus groups. All groups had mixed gender representation, with a total of 8 women and 10 men. In Case B, 34 individ- ual and semi-structured interviews were conducted with managers, healthcare staff and coordinators. Focus groups and interviews for both cases were recorded after verbal consent had been collected and transcribed verbatim after the interviews. See Table 1 for respondents’ backgrounds and data collection. Thematic analysis for the two cases was carried out, similar to the procedure of template analy- sis (Brooks et al., 2015; King, 2012) with seven steps: (1) familiarization with the data in which transcripts and recordings were listened to and discussed; (2) preliminary coding, categorizing data based on similarities and in relation to pur- pose, research questions and close to expressions used by respondents and also in developing a priori themes (tentatively defined themes based on theoretical interest; in this paper, the value configuration literature; e.g. Stabell and Fjeldstad, 1998); (3) clustering themes based on similarities to and differences from second-order themes; (4) producing an initial template that links clusters together; (5) developing the template by applying it to further transcripts and modifying themes in an iterative way; (6) applying the final template on the remaining material; and (7) writing up (King and Brooks, 2017). Deviating from the procedure, in © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management. © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management Findings In this section, different value configurations in the cases are illustrated. More specifically, the di- mensions and themes in Figure 1 are presented. First, the shop dimension and connected themes will be presented for each case, followed by the chain and network dimensions and their respec- tive connected themes, which are also presented for each case. By way of introduction, Table 2 of- fers typical examples of quotes from the qualita- tive data collection: the focus group discussions (Case A) and individual interviews (Case B). © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management. © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management. Shop: Professional knowledge Shop: Professional knowledge Shop: Professional knowledge In Case A (cancer care), an explicit reason for launching process ownership was to move away 8 E. Eriksson et al. Figure 1. Data analysis Figure 1. Data analysis from managerialism and let the professionals in healthcare have a greater say. The process-owners were supposed to be clinically active physicians with responsibility for developing and disseminat- ing knowledge about their particular field. Other than that, the cancer centre let the process-owners themselves decide how to run their respective pro- cesses and put together their process team, of- ten represented by various professions from differ- ent hospitals in western Sweden. The cancer cen- tre’s staff supported the process-owners by such means as providing statistics, training and edu- cation, and facilitating networking meetings, as well as administering travel expenses and so on. The professional knowledge applied not only to the process-owners themselves. Many responding process-owners said that an important task was to ‘lend a helping hand’ to the hospitals, enabling local clinicians to have a greater impact on their job. In Case B (elderly care), concerning professional knowledge, managers mentioned that they should not ‘interfere with the process’, but should rely on the professionals on the floor to do their work. However, it was also mentioned that traditional ways of working made it difficult and ‘unnatural’ for general practitioners to work at older patients’ homes, because they were trained to work with di- verse patients – not only older patients – and to have colleagues to discuss issues with. This was a Value Propositions in Public Collaborations 9 Table 2. Quotes from the two cases Dimension Quote Shop … we work with very engaged people, who want to do their best, and therefore you don’t need to force them to do things… if we can show and convince them that it is good for our patients, then we don’t need any imposed regulation. (Process-owner and physicians, Case A) It is hard to reach teamwork if we are always at different physical places. The closeness in everyday work, just to sit down with a cup of coffee and discuss patients… that does not really work. (Manager, Case B) We should not interfere with the process. Shop: Teamwork In Case A, teamwork mainly addressed their own process team, consisting of various professions. In Case A, teamwork mainly addressed their own process team, consisting of various professions. © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management. Shop: Professional knowledge (Manager, Case B) Chain … her boss says that this [reporting to the register] is of a low priority and then one starts to wonder what kind of mandate that one is having. (Process-owner and physicians, Case A) … some think we are nagging […] they all have lots to do […] so when they don’t manage to report [performance measures] it is because they don’t have time for it. (Process-owner and physicians, Case A) We physicians are very competitive, we want to be the best in class, right? There’s nothing wrong in being compared with others. (Process-owner and physicians, Case A) It’s a darn measuring of minutes and seconds all the time, we are clocked all the time. (Nurse assistant, Case B) Network … when talking to people one realizes that seeds have been sown and things start to happen […] people think more over organizational borders now. (Process-owner and physicians, Case A) … we have some very active ones and I always get a little thoughtful about these people that always comes back, that always wants to participate […] those I call ‘professional patients’ that I am not too fond of. How do you find the ‘right’ persons? (Process-owner and physicians, Case A) When relations are established and you know your role, then I believe that this kind of work runs rather smoothly, both management and on the field. You cannot just write a manual and everything will work. (Manager, Case B) It is so much and eventually one does not know what is what. I, who sits in the middle of this system, hardly understand anything. (Manager, Case B) Value Propositions in Public Collaborations 9 It is so much and eventually one does not know what is what. I, who sits in the middle of this system, hardly understand anything. (Manager, Case B) It is so much and eventually one does not know what is what. I, who sits in the middle of this system, hardly understand anything. (Manager, Case B) reason why it had been difficult to recruit general practitioners to these teams. ‘we are at the bottom of the ladder’. More team meetings between assistants, nurses and physicians were required, as were more rehabilitation staff, to address a holistic view of clients that incorporated social, medical and spiritual aspects. Chain: Standardization of work processes p g p In Case B, physicians, nurses and nurse assistants from hospitals, primary care and mu- nicipalities would ideally treat patients at home in collaboration. One physician mentioned the im- portance of the collaborations and relationships with other staff at patients’ homes, but another said it was difficult for physicians to be part of the group of nurses. However, working in teams was essential for the patient group with multiple and chronic illnesses. When all staff participated, most nurses and nurse assistants considered the collab- oration to be good. However, even though official documents stated that nurse assistants should participate in these meetings, many of them were not involved, and some interviewed nurse assis- tants did not know that the teams existed and had bumped into the nurse/physician at a patient’s home by coincidence. The nurse assistants said, ‘the physicians do not really exist in our world’ or In the cancer case, traditional healthcare organi- zation based on medical specialization – and thus human anatomy – caused various problems, not least an inability to address the patient’s holistic medical situation. Introducing processes through training and education to the newly appointed process-owners was an attempt to bridge units and cut across silos. However, many process-owners mentioned that their role sometimes came into conflict with traditional, more ‘bureaucratic’ or ‘chain-of-command’, aspects of healthcare orga- nization. These process-owners felt they lacked a mandate because they were not responsible for either economy or staff at the hospitals. Others argued that this was an enabler for value creation since they could focus on ‘creating dialogue’. It was standardized that there should be processes run by clinically active physicians as process-owners, but 10 E. Eriksson et al. more qualitative ways. For instance, some process- owners had recruited patient representatives in their teams, which had been important for identify- ing areas in need of improvement; for example, by bringing attention to situations in which patients risked falling through the cracks between organi- zational units, as well as providing feedback on printed information to patients. However, finding patients to involve was difficult and those willing to participate were ‘not exactly the weak patients’. Some of the process-owners were more sceptical about patient involvement, arguing that collecting patient-reported data was a ‘political thing’ or ‘po- litically correct’ rather than being based on medi- cal evidence. not how they were supposed to run their respective team. © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management. Chain: Standardization of work processes However, it was standardized on the next level of abstraction in which the process-owners of the different cancer processes met continuously to exchange experiences and learn from each other. There was a sense of ‘connection’ and ‘creating identity’ in these meetings, that there were others doing the same thing. Many had created similar arenas for their respective cancer process. p p In the case of elderly care, the different orga- nizations had tried to collaborate for years, but when they started to map the process, it became ev- ident that no organization could see the complete patient journey and that collaboration was neces- sary if they were to have a holistic approach to care of the elderly. The model had started in a few municipalities and then spread in the rest of the area. Some of the municipalities mentioned that the standardization was good, since it clearly put the patient at the centre and was a model that had obviously worked elsewhere. Others were critical of the adopted model, arguing that little consider- ation was given to local prerequisites and already established collaborations. In Case B, official documents and coordinators also mentioned the need to work in a ‘person- centred’ manner and to see the whole person, not only the diagnosis, and with patient involvement in the teams. The nurse assistants explained that the broader life situation, everyday situations, relatives and so on had to be taken into account in their ev- eryday work, but that no one had asked them how the teams should work in a ‘person-centred’ way. Network: Trust and relationship In the cancer case, the local clinicians were ac- countable for registering data, but reporting rates among the local clinicians were sometimes too low. Because the process-owners did not have a man- date, they felt that they were not in a position to require local clinicians to improve data reporting. Some clinicians did not report sufficiently because of a heavy workload, and asking for data could not only add to the stress but also take the fo- cus off treating patients. Being active physicians themselves, the process-owners understood every- day work and could be careful not to place a bur- den on their colleagues: ‘It is easy to measure, and it can be misused’. In the cancer case, the first year as a process-owner had been spent travelling around western Sweden to meet the local hospital representatives in or- der to ‘get an understanding’ of how they worked and to establish dialogue with the local physicians to create trust and relationship. That the process- owners themselves worked clinically ‘made dia- logue easier’ and created ‘trust among colleagues’. Ideally, the collaboration with local clinicians cre- ated a sense of ‘doing something together’ and enabled more direct and honest communication. To some, having ‘history in the field’ helped them gain ‘authority’ in contacts with other clinicians, whereas others felt that having a ‘history’ meant they had ‘more enemies than friends’ among the local professionals. In the elderly care case, the decentralization of staff working close to the patients (physicians, nurses, nurse assistants, rehabilitation personal) was an important aspect of decentralization of accountability. In the elderly care case, the collaboration be- tween the three organizations was said to have developed over time. In the first years, the organizations were stuck discussing organizational responsibilities and boundaries. Over time, a re- lationship developed between the participants and trust grew, which made the process of address- ing issues and improving services much smoother. It was argued that the organizations had to © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management. Network: Systems approach In the cancer case, the cancer centre emphasized the cancer care system, often by using a picture of an aqueduct: the patient’s journey in the upper fur- row, supported by clinicians in the upper vault and continuously, with each vault existing to support the level above. Many process-owners agreed with the interconnectedness and how things ‘hung to- gether’ at the same time, highlighting that ‘knowl- edge and interest derive from the floor’ and that ‘we are supposed to do things for the patient’. The shop is likely to be more appropriate when the problem to be solved is diffuse and a consid- erable amount of expertise is required to quickly identify the causes of the problem (Christensen, Grossman and Hwang, 2009). Empirical examples of this are found in both cases, but most promi- nently in Case B, in which physicians, nurses and nurse assistants worked around elderly patients for optimal treatment in their homes rather than sending patients back and forth between health- care specializations. It is likely that the professional knowledge of the shop is more prominent when knowledge that is more specialized is required to define the needs and problems of public service (Ferlie, 2017). Management’s trust in the profes- sionals’ expertise ethos (Denhardt and Denhardt, 2015) has been central in the Swedish discourse over the last decade (e.g. Statens offentliga utred- ningar, 2019), in which the need to increase the professionals’ autonomy in the public sector has been emphasized. Consequently, professional-led collaborations have been encouraged. The empha- sis on trust in our Case B in cancer care shows similarities with the managed clinical networks in UK cancer care (Addicott, McGivern and Fer- lie, 2007), in which professional collaborations are ‘managed’ in both cases by the professionals them- selves and in which trust is a critical component. The empirical data illustrate how the creation of the value propositions is contingent on the reliance on professional knowledge and teamwork, which highlights the prospect that the shop configuration may hold in practice. In Case B, the system supporting care of the el- derly consisted of various networks: management network represented by managers and politicians (local and ‘regional’ networks existed, but the dis- tinction is not important here). These networks were important for discussing problems at the local level and understanding each other’s perspectives. Network: Systems approach In learning networks, staff working at homes met to share experiences, and at local levels, the teams themselves met. Coordinators were supposed to hold together both horizontally and vertically and were described as a bridge between managers and teams. This was especially important in the begin- ning – to hold the collaboration between organi- zations and professions together – but it is impor- tant not to be a burden or ‘control apparatus’, but rather to absorb difficulties at local levels and pass on updated information. Network: Patient involvement In Case A, the process-owners had also added patient-reported data to the traditional medical data. Patient involvement was also included in Value Propositions in Public Collaborations 11 value configuration serves a different purpose, and therefore enables different ‘building blocks’ for po- tential value creation through value propositions of PSO collaborations. By utilizing the ideal value configurations suggested by Stabell and Fjeldstad (1998), the findings of the present paper propose that public service collaborations – and hence re- search concerning the newly emerged PSL (Engen et al., 2021; Osborne, Nasi and Powell, 2021) – must acknowledge that distinct value configura- tions influence each other and describe different organizational processes that, in concert, hold po- tential to facilitate the creation of the value propo- sitions for the end user/citizen. continually nurture the relationship by discussing and understanding each other’s rules and tasks to maintain trust. When functioning ideally, the col- laboration meant that clients received better care and treatment by meeting many professionals from different organizations at the same time, not least the nurse assistants, who were often the staff mem- bers the clients knew best. © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management. Discussion Instead, the focus of PSL is either on citizen/public service user or diffuse public service ecosystems (e.g. Osborne, 2020). In doing so, important as- pects regarding the way in which the organization – either alone or jointly with other organizations – develops its value propositions, as potential value that the actors concretize to real value (Eriksson et al., 2020; Skålén et al., 2018), have been left un- elaborated, both in theory and in practice. y p The PSL literature is often overly positive about the notion of value creation (Cluley, Parker and Radnor, 2021; Dudau, Glennon and Verschuere, 2019) and we believe that the value configurations may help to increase understanding of the com- plex factors that influence organizations’ ability to develop joint value propositions. For instance, the assumptions that value is mutually created in public service ecosystems by resource-integrating actors (Eriksson and Hellström, 2021) builds on an ideal that is also prominent in the general col- laboration literature, in which non-hierarchy and informality are assumed (Span et al., 2012). How- ever, the empirical cases show that the presence of bureaucratic structures, managerial top-down as- pects and standardized one-size-fits-all solutions is a reality in these collaborations. In this sense, different value configurations might both facilitate and hinder value propositions through PSO col- laborations (cf. Cluley, Parker and Radnor, 2021). As Provan and Kenis (2008) have illustrated, formal structures are often needed to support collaboration, and it is important that formaliza- tion supports collaboration rather than making it more difficult. A systems perspective is central in the networked configuration, which recognizes the active role of a multiplicity of actors (Fjeldstad et al., 2020), as seen in both cases. This is deemed necessary in or- der to gather resources provided by the responsi- ble PSO, but also when knowledge and skills are required from other actors or when responsibility is unclear, such as for many contemporary chal- lenges (Sørensen and Torfing, 2011). The required knowledge and skills may also include the citizens or public service users. This perspective may not be easily combined with the professional knowl- edge of the shop, not least because it may interfere with expectations about what public employees and public service users should do (Eriksson, 2019; Osborne, 2020). Discussion We analyse how the value configurations influ- ence value propositions through collaborations. It is highlighted that a strict focus on the network value configuration is too narrow when aiming to develop potential value in public service collabo- rations. When focusing on the organizational level of the studied PSOs, it becomes apparent that the development of potential value is also dependent on the shop and chain value configurations. Each However, this has proved easier said than done. The traditional public administration, with its hierarchies and rules, has not always been eas- ily combined with professional autonomy; fea- tures of the chain brought about by the NPM 12 E. Eriksson et al. paradigm – in which standardization of processes has sought a one-size-fits-all model at the same time – hinder autonomy (Hellström, Lifvergren and Quist, 2010), as seen to various degrees in both empirical cases. The administrative burden of staff to report output (Moynihan, Herd and Harvey, 2015), typical of the chain configuration, may be somewhat counteracted if those with ex- pert knowledge rather than managers also decide what to report and what may not even need to be reported (Ferlie et al., 2016). However, the empir- ical data also highlight the benefits of the chain configuration in its ability to convey, both within the studied PSOs as well as across organizational boundaries, a clearer understanding of how vari- ous actors’ actions have to be efficiently connected in order to successfully cater for the needs of the service user. paradigm – in which standardization of processes has sought a one-size-fits-all model at the same time – hinder autonomy (Hellström, Lifvergren and Quist, 2010), as seen to various degrees in both empirical cases. The administrative burden of staff to report output (Moynihan, Herd and Harvey, 2015), typical of the chain configuration, may be somewhat counteracted if those with ex- pert knowledge rather than managers also decide what to report and what may not even need to be reported (Ferlie et al., 2016). However, the empir- ical data also highlight the benefits of the chain configuration in its ability to convey, both within the studied PSOs as well as across organizational boundaries, a clearer understanding of how vari- ous actors’ actions have to be efficiently connected in order to successfully cater for the needs of the service user. © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management. © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management Discussion In relation to this, the choice to lead, not only from professionals, but also from those with long-term careers in the collaborations (Ferlie et al., 2011), may be important to maintain the organization learning and retention of important values and norms that are important in order for collaborative networks to last over time (Anders- son, Stockhult and Tengblad, 2021; Pollitt, 2009). Our study shows how organizational learning and retention of important values are essential intra-organizational processes that support value creation through collaboration over time. sitions. Each configuration highlights different inter- and intra-organizational processes, within and between organizations, which serve different purposes in forming the value proposition. The shop configuration underscores the need to ac- knowledge professional knowledge and teamwork as central; the chain configuration contributes by accentuating the need for actors’ actions to be connected; and the network configuration is vital for facilitating trusting relationships and patient involvement. Despite the configurations’ inherent differences, they all influence value propositions that are later realized into real value by the public service users (and other actors in the public ser- vice ecosystem). Focusing on the organizational level in value creation is an essential perspective when value creation offered by PSL emphasizes abstract public service ecosystems or the citizen. The contribution to practice and policy is the importance of recognizing a multiplicity of loci for value creation in collaborations. This paper has several limitations. First, the focus has been on value creation, not the central issue in general PAM literature (Moore, 1994), as well as the developments of PSL (Alford, 2016), of what value is in a public administration context. Thus, future research could connect the value configurations to perceptions of value at different levels: public, individual and potential in-between levels (e.g. Eriksson and Nordgren, 2018). Sec- ond, the empirical material draws from a Swedish context only, and only cases from healthcare. The decentralized healthcare system, relatively long-term NPM implementation and consensus orientation in public administration (Christensen and Lægreid, 2002; Wiborg, 2015) are but a few aspects that make the Swedish case less typical, in some respects at least. Thus, similar research on value creation in collaborations in other public ser- vices and in other countries is likely to be needed. PSL makes little mention of leadership, and it is clear that, in collaborations, another type of lead- ership is required. Discussion Relatedly, the notion of individu- alization in public services – as in modern society overall (Beck, 1992) – promoted by sector-specific concepts (such as patient or person-centredness in healthcare; Andersson and Liff, 2012), is not easily combined with the standardization feature of the chain (Stabell and Fjeldstad, 1998). Despite these inherent challenges, the findings of this paper show that the network configuration has potential to develop the value proposition for the user/citizen through facilitating a systematic approach towards service delivery characterized by trusting relation- ships and patient involvement. As the inter-organizational collaborations are not always self-organized, coordination may often be essential. In both cases, a new administrative unit was created (the process ownership and the coordinators in Cases A and B, respectively) to govern the network (Provan and Kenis, 2008). The idea that this type of centralized collabora- tion entails more traditional managerial features (Markovic, 2017) is shown, to a certain extent, in the empirical material, in which reporting of the value chain’s output was central in the narratives, but it also avoids one single organization fully controlling the network and thereby creates better conditions for meeting the interests of all organi- zations, as it avoids too much competition between different organizational value configurations. Collectively, the shop, chain and network con- figurations emphasize that processes within and between organizations are essential for developing potential value in collaborations. This focus on the organizational level is typically and deliberately avoided by PSL (Grönroos, 2019; Osborne, 2018). Moreover, the under-theorized (Cristofoli, Meneguzzo and Riccucci, 2017) interpersonal Value Propositions in Public Collaborations 13 dimensions of collaborations were also clear in both cases. Shop configuration and profession- alization may be an ideal, but it may also be difficult for some professions to be involved in the teamwork (such as the physician ‘being part of the gang’ with the nurses), as well as for some profes- sions to access leadership (process ownership was for physicians, not nurses). Thus, the differences of mandate and power in collaborations (Agranoff and McGuire, 2004) within organizations and between organizations should not be neglected, as these are likely to impact potential value cre- ation. Discussion In both cases, an ‘integrative leadership’ (Crosby and Bryson, 2010) was found among the managers, arguing that it was impor- tant to facilitate interaction between participants. The process-owners mentioned that they often lacked a formal mandate and that this ‘distributed leadership’ (Crosby, Hart and Torfing, 2017) was sometimes difficult, but also an enabler as it could focus more on creating trust – an essential feature in a network configuration (Fjeldstad et al., 2020). Whereas Crosby and Bryson (2010) emphasized integrative leadership as an inter-organizational phenomenon, our study adds that integrative lead- ership also constitutes intra-organizational pro- cesses that might facilitate value creation through collaboration. © 2021 The Authors. British Journal of Management published by John Wiley & Sons Ltd on behalf of British Academy of Management. Conclusion, limitations and future research ‘Collaborative governance in the- ory and practice’, Journal of Public Administration Research and Theory, 18, pp. 543–571. Crosby, B., T. P. Hart and J. 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He also serves as scientific co-director at the Centre for Healthcare Improvement at Chalmers University of Technology.
https://openalex.org/W2562328459	https://journals.ssau.ru/index.php/vestnik/article/download/2075/2088	Russian	null	Specific electrical conductivity of chromium plating and nickel plating electrolytes	Vestnik Samarskogo universiteta. Aèrokosmičeskaâ tehnika, tehnologii i mašinostroenie	2,015	cc-by	4,324	Ульяновский научно-технологический центр Всероссийского института авиационных материалов (УНТЦ ВИАМ) Представлены результаты измерений удельной электропроводности (УЭП) электролитов хроми- рования и никелирования с помощью бесконтактного кондуктометра типа КРАБ-Д № 064 в температур- ных интервалах, соответствующих рабочим диапазонам электрохимических процессов осаждения по- крытий. Показана принципиальная схема установки для измерений УЭП электролитов. В качестве объ- ектов исследования выбраны электролиты на основе соединений хрома различной степени валентности (Cr3+,Cr6+) и стандартный электролит никелирования (электролит Уотса). Исследована зависимость УЭП электролитов от их температуры, дана сравнительная оценка влияния температурного фактора на вели- чину электропроводности различных электролитов хромирования. Изучено влияние добавок наноразмерных частиц оксида алюминия и карбида кремния дисперсно- стью 40-100 нм и удельной поверхностью 23-32 м2/г на величину электропроводности оксалатно- сульфатного электролита хромирования при заданных значениях температуры. Исследованием наносус- пензий установлено, что добавка в электролиты наноразмерных частиц различной природы концентра- цией 5-10 г/л не оказывает существенного влияния на величину УЭП. Для оценки взаимосвязи электропроводности электролитов с технологическими параметрами про- цесса электроосаждения хромовых и никелевых покрытий была проведена количественная оценка выхо- да по току хрома и никеля и представлены гистограммы, показывающие соотношение величин электро- проводности электролита и выхода металла по току. Установлено, что электропроводность электролитов и выход металла по току являются независимыми друг от друга параметрами. Удельная электропроводность, кондуктометр, электролит хромирования, электролит никелирова- ния, выход по току, наноразмерные частицы. свящённых изучению природы электро- проводности электролитов, до сих пор окончательно не выяснены и не объясне- ны важнейшие экспериментально наблю- даемые закономерности изменения элек- тропроводности электролитов в зависимо- сти от температуры, природы растворите- ля и концентрации компонентов. Изуче- ние зависимостей электропроводности от температуры и состава раствора позволяет получить данные, которые делают воз- можным рациональный выбор электроли- тов, конструкции электролизёров и источ- ников питания [3]. Имеющиеся в литера- туре данные по исследованию электро- проводности посвящены в основном срав- нительно разбавленным системам [4–6]. В случае изучения концентрированных систем, к которым относятся исследуемые электролиты хромирования и никелиро- № 3(45) 2014 г. № 3(45) 2014 г. Вестник Самарского государственного аэрокосмического университета тник Самарского государственного аэрокосмичес УДК 621.357.7 ИССЛЕДОВАНИЕ УДЕЛЬНОЙ ЭЛЕКТРОПРОВОДНОСТИ ЭЛЕКТРОЛИТОВ ХРОМИРОВАНИЯ И НИКЕЛИРОВАНИЯ © 2014 Р. К. Салахова, В. В. Семенычев, А. Б. Тихообразов Ульяновский научно-технологический центр Всероссийского института авиационных материалов (УНТЦ ВИАМ) Введение Защитные и функциональные галь- ванические покрытия являются наиболее распространёнными в машиностроении и нашли массовое применение в различных отраслях промышленности. Вместе с этим постоянно растут и требования, предъяв- ляемые к вновь разрабатываемым покры- тиям по их пористости, износостойкости, коррозионной стойкости и по экологиче- ской безопасности процессов их осажде- ния [1]. В связи с этим интенсивно разра- батываются новые технологические про- цессы осаждения покрытий, обладающих необходимыми свойствами [2]. Одной из важнейших физико- химических характеристик электролитов является их электропроводность. Несмот- ря на значительное количество работ, по- 70 Вестник Самарского государственного аэрокосмического университета № 3(45) 2014 г. ваника», объектами исследований которо- го являются процессы электроосаждения покрытий в электролитах, содержащих наноразмерные частицы [9, 10]. вания, необходимо учитывать процессы комплексообразования и структурные пе- рестройки в растворах, как это делается для органических сред. Несмотря на ряд попыток ввести дополнения и поправки на учёт активности ионов и ионных ассоциа- тов, вопрос об интерпретации свойств и особенностях строения концентрирован- ных водных растворов сильных электро- литов остаётся до сих пор открытым. Методическая часть Измерения удельной электропро- водности (УЭП) электролитов проводили с помощью кондуктометра радиочастот- ного бесконтактного типа КРАБ-Д № 0647. Погрешность показаний прибора, проверенная на стандартном растворе хлористого калия в соответствии с ГОСТ 22171, составила 6 %. Исследуемый тем- пературный интервал электролитов был выбран исходя из рабочих диапазонов электрохимических процессов осаждения покрытий, а за величину УЭП принимали среднее значение измеренной УЭП, полу- ченной в режиме нагрев-охлаждение. Со- ставы исследуемых электролитов пред- ставлены в табл. 1. В качестве объектов исследования были использованы элек- тролиты хромирования на основе соеди- нений Cr6+: стандартный – I, саморегули- рующийся концентрированный – II, само- регулирующийся разбавленный – III; на основе соединений Cr3+ (оксалатно- сульфатный) – IV; стандартный электро- лит никелирования (электролит Уотса) – V по ГОСТ 9.305. В качестве количественной меры способности раствора электролита прово- дить электрический ток обычно исполь- зуют удельную электропроводность – ве- личину, обратную удельному сопротивле- нию. Известно, что величина удельной электропроводности электролита зависит от целого ряда факторов: природы элек- тролита, температуры, концентрации рас- твора [7, 8]. В данной работе рассматри- вается взаимосвязь (корреляция) между удельной электропроводностью (УЭП) электролитов хромирования и никелиро- вания и выходом металла по току, а также оценивается влияние вводимых в электро- литы наноразмерных частиц на величину УЭП. Исследования электропроводности электролитов-суспензий с добавками на- ночастиц представляет особый интерес, так как являются частью исследований в рамках существующего в УНТЦ ВИАМ научного направления «кластерная галь- Таблица. 1. Составы электролитов Состав электролита Концентрация, г/л №/№ I II III IV V 1 СrO3 230 – 270 230 – 270 140 – 170 - - 2 H2SO4 23 – 27 - - - - 3 K2SiF6 - 18 – 22 - - - 4 Sr2SO4 - 3-5 3 – 5 - - 5 Cr2(SO4)3 - - - 200 – 250 - 6 Al2(SO4)3 18Н2O - - - 90 – 120 - 7 Na2SO4 - - - 120 – 140 - 8 NaF - - - 20 – 30 - 9 Na2C2O4 - - - 20 – 30 - 10 NiSO4 - - - - 280 – 330 11 NiCl2 - - - - 35 – 50 12 H3BO3 - - - - 25 – 35 13 Температура,0C 43 – 65 43 – 65 43 – 65 30 – 51 18 – 45 Таблица. 1. Вестник Самарского государственного аэрокосмического университета № 3(45) 2014 г. Вестник Самарского государственного аэрокосмического университета Выход металла (хрома, никеля) по току, т.е. отношение фактически оса- ждённого определённым количеством электричества металла к его теоретиче- скому эквиваленту (отнесённому к тому же количеству электричества), выражен- ное в процентах, определяли по уравне- нию [11,12]: а – масса электрода до покрытия, г; b – масса электрода после покрытия, г; ЭМе – электрохимический эквивалент металла. В качестве электрода для оценки ве- личины выхода по току использовали стальной шарик диаметром 19 мм (пло- щадь поверхности равна 0,1 дм2). Была исследована электропровод- ность оксалатно-сульфатного электролита хромирования с добавками наноразмер- ных частиц оксида алюминия и карбида кремния. Характеристики используемых наночастиц представлены в табл. 2. η = 6000(b - a)/ЭМеIT, где η – выход по току, %; I – сила тока, А; T – продолжительность электролиза, мин.; Таблица 2. Характеристики наноразмерных частиц Al2O3 и SiC Вид нанопорошка Форма частиц Размер частиц, нм Среднее значение дисперсности, нм Удельная поверхность, м2/г Хим. состав основных веществ, % Al2O3 сферическая 5 – 100 40 32 > 99 SiC кубическая, осколочная 50 – 250 100 23 > 99 Таблица 3. Результаты измерений УЭП № электролита Температура,0С I II III IV V 31 - - - 6,1 5,6 33 - - - 6,35 5,8 35 - - - 6,65 6,1 37 - - - 6,95 6,3 39 - - - 7,3 6,5 41 - - - 7,55 6,8 43 55,5 52,6 42,9 7,85 7,0 45 56,35 53,25 43,7 8,2 7,2 47 57,15 53,95 44,4 8,5 7,5 49 57,9 54,8 45,0 8,9 7,7 51 58,6 55,55 45,6 9,25 7,9 53 59,4 56,3 46,1 - - 55 60,1 57,05 46,7 - - 57 60,8 57,7 47,3 - - 59 61,5 58,4 47,8 - - 61 62,2 59,1 48,3 - - 63 62,65 59,7 48,8 - - 65 63,2 60,3 49,2 - - Таблица 2. Характеристики наноразмерных частиц Al2O3 и SiC Вид нанопорошка Форма частиц Размер частиц, нм Среднее значение дисперсности, нм Удельная поверхность, м2/г Хим. состав основных веществ, % Al2O3 сферическая 5 – 100 40 32 > 99 SiC кубическая, осколочная 50 – 250 100 23 > 99 Таблица 2. Характеристики наноразмерных частиц Al2O3 и SiC Таблица 3. Методическая часть Составы электролитов Состав электролита Концентрация, г/л №/№ I II III IV V 1 СrO3 230 – 270 230 – 270 140 – 170 - - 2 H2SO4 23 – 27 - - - - 3 K2SiF6 - 18 – 22 - - - 4 Sr2SO4 - 3-5 3 – 5 - - 5 Cr2(SO4)3 - - - 200 – 250 - 6 Al2(SO4)3 18Н2O - - - 90 – 120 - 7 Na2SO4 - - - 120 – 140 - 8 NaF - - - 20 – 30 - 9 Na2C2O4 - - - 20 – 30 - 10 NiSO4 - - - - 280 – 330 11 NiCl2 - - - - 35 – 50 12 H3BO3 - - - - 25 – 35 13 Температура,0C 43 – 65 43 – 65 43 – 65 30 – 51 18 – 45 71 Вестник Самарского государственного аэрокосмического университета Результаты измерений УЭП № электролита Температура,0С I II III IV V 31 - - - 6,1 5,6 33 - - - 6,35 5,8 35 - - - 6,65 6,1 37 - - - 6,95 6,3 39 - - - 7,3 6,5 41 - - - 7,55 6,8 43 55,5 52,6 42,9 7,85 7,0 45 56,35 53,25 43,7 8,2 7,2 47 57,15 53,95 44,4 8,5 7,5 49 57,9 54,8 45,0 8,9 7,7 51 58,6 55,55 45,6 9,25 7,9 53 59,4 56,3 46,1 - - 55 60,1 57,05 46,7 - - 57 60,8 57,7 47,3 - - 59 61,5 58,4 47,8 - - 61 62,2 59,1 48,3 - - 63 62,65 59,7 48,8 - - 65 63,2 60,3 49,2 - - Таблица 3. Результаты измерений УЭП 72 № 3(45) 2014 г. Вестник Самарского государственного аэрокосмического университета Результаты и их обсуждение никелирования. Исходя из этого, можно утверждать, что «шестивалентные» элек- тролиты хромирования являются более сильными электролитами, чем электроли- ты IV и V. Для всех видов электролитов характерно увеличение электропроводно- сти с повышением температуры, что со- гласуется с теоретическими основами электрохимии и данными, приведёнными в [13–16]. Однако стоит отметить, что температурный фактор оказывает мень- шее влияние на электролиты хромирова- ния на основе соединений Cr6+ (увеличе- ние УЭП на 15 %), чем на остальные элек- тролиты (увеличение УЭП на 40–50 %). В табл. 3 представлены результаты измерений УЭП исследуемых электроли- тов в температурном диапазоне, соответ- ствующем оптимальным значениям тем- пературы в процессе электроосаждения покрытий. Графическая интерпретация зависимости УЭП электролитов от темпе- ратуры растворов показана на рис. 1, 2. Видно, что электролиты на основе хромо- вой кислоты (I – III) имеют самую высо- кую удельную электропроводность, кото- рая почти на порядок выше значений УЭП оксалатно-сульфатного электролита хро- мирования и стандартного электролита Рис. 1. УЭП электролитов хромирования на основе Cr6+ Рис. 2. УЭП оксалатно-сульфатного электролита (IV) и электролита Уотса (V) Рис. 1. УЭП электролитов хромирования на основе Cr6+ Рис. 2. УЭП оксалатно-сульфатного электролита (IV) и электролита Уотса (V) Рис. 2. УЭП оксалатно-сульфатного Рис. 2. УЭП оксалатно-сульфатного электролита (IV) и электролита Уотса (V) Рис. 1. УЭП электролитов хромирования на основе Cr6+ Если вопрос о зависимости электро- проводности электролитов от их темпера- туры и концентрации изучен достаточно хорошо, то публикаций об исследовании электропроводности электролитов- суспензий практически не встречается. В связи с этим особый интерес представляет получение экспериментальных данных о зависимости электропроводности элек- тролитов от концентрации вводимых в них наноразмерных частиц различной природы. В качестве добавок использова- ли наночастицы Al2O3, которые являются диэлектриками, и наночастицы SiC, отно- сящиеся к полупроводникам. Измерение электропроводности проводили при тем- пературах 37°С, 41°С, 45°С, соответству- ющих рабочему температурному диапазо- ну процесса «трёхвалентного» хромиро- вания. Установлено, что добавка в элек- тролиты наноразмерных частиц концен- трацией 5–10 г/л не оказывает существен- ного влияния на величину УЭП, что пока- зано на примере исследования наносус- пензии оксалатно-сульфатного электроли- та хромирования (рис. 3). Это свидетель- ствует о том, что наночастицы не оказы- вают влияния на степень диссоциации электролита и подвижность ионов в рас- творе и их функциональная роль, как бы- ло доказано исследованиями [17–20], сво- дится в основном к адсорбции на своей поверхности ионов покрываемого металла и доставке образовавшихся кластеров в прикатодное пространство. ну процесса «трёхвалентного» хромиро- вания. Заключение Содержание в электролитах хроми- рования и никелирования наноразмерных частиц Al2O3 и SiC в рабочем диапазоне концентраций 5–10 г/л не оказывает су- щественного влияния на величину УЭП. С повышением температуры удель- ная электропроводность стандартного и саморегулирующегося электролитов хро- мирования увеличивается на 15 %, окса- латно-сульфатного электролита хромиро- вания и стандартного электролита нике- лирования – на 40–50 %. Не установлена корреляция между электропроводностью исследуемых элек- тролитов и выходом металла по току. Результаты и их обсуждение Установлено, что добавка в элек- тролиты наноразмерных частиц концен- трацией 5–10 г/л не оказывает существен- ного влияния на величину УЭП, что пока- зано на примере исследования наносус- пензии оксалатно-сульфатного электроли- та хромирования (рис. 3). Это свидетель- ствует о том, что наночастицы не оказы- вают влияния на степень диссоциации электролита и подвижность ионов в рас- творе и их функциональная роль, как бы- ло доказано исследованиями [17–20], сво- дится в основном к адсорбции на своей поверхности ионов покрываемого металла и доставке образовавшихся кластеров в прикатодное пространство. 73 Вестник Самарского государственного аэрокосмического университета № 3(45) 2014 г. а б Рис. 3. УЭП электролита хромирования IV при температурах 37°С, 41°С, 45°С: а – с добавками наночастиц Al2O3; б – с добавками наночастиц SiC б Рис. 3. УЭП электролита хромирования IV при температурах 37°С, 41°С, 45°С: а – с добавками наночастиц Al2O3; б – с добавками наночастиц SiC На рис. 4 представлена гистограмма, демонстрирующая соотношение величин электропроводности электролита и выхо- да металла по току. Как видно из рис. 4, электропроводность электролита и выход по току не коррелируют между собой, т.е. являются независимыми друг от друга па- раметрами. Полученные результаты сви- детельствуют о том, что стадия массопе- реноса реагирующих веществ не является лимитирующей стадией электроосажде- ния хромовых и никелевых покрытий, а выход металла по току определяется в ос- новном величиной перенапряжения выде- ления водорода, которая, по-видимому, не связана с УЭП электролитов. Рис. 4. Соотношение величин электропроводности электролита и выхода металла по току Рис. 4. Соотношение величин электропроводности электролита и выхода металла по току 74 № 3(45) 2014 г. Вестник Самарского государственного аэрокосмического университета Библиографический список 1. Каблов Е.Н. Стратегические направления развития материалов и тех- нологий их переработки на период до 2030 года // Авиационные материалы и технологии. 2012. № S. С. 7-17. 8. Артёмкина Ю.М., Плешкова Н.В., Седдон K.P., Щербаков В.В. Электропро- водность некоторых ионных жидкостей // Физико-химические свойства растворов и неорганических веществ: сб. научных трудов. Вып. 182. М.: РХТУ им. Д.И. Менделеева, 2008. С. 139-144. 8. Артёмкина Ю.М., Плешкова Н.В., Седдон K.P., Щербаков В.В. Электропро- водность некоторых ионных жидкостей // Физико-химические свойства растворов и неорганических веществ: сб. научных трудов. Вып. 182. М.: РХТУ им. Д.И. Менделеева, 2008. С. 139-144. 2. Семенычев В.В., Салахова Р.К., Тюриков Е.В., Ильин В.А. Защитные и функциональные гальванические покры- тия, получаемые с применением нанораз- мерных частиц // Авиационные материалы и технологии. 2012. № S. С. 335-342. 2. Семенычев В.В., Салахова Р.К., Тюриков Е.В., Ильин В.А. Защитные и функциональные гальванические покры- тия, получаемые с применением нанораз- мерных частиц // Авиационные материалы и технологии. 2012. № S. С. 335-342. 9. Ильин В.А. Нанотехнологии нане- сения кластерных гальванических покры- тий // Авиационные материалы и техноло- гии. 2009. № 2. С. 3-6. 9. Ильин В.А. Нанотехнологии нане- сения кластерных гальванических покры- тий // Авиационные материалы и техноло- гии. 2009. № 2. С. 3-6. 3. Антропов Л.И. Теоретическая электрохимия. М.: Высшая школа, 1969. 510 с. 10. Налётов Б.П. Кластерная гальва- ника – нанотехнология электрохимиче- ских коллоидных систем // Авиационные материалы и технологии. 2009. № 1. С. 27- 32. 4. Щербаков В.В., Артемкина Ю.М., Понамарева Т.Н., Кириллов А.Д. Элек- тропроводность системы аммиак-вода // Журнал неорганической химии. 2009. Т. 54, № 2. С. 321-323. 11. Вячеславов П.М., Шмелёв Н.М. Контроль электролитов и покрытий. Л: Машиностроение, 1985. 96 с. р 12. Нагаев В.В. Исследование элек- тролитов никелирования, содержащих нанопорошки // Авиационные материалы и технологии. 2009. № 1. С. 18-19. р 12. Нагаев В.В. Исследование элек- тролитов никелирования, содержащих нанопорошки // Авиационные материалы и технологии. 2009. № 1. С. 18-19. 5. Щербаков В.В., Артемкина Ю.М., Плешкова Н.В., Седдон К.Р. Электриче- ская проводимость растворов некоторых ионных жидкостей в ацетонитриле // Хи- мическая промышленность сегодня. 2011. № 12. С. 36-41. 5. Щербаков В.В., Артемкина Ю.М., Плешкова Н.В., Седдон К.Р. Электриче- ская проводимость растворов некоторых ионных жидкостей в ацетонитриле // Хи- мическая промышленность сегодня. 2011. № 12. С. 36-41. 13. Багоцкий В.С. Основы электро- химии. М.: Химия, 1988. 400 с. 14. Бардышев И.И., Ляхов Б.Ф., По- лукаров Ю.М, Котенев В.А., Цивад- зе А.Ю. Информация об авторах Салахова Розалия Кабировна, кандидат технических наук, начальник лаборатории, Ульяновский научно- технологический центр Всероссийского института авиационных материалов. E- mail: salahovark@viam.ru. Область науч- ных интересов: электрохимия, коррозия и защита металлов, электроосаждение галь- ванических покрытий. Салахова Розалия Кабировна, кандидат технических наук, начальник лаборатории, Ульяновский научно- технологический центр Всероссийского института авиационных материалов. E- mail: salahovark@viam.ru. Область науч- ных интересов: электрохимия, коррозия и защита металлов, электроосаждение галь- ванических покрытий. Всероссийского института авиационных материалов. E-mail: untcviam@viam.ru. Область научных интересов: конструкци- онные сплавы, коррозионная стойкость, защитные и функциональные покрытия, свойства сплавов. Тихообразов Андрей Борисович, ведущий инженер-технолог, Ульяновский научно-технологический центр Всерос- сийского института авиационных матери- алов. E-mail: untcviam@viam.ru. Область научных интересов: кластерные гальвани- ческие покрытия, нанотехнологии. Семенычев Валентин Владимиро- вич, кандидат технических наук, главный научный сотрудник лаборатории, Улья- новский научно-технологический центр Библиографический список Исследование служебных свойств покрытий, получен- ных в саморегулирующемся электролите хромирования, содержащем нанопорошок Библиографический список Влияние абсорбции водорода на атомно-электронную структуру PDH по данным аннигиляции позитронов // Физи- кохимия поверхности и защита материа- лов. 2011. Т. 47, № 5. С. 550-553. 14. Бардышев И.И., Ляхов Б.Ф., По- лукаров Ю.М, Котенев В.А., Цивад- зе А.Ю. Влияние абсорбции водорода на атомно-электронную структуру PDH по данным аннигиляции позитронов // Физи- кохимия поверхности и защита материа- лов. 2011. Т. 47, № 5. С. 550-553. 6. Артемкина Ю.М. Закономерности в электропроводности некоторых раство- ров ассоциированных электролитов в воде и в ацетонитриле: Автореф. дис. канд. хим. наук. М., 2008. С. 16. 6. Артемкина Ю.М. Закономерности в электропроводности некоторых раство- ров ассоциированных электролитов в воде и в ацетонитриле: Автореф. дис. канд. хим. наук. М., 2008. С. 16. 7. Артёмкина Ю.М., Ворошилова Ю.В., Плешкова Н.В. и др. Ассоциация некоторых ионных жидкостей в ацетонит- риле по данным кондуктометрических измерений // Успехи в химии и химиче- ской технологии. 2008. Т. 22, № 3. С. 11- 16. 15. Петухов И.В., Медведева Н.А., Шестакова А.А. О временной зависимо- сти скорости коррозии Ni-P-покрытий в сульфатных средах // Коррозия: материа- лы, защита. 2013. № 5. С. 28-34. 75 № 3(45) 2014 г. Вестник Самарского государственного аэрокосмического университета 16. Кузнецов Ю.И. Прогресс в инги- бировании коррозии металлов и модифи- кация нанослоёв на металлах // Коррозия: материалы, защита. 2011. № 1. С. 1-10. оксида алюминия с размером частиц 5-50 нм // Авиационные материалы и техноло- гии. 2009. № 1. С. 13-17. оксида алюминия с размером частиц 5-50 нм // Авиационные материалы и техноло- гии. 2009. № 1. С. 13-17. оксида алюминия с размером частиц 5-50 нм // Авиационные материалы и техноло- гии. 2009. № 1. С. 13-17. 19. Салахова Р.К. Хромирование в электролите, содержащем соли трёхва- лентного хрома и нанопорошок оксида алюминия // Авиационные материалы и технологии. 2009. № 2. С. 19-24. 17. Тюриков Е.В., Семёнычев В.В., Ильин В.А. О роли наноразмерных частиц оксида алюминия в саморегулирующемся электролите хромирования // Известия Самарского научного центра РАН. 2012. Т.14, № 4 (3). С. 802-807. 17. Тюриков Е.В., Семёнычев В.В., Ильин В.А. О роли наноразмерных частиц оксида алюминия в саморегулирующемся электролите хромирования // Известия Самарского научного центра РАН. 2012. Т.14, № 4 (3). С. 802-807. 20. Салахова Р.К., Жирнов А.Д., Ильин В.А. Свойства и структура трёхва- лентных хромовых покрытий, сформиро- ванных в присутствии наноразмерных ча- стиц оксидов металлов // Коррозия: мате- риалы, защита. 2009. № 10. С. 140-141. ( ) 18. Тюриков Е.В. Вестник Самарского государственного аэрокосмического университета To evaluate the relationship between the electrolyte conductivity and process parameters of electrodeposi- tion of chromium and nickel coatings a qualitative assessment of chromium and nickel current output was per- formed and bar graphs showing the ratio of the values of the electrolyte electrical conductivity and the metal current output are presented. It has been established that the electrical conductivity of electrolytes and the metal current output are parameters that do not depend on each other. Electrical conductivity, conductivity meter, chromium electrolyte, nickel electrolyte, current output, nano particles. References vances in chemistry and chemical technolo- gy. 2008. V. 22, no. 3. P. 11-16. (In Russ.) 1. Kablov E.N. Strategic directions for the development of materials and tech- nologies to process them for the period up to the year 2030 // Aviation materials and tech- nologies. 2012. No. S. P. 7-17. (In Russ.) 8. Artemkina Yu.M., Pleshkova N.V., Seddon K.R., Shcherbakov V.V. Elec- trical conductivity of some ionic liquids // Cbornik nauchnykh trudov «Fiziko- khimicheskie svoystva rastvorov i neor- ganicheskikh veshchestv». Iss. 182. Moscow: RCTU them. D.I. Mendeleev Publ., 2008. P. 139-144. (In Russ.) 2. Semenychev V.V., Salakhova R.K., Tyurikov E.V., Ilyin V.A. Protective and functional galvanic coatings, obtained with the use of nano-sized particles // Avia- tion materials and technologies. 2012. No. S. P. 335-342. (In Russ.) 9. Ilyin V.A. Nanotechnology appli- cation cluster of galvanic coatings // Aviation materials and technologies. 2009. No. 2. P. 3-6. (In Russ.) 3. Antropov L.I. Teoreticheskaya el- ektrokhimiya [Theoretical electrochemistry]. Moscow: Vysshaya Shkola Publ., 1969. 510 p. 10. Nalyotov B.P. Cluster electroplat- ing-nanotechnology electrochemical colloi- dal systems // Aviation materials and tech- nologies. 2009. No. 1. P. 27-32. (In Russ.) 4. Shcherbakov V.V., Artemkina Yu.M., Ponomaryova T.N., Kirilov A.D. Electrical conductivity of the ammonia-water system // Russian journal of inorganic chem- istry. 2009. V. 54, no. 2. P. 277-279. 11. Vyacheslavov P.M., Shmelyov N.M. Kontrol' elektrolitov i pokrytiy [Con- trol of electrolytes and coatings]. Leningrad: Mashinostroenie Publ., 1985. 96 p. 5. Shcherbakov V.V., Artemkina Yu.M., Pleshkova N.V., Seddon K.R. Elec- trical conductivity of solutions of some ionic liquids in acetonitrile // Chemical industry today. 2011. No. 12. P. 36-41. (In Russ.) 12. Nagayev V.V. Research of nickel nanopowders containing electrolytes // Avia- tion materials and technologies. 2009. No. 1. P. 18-19. (In Russ.) 6. Artemkina Yu.M. Zakonomernosti v elektroprovodnosti nekotorykh rastvorov assotsiirovannykh elektrolitov v vode i v atsetonitrile: Avtoref. dis. kand. khim. nauк. [Consistent patterns of electrical conductivity of certain solutions of associated electrolytes in water and acetonitrile. Author’s abstract of the Candidate’s dissertation in chemistry]. Moscow, 2008. P. 16. 13. Bagocky V.S. Osnovy el- ektrokhimii [Fundamentals of electrochemis- try]. Moscow: Chemistry Publ., 1988. 400 p. 14. Bardyshev I.I., Lyakhov B.F., Polukarov Y.M., Kotenev V.A., Tsivadze A.Y. Effect of hydrogen absorption on atom- ic-electronic structure of PdH system accord- ing to positron annihilation data // Protection of metals and physical chemistry of surfaces. 2011. V. 47, no. 5. P. 680-683. 14. © 2014 R. K. Salakhova, V. V. Semenychev, A. B. Tihoobrazov © 2014 R. K. Salakhova, V. V. Semenychev, A. B. Tihoobrazov Ulyanovsk Science and Technology Center y gy of the All-Russian Institute of Aviation Materials USTC AIAM, Russian Federation Russian Institute of Aviation Materials USTC AIAM, Russian Federation The paper presents the results of measuring specific electrical conductivity (SEC) of chromium and nickel plating electrolytes with the help of a non-contacting conductivity meter of the CRAB-d 064 type in temperature intervals corresponding to the working ranges of electrochemical deposition processes. A schematic diagram of the apparatus for the measurement of electrolyte SEC is presented. Electrolytes based on chromium compounds of varying degrees of valence (Cr3+ Cr6 +) and a standard nickel electrolyte (Watts electrolyte) were chosen as the objects of study. The dependence of the SEC of electrolytes on their temperature is analyzed, a comparative as- sessment of the impact of temperature on the electrical conductivity of various chroming electrolytes is given. The influence of addition of nanoparticles of aluminum oxide and silicon carbide with the dispersion of 40-100 nm and a specific surface area of 23-32 m2 / g on the amount of electrical conductivity of an oxalate- sulfate electrolyte for chromium plating at a specified temperature is investigated. The study of nanosuspensions showed that the addition of nanoparticles of different nature with the concentration of 5-10 g / l to an electrolyte has no significant effect on the value of SEC. 76 76 № 3(45) 2014 г. References Bardyshev I.I., Lyakhov B.F., Polukarov Y.M., Kotenev V.A., Tsivadze A.Y. Effect of hydrogen absorption on atom- ic-electronic structure of PdH system accord- ing to positron annihilation data // Protection of metals and physical chemistry of surfaces. 2011. V. 47, no. 5. P. 680-683. 7. Artemkina Yu.M., Voroshilova Yu.V., Pleshkova N.V. Association of some ionic liquids in acetonitrile according to the data of conductometer measurements // Ad- 15. Petukhov I.V., Medvedeva I.V., Shestakova A.A. Time dependence of Ni-P coating corrosion rate in sulfate media // Cor- 15. Petukhov I.V., Medvedeva I.V., Shestakova A.A. Time dependence of Ni-P coating corrosion rate in sulfate media // Cor- 77 № 3(45) 2014 г. Вестник Самарского государственного аэрокосмического университета rosion: materials, protection. 2013. No. 5. P. 28-34. (In Russ.) chromium electrolyte containing aluminium oxide nanopowder particles with the size of 5-50 nm // Aviation materials and technolo- gies. 2009. No.1. P. 13-17. (In Russ.) 16. Kuznetsov Y.I. Advances in metal corrosion inhibition and modification of nanolayers on metals // Corrosion: materials, protection. 2011. No.1. P. 1-10. (In Russ.) 19. Salakhova R.K. Chrome-plating electrolyte containing salt trivalent of chro- mium and aluminium oxide nanopowder // Aviation materials and technologies. 2009. No. 2. P. 19-24. (In Russ.) 17. Tyurikov E.V., Semenychev V.V., Ilyin V.A. About the role of nanosized alu- minum oxide particles in the automatically adjusting chrome plating electrolyte // Izvestiya Samarskogo nauchnogo tsentra RAN. 2012. V.14, no.4 (3). P. 802-807. (In Russ.) 20. Salakhova R.K., Zhirnov A.D., Ilyin V.A. Properties and structure of triva- lent chrome plating formed in the presence of nanoscale metal oxide particles // Corrosion: materials, protection. 2009. No. 10. P. 140- 141. (In Russ.) 20. Salakhova R.K., Zhirnov A.D., Ilyin V.A. Properties and structure of triva- lent chrome plating formed in the presence of nanoscale metal oxide particles // Corrosion: materials, protection. 2009. No. 10. P. 140- 141. (In Russ.) 18. Tyurikov E.V. Study of properties of coatings obtained in a self-regulating About the authors Salakhova Rozalia Kabirovna, Can- didate of Science (Engineering), Chief of la- boratory, USTC AIAM. E-mail: salahovark@viam.ru. Area of Research: elec- trochemistry, corrosion and protection of metals against corrosion, deposition of elec- troplated coatings. untcviam@viam.ru. Area of Research: struc- tural alloys, corrosion resistance, protective and functional coatings, properties of alloys. Tihoobrazov Andrey Borisovich, leading production engineer, USTC AIAM, Ulyanovsk, Russian Federation. E-mail: untcviam@viam.ru. Area of Research: clus- tered electrolyte coatings, nanotechnologies. Semenychev Valentin Vladimirovich, Candidate of Science (Engineering), Chief Researcher, USTC AIAM. E-mail: Semenychev Valentin Vladimirovich, Candidate of Science (Engineering), Chief Researcher, USTC AIAM. E-mail: 78
https://openalex.org/W3005304761	https://journal.ilininstitute.com/index.php/caradde/article/download/374/244	Indonesian	null	Pendampingan Pemanfaatan Duolingo melalui Smartphone Sebagai Alat Pengajaran dan Pembelajaran Bahasa Inggris Bagi Masyarakat	Caradde	2,020	cc-by	3,498	Pendampingan Pemanfaatan Duolingo melalui Smartphone Sebagai Alat Pengajaran dan Pembelajaran Bahasa Inggris Bagi Masyarakat Raden Agus Budiharto 1 , Abdul Wahab Syahroni 2 Abstrak. Kegiatan pengabdian masyarakat ini bertujuan untuk memberikan pengetahuan pada guru bahasa Inggris dan siswa MA Miftahul Ulum Pagendingan-Pamekasan akan manfaat aplikasi Duolingo yang bisa digunakan sebagai alat pengajaran dan pembelajaran bahasa Inggris. Metode yang digunakan dalam kegiatan ini adalah berbentuk pelatihan dan pendampingan. Adapun pelatihan dan pendampingan yang diberikan dalam kegiatan ini adalah pemanfaatan aplikasi Duolingo melalui smartphone. Kegiatan dari pengabdian ini memberikan banyak pengetahuan pedagogi pada seluruh peserta sehingga mereka tahu bagaimana belajar atau mengajarkan bahasa Inggris dengan cara yang menyenangkan dengan menggunakan aplikasi Duolingo. Para peserta merasa bahwa aplikasi Duolingo sebagai aplikasi edukasi sangat bermanfaat ketika digunakan dalam kegiatan pengajaran dan pembelajaran bahasa Inggris di kelas. Keywords : Smartphone; Kemajuan Teknologi; Aplikasi Duolingo. Corespondensi Author 1 Program Studi Pendidikan Bahasa Inggris, Universitas Madura 2 Program Studi Teknik Informatika, Universitas Madura Jl. Raya Panglegur KM 3,5 Pamekasan-Madura Email: budiharto@unira.ac.id History Article Received: 21-Januari-2020; Reviewed: 14-Januari-2020; Accepted: 02-Februari-2020; Avalaible Online: 02-Februari-2020; Published: 14-Februari-2020 History Article Received: 21-Januari-2020; Reviewed: 14-Januari-2020; Accepted: 02-Februari-2020; Avalaible Online: 02-Februari-2020; Published: 14-Februari-2020 History Article Received: 21-Januari-2020; Reviewed: 14-Januari-2020; Accepted: 02-Februari-2020; Avalaible Online: 02-Februari-2020; Published: 14-Februari-2020 Abstract. The community service activity is aimed at providing a great command of Duolingo application for English teacher and MA Miftahul Ulum Pagendingan students where the application can be utilized as an interesting English teaching and learning tool. The method employed in this activity is training and mentoring regarding the utilization of Duolingo application through Smartphone. The activity of the service is desperately provide pedagogical commands for all participants how to teach or learn English in an interesting way that is by using Duolingo application. The participants consider that educational Duolingo application is extremely beneficial when to be utilized in English teaching and learning process in classroom. This work is licensed under a Creative Commons Attribution 4.0 International License This work is licensed under a Creative Commons Attribution 4.0 International License CARADDE: Jurnal Pengabdian Kepada Masyarakat https://journal.ilininstitute.com/index.php/caradde Volume 2 \| Nomor 2 \| Februari \|2020 e-ISSN: 2621-7910 dan p-ISSN: 2621-7961 DOI: https://doi.org/10.31960/caradde.v2i2.374 CARADDE: Jurnal Pengabdian Kepada Masyarakat https://journal.ilininstitute.com/index.php/caradde Volume 2 \| Nomor 2 \| Februari \|2020 e-ISSN: 2621-7910 dan p-ISSN: 2621-7961 DOI: https://doi.org/10.31960/caradde.v2i2.374 CARADDE: Jurnal Pengabdian Kepada Masyarakat https://journal.ilininstitute.com/index.php/caradde Volume 2 \| Nomor 2 \| Februari \|2020 e-ISSN: 2621-7910 dan p-ISSN: 2621-7961 DOI: https://doi.org/10.31960/caradde.v2i2.374 CARADDE: Jurnal Pengabdian Kepada Masyarakat https://journal.ilininstitute.com/index.php/caradde Volume 2 \| Nomor 2 \| Februari \|2020 e-ISSN: 2621-7910 dan p-ISSN: 2621-7961 DOI: https://doi.org/10.31960/caradde.v2i2.374 Pendampingan Pemanfaatan Duolingo melalui Smartphone Sebagai Alat Pengajaran dan Pembelajaran Bahasa Inggris Bagi Masyarakat PENDAHULUAN demikian masih ada masyarakat yang enggan memanfaatkan teknologi smartphone dalam pengajaran dan pembelajaran di tempat pendidikan (McNeal, and Van’t Hooft, 2006). Hal ini bisa ditemukan pada masyarakat MA Miftahul Ulum Pagendingan-Pamekasan. Para guru termasuk guru bahasa Inggris Akhir-akhir ini banyak penelitian yang mengamati dan meneliti pemanfaatan teknologi, seperti pemanfaatan teknologi smartphone, dalam pengajaran dan pembelajaran (El Hariry, 2015). Walaupun 339 Caradde: Jurnal Pengabdian Kepada Masyarakat Vol 2 No 2, Februari 2020 Caradde: Jurnal Pengabdian Kepada Masyarakat Vol 2 No 2, Februari 2020 beserta siswa-siswa disana banyak yang memiliki smartphone dan sering membawanya ke sekolah. Nalliveettil & Alenazi (2016) berpendapat bahwa guru bahasa Inggris harus mempunyai banyak pengetahuan akan manfaat teknologi smartphone dan dapat memanfaatkannya ketika sedang mengajar. Minimnya pengetahuan dan pelatihan akan manfaat teknologi smartphone bagi para guru bahasa Inggris membuat mereka lebih memanfaatkannya untuk tujuan yang tidak berhubungan dengan pedagogi seperti chatting, mengecek WhatsApp (WA), Facebook (FB) Instagram dan lain-lain. Hal senada juga dinyatakan dalam penelitian Tindell & Bohlander (2012) bahwa banyak murid menggunakan smartphone hanya untuk texting, browsing ataupun chatting online daripada memperhatikan penjelasan guru. Demikian pula dengan para siswa MA yang lebih cenderung memanfaatkan smartphone untuk bermain game daripada memanfaatkannya sebagai alat pembelajaran untuk belajar bahasa Inggris. pembelajaran yang pasif, yang dapat membuat siswa merasa tidak begitu termotivasi untuk belajar bahasa Inggris. j gg Mengacu kepada fenomena bahwa guru bahasa Inggris dan siswa MA Miftahul Ulum Pagendingan-Pamekasan tidak bisa memanfaatkan smartphone dalam KBM, maka Tim pengabdian masyarakat melakukan analisis situasi dan kebutuhan yang menunjukkan bahwasannya para guru bahasa Inggris MA Miftahul Ulum Pagendingan seharusnya menjadi guru dinamis dan kreatif dengan menggunakan alat pembelajaran yang variatif dan inovatif dimana alat tersebut sangat dibutuhkan untuk menciptakan suasana pembelajaran yang menyenangkan, yang dapat memotivasi para siswanya untuk belajar bahasa Inggris di kelas. Dan siswa kelas 3 MA Miftahul Ulum Pagendingan sebetulnya membutuhkan guru bahasa Inggris yang melek teknologi. Karena dalam mengajarkan ketrampilan bahasa, guru harus memiliki pengetahuan teknologi (Gilakjani, 2017., Shyamlee & Phil, 2012). Siswa kelas 3 juga butuh alat pembelajaran yang inovatif yang dapat membuat mereka bisa belajar bahasa Inggris secara mandiri tanpa selalu menggantungkan pada penjelasan guru. Karena menurut mereka guru bahasa Inggris MA masih menggunakan metode konvensioanal yaitu menjelaskan materi dengan sistem ceramah. Ini menunjukkan guru bahasa Inggris MA Miftahul Ulum Pagendingan dituntut untuk bisa menjadi guru inovatif, kreatif saat menjalankan proses KBM di kelas dengan memanfaatkan kemajuan teknologi. METODE Metode yang dilakukan dalam pengabdian kepada masyarakat ini adalah pelatihan dan pendampingan dengan sasaran utamanya adalah guru–guru bahasa Inggris MA sebanyak 2 orang beserta beberapa siswa kelas 3 MA Miftahul Ulum Pagendingan- Pamekasan. Siswa yang terlibat dalam kegiatan pelatihan dan pendampingan ini hanya dibatasi pada siswa kelas 3 MA saja yang terdiri dari 2 kelas yaitu kelas 3 IPA berjumlah 12 siswa dan kelas 3 IPS berjumlah 10 siswa. Alasan siswa kelas 3 MA dillibatkan dalam kegiatan ini agar kemampuan bahasa Inggris mereka lebih baik dan tambah meningkat dengan harapan setelah lulus dari sekolah MA Miftahul Ulum Pagendingan- Pamekasan, hasil pelatihan ini bisa digunakan sebagai bekal untuk mempersiapkan diri mereka untuk bisa melanjutkan ke perguruan tinggi favorit atau untuk mendapatkan pekerjaan yang lebih baik. y g Berdasarkan penjelasan di atas dan permasalahan yang sudah teridentifikasi maka tampaknya perlu Tim pengabdian masyarakat melakukan kegiatan pengabdian masyarakat dengan memberikan pelatihan dan pendampingan pada para guru bahasa Inggris beserta siswa MA Miftahul Ulum Pagendingan-Pamekasan melalui alat teknologi modern yaitu smartphone dengan menggunakan aplikasi Duolingo sebagai alat pengajaran dan pembelajaran bahasa Inggris. Alat virtual smartphone (yaitu telepon ‘pinter’ yang penggunaannya seperti komputer) dipilih dalam kegiatan pelatihan dan pendampingan ini karena selain berfungsi sebagai alat komunikasi canggih dan modern yang banyak dimiliki oleh orang di berbagai negara juga memiliki sifat yang fleksibel, portable (yaitu dirancang ringan, cukup kecil sehingga mudah untuk dibawa ke mana-mana), serta memiliki berbagai aplikasi pembelajaran, seperti aplikasi Duolingo. Sedangkan alasan Tim pengabdian masyarakat memilih aplikasi Duolingo untuk dimanfaatkan dalam kegiatan pelatihan dan pendampingan ini karena aplikasi ini bisa diakses dengan mudah, bisa didownload, dan bisa diinstal melalui smartphone android tanpa harus membayar atau mengeluarkan biaya. Sedangkan guru-guru bahasa Inggris MA dilibatkan untuk mengikuti kegiatan pelatihan dan pendampingan ini agar mereka melek teknologi, mengerti tentang cara-cara memanfaatkan teknologi sehingga dapat membantu memajukan pendidikan siswa (Goundar, 2011). Disamping itu mereka dapat memanfaatkan kemajuan teknologi smartphone dengan aplikasi Duolingo saat menjalankan proses KBM bahasa Inggris di kelas sehingga dapat menciptkan KBM yang menyenangkan, inovatif, dan dapat memotivasi siswa. Tempat pelaksanaan kegiatan ini dilaksanakan di ruang laboratorium komputer MA Miftahul Ulum Pagendingan-Pamekasan. Dan kegiatan pelatihan dan pendampingan ini dilaksanakan dengan tahapan – tahapan sebagai berikut: 1) Ceramah / presentasi materi pelatihan melalui power point. PENDAHULUAN Anshari dkk (2017) menegaskan banyak guru tidak sepunuhnya menggunakan smartphone untuk tujuan pembelajaran dan pengajaran. Oleh karena itu fenomena yang ditemukan di masyarakat MA Miftahul Ulum Pamekasan menunjukkan bahwa keberadaan smartphone disekitar mereka tidak bisa dijadikan sebagai alat pengajaran dan pembelajaran yang bermanfaat bagi mereka dalam membantu dan menunjang kegiatan belajar mengajar (KBM) bahasa Inggris di kelas. Bagi mereka smartphone hanya sebagai alat virtual untuk bersosial atau sekedar untuk mengisi waktu senggang sambil bermain game dengan alat itu. Maka daripada itu, smartphone sangat perlu untuk dimanfaatkan keberadaannya utamanya sebagai alat pengajaran dan pembelajaran bahasa Inggris bagi guru dan siswa MA Mifthahul Ulum. Selain itu, kurangnya pelatihan tentang pengetahuan kemajuan teknologi smartphone yang dapat diintegrasikan dengan pengajaran dan pembelajaran bahasa Inggris di kelas, membuat para guru bahasa Inggris tidak melek teknologi. Sedangkan pada era ini guru dituntut untuk mempunyai keahlian menerapkan teknologi dalam pembelajaran dan pengajaran (Williams dkk, 2000) agar guru bisa dinamis dan inovatif. Karena guru statis dan tidak inovatif bisa menciptakan suasana Menurut Larsen-Freeman & Anderson (2011) bahwa kemajuan teknologi dapat memberikan sumber pengajaran, dapat meningkatkan pengalaman pembelajaran bahasa, dapat memberikan kesempatan pada pembelajar untuk bekerja sendiri dan dapat memilih kapan dan dimana untuk belajar. Eggen & Kauchak (2011) juga menegaskan bahwa pendidikan masa kini sangat memerlukan dan menekankan adanya pemanfaatan kemajuan teknologi. Salah satu contoh dari kemajuan teknologi di dunia pendidikan adalah Duolingo yaitu suatu aplikasi pembelajaran bahasa yang dapat digunakan dengan menggunakan smartphone, yang diciptakan oleh Luis Von Ahn and Severin Hacker pada November 2011 dengan 340 R. Agus Budiharto , Abd Wahab Syahroni. Pendampingan Pemanfaatan Duolingo R. Agus Budiharto , Abd Wahab Syahroni. Pendampingan Pemanfaatan Duolingo tujuan untuk memberikan kesempatan pada pengguna untuk belajar bahasa dengan bebas dan membuat pembelajaran bahasa lebih menyenangkan. METODE Materi pelatihan yang diberikan meliputi pengenalan dan pengetahuan tentang aplikasi Duolingo sebagai alat pengajaran dan pembelajaran bahasa Inggris, penayangan audio visual mengenai mudahnya penggunaan aplikasi tersebut, penjelasan tahapan-tahapan penggunaan aplikasi Duolingo, serta diskusi atau sesi tanya jawab mengenai meteri yang telah diberikan. 2) Pelatihan mengenai cara menggunakan aplikasi Duolingo. Kegiatan pelatihan ini diawali dengan demonstrasi tentang bagaimana menggunakan aplikasi Duolingo sebagai alat pengajaran dan y g y Selain penjelasan tadi, Duolingo juga mempunyai aplikasi yang menarik dan menyenangkan bagi para penggunanya yaitu ‘gamefied application’ atau dikenal dengan istilah ‘gamification’ dimana dalam aplikasi ini terdapat gabungan audio dengan visual yang menarik juga pertanyaan mengenai English Grammar dalam bentuk permainan. Dengan begitu pengguna dari pada aplikasi Duolingo ini bisa belajar bahasa Inggris sambil bermain layaknya seperti bermain game. Hal ini tentunya bisa memudahkan dan menyenangkan bagi si pengguna aplikasi Duolingo saat belajar bahasa Inggris karena pengguna bisa belajar bahasa Inggris secara santai, tidak harus serius atau tertekan sehingga pengguna tidak cepat merasa bosan belajar bahasa Inggris saat menggunakan aplikasi ini. 341 Caradde: Jurnal Pengabdian Kepada Masyarakat Vol 2 No 2, Februari 2020 Caradde: Jurnal Pengabdian Kepada Masyarakat Vol 2 No 2, Februari 2020 pembelajaran bahasa Inggris. Kemudian pembimbingan secara teknis pada guru – guru bahasa Inggris dan siswa kelas 3 MA Miftahul Ulum Pagendingan-Pamekasan untuk bisa menggunakan aplikasi tersebut secara mandiri. 3) Pendampingan penggunaan aplikasi Duolingo. Kegiatan pendampingan ini dilakukan untuk mengetahui bagaimana respon para guru bahasa Inggris dan siswa kelas 3 MA saat mendapatkan permasalahan ketika mempraktekkan aplikasi Duolingo yang sudah diajarkan. Pendampingan ini juga bertujuan untuk memantapkan bahwa para guru bahasa Inggris dan siswa kelas 3 MA Miftahul Ulum Pagendingan–Pamekasan Madura mampu menggunakan aplikasi Duolingo dengan baik dan benar. Dan kegiatan pendampingan ini dilakukan secara langsung yaitu melalui tatap muka dengan peserta ataupun secara tidak langsung yaitu melalui media komunikasi whatsapp atau e-mail. 4) Melakukan evaluasi dengan menyebarkan lembar angket Pretest & dan Post- test pada semua peserta. nyaman, cukup luas, juga terpasang jaringan internet Wifi. Pelatihan ini dipandu oleh dua dosen Universitas Madura yang telah berpenglaman yaitu dosen dari Program Studi Pendidikan Bahasa Inggris: R. Agus Budiharto, S.S., M.Pd dan dosen dari Program Studi Tehnik Informatika: Abd. Wahab Syahroni, M.Kom. Pada hari pertama, hari Jum’at jam 07.00 WIB tanggal 6 September 2019, pelaksanaan kegiatan pelatihan diawali dengan sambutan oleh ketua pelaksana program yang memberikan gambaran mengenai tujuan dan agenda kegiatan pengabdian masyarakat. METODE Kemudian diteruskan dengan mendistribusikan kuesioner yang terdiri dari 6 pertayaan pada seluruh peserta mengenai pengetahuan “Aplikasi Duolingo” yang dapat dilihat di table 1. Table 1: Hasil Pretest tentang Pengetahuan Aplikasi Duolingo 2 Table 1: Hasil Pretest tentang Pengetahuan Aplikasi Duolingo No Pertanyaan Ya Tidak 1 Pernahkah Bapak, Ibu, Saudara mendengar tentang aplikasi Duolingo ? 5% 95% 2 Apakah Bapak, Ibu, Saudara mengetahui cara menginstal aplikasi tersebut melalui smartphone ? 0% 100% 3 Apakah Bapak, Ibu, Saudara dapat menggunakan aplikasi tersebut melalui smartphone ? 0% 100% 4 Apakah Bapak, Ibu, Saudara mengetahui manfaat dari aplikasi tersebut ? 0% 100% 5 Pernahkah Bapak, Ibu, Saudara menggunakan aplikasi Duolingo melalui smartphone saat ada KBM bahasa Inggris di kelas ? 0% 100% 6 Apakah Bapak, Ibu, Saudara mempunyai keinginan untuk menggunakan aplikasi tersebut sebagai alat pengajaran dan pembelajaran bahasa Inggris di kelas ? 0% 100% Hasil kuesioner pada table 1 menunjukkan bahwasannya peserta yang pernah mendengar istilah aplikasi Duolingo hanya 5%, sedangakan sisanya 95% tidak HASIL DAN PEMBAHASAN Pelaksanaan kegiatan pelatihan dan pendampingan dilakukan pada hari Jumat tanggal 6 dan hari Sabtu tanggal 7 di bulan September 2019. Kegiatan ini diawali dengan mendistribusikan undangan kepada perserta yang terdiri dari Kepala sekolah MA, guru– guru bahasa Inggris MA serta siswa kelas 3 MA Miftahul Ulum Pagendingan– Pamekasan. Dalam undangan tersebut, diinformasikan juga bahwa setiap peserta diwajibkan membawa smartphone android. Hasil dari pendistibusian undangan direspon dengan sangat positif oleh peserta, hal ini bisa dilihat dari antusias para peserta saat mendaftar dan menghadiri pada hari pelaksanaan karena jumlah peserta ternyata melampaui target dari jumlah yang direncanakan yaitu dari jumlah 24 orang menjadi 25 orang. Karena Kepala Sekolah MA yang berpendidikan S1 tehnik informatika yaitu Bapak Hasan Basri, S.Kom tertarik untuk menghadiri mengikuti kegiatan pengabdian ini. Kegiatan pelatihan dan pendampingan ini dilaksanakan di ruang laboratorium MA Miftahul Ulum karena selain ruangannya yang 342 R. Agus Budiharto , Abd Wahab Syahroni. Pendampingan Pemanfaatan Duolingo bahasa Inggris. Setelah mendistribusikan kuesioner Tim pengabdian masyarakat melakukan presentasi pemaparan materi mengenai pengenalan dan pengetahuan aplikasi Duolingo, melakukan penayangan audio visual tentang mudahnya penggunaan aplikasi tersebut, serta memberikan penjelasan melalui power point tahapan-tahapan tentang bagaimana menggunakan aplikasi Duolingo melalui smartphone (seperti: menginstal, mengaktifkan, memilih item, mengoperasionalkan, dsb) yang dapat dilihat di Gambar 1. pernah mendengar istilah aplikasi tersebut. Hal ini di perburuk dengan temuan bahwa 100% atau seluruh peserta tidak pernah sama sekali menggunakan aplikasi tersebut sebagai alat pengajaran dan pembelajaran bahasa Inggris saat KBM berlangsung di kelas. Ini menandakan bahwa peserta tidak pernah menginstal aplikasi Duolingo, tidak tahu manfaat kegunaannya dan tidak pernah memanfaatkan teknologi aplikasi tersebut sebagai alat pengajaran dan pembelajaran, sedangkan memanfaatkan teknologi merupakan bagian yang paling penting dalam lingkungan pembelajaran dan pengajaran (Ahmadi, 2018). Ini dapat dinyatakan bahwa mereka tidak tahu bahwasannya aplikasi Duolingo yang merupakan bagian dari Mobile – Assisted Language Learning (MALL) bisa digunakan untuk belajar kosakata (vocabulary), berlatih tata bahasa (grammar), dan menurut Kim & Kwon (2012) juga bisa meningkatkan writing proficiency: kemampuan untuk menulis serta bisa meningkatkan reading proficiency: kemampuan untuk membaca. bahasa Inggris. Setelah mendistribusikan kuesioner Tim pengabdian masyarakat melakukan presentasi pemaparan materi mengenai pengenalan dan pengetahuan aplikasi Duolingo, melakukan penayangan audio visual tentang mudahnya penggunaan aplikasi tersebut, serta memberikan penjelasan melalui power point tahapan-tahapan tentang bagaimana menggunakan aplikasi Duolingo melalui smartphone (seperti: menginstal, mengaktifkan, memilih item, mengoperasionalkan, dsb) yang dapat dilihat di Gambar 1. HASIL DAN PEMBAHASAN Selesai menjelasakan tahapan-tahapan tadi, peserta kemudian diajak untuk menerapkan aplikasi Duolingo dengan cara menginstalnya terlebih dahulu melalui playstore di smartphone mereka masing-masing dengan mengikuti tahapan-tahapan yang sudah dijelaskan sebelumnya. Kemudian kegiatan diteruskan dengan sesi tanya jawab. Selanjutnya Tim pengabdian masyarakat menginformasikan pada seluruh peserta bahwa kegiatan pada hari pertama berakhir pada jam 10.20 WIB dan meminta para peserta untuk berlatih menerapkan aplikasi Duolingo di rumah mereka masing-masing. Berkenaaan dengan temuan-temuan tadi maka dapat ditarik kesimpulan bahwa kegiatan pelatihan ini sangat diperlukan untuk memberikan pengetahuan pada para peserta adanya manfaat penggunaan aplikasi Duolingo sebagai alat pengajaran dan pembelajaran g p g j p j Gambar 1: Tahapan-Tahapan Menggunakan Aplikasi Duolingo Gambar 1: Tahapan-Tahapan Menggunakan Aplikasi Duolingo Pada hari kedua, hari Sabtu jam 07.00 WIB tanggal 7 September 2019, peserta kembali hadir untuk mengikuti pelatihan pemanfaatan aplikasi Duolingo. Mengingat dari semua peserta hanya beberapa orang yang memiliki fasilitas internet dirumah, sehingga mereka melanjutkan untuk berlatih menerapkan aplikasi Duolingo di tempat pelatihan. Selama proses pelatihan, para peserta didampingi oleh Tim pengabdian masyarakat dengan tujuan agar peserta merasa terbantu apabila mereka mengalami kesulitan atau ada hal-hal yang masih dianggap belum dimengerti berkenaan dengan cara 343 Caradde: Jurnal Pengabdian Kepada Masyarakat Vol 2 No 2, Februari 2020 Caradde: Jurnal Pengabdian Kepada Masyarakat Vol 2 No 2, Februari 2020 memanfaatkan aplikasi Duolingo. Setelah dari peserta tidak ada yang bertanya lagi, kegiatan dilanjutkan dengan meminta beberapa peserta yaitu perwakilan dari guru dan perwakilan dari siswa untuk menampilkan cara memanfaatkan aplikasi Duolingo sebagai alat pengajaran dan pembelajaran bahasa Inggris. Selesai melakukan presentasi, peserta mendapatkan feedback berupa komentar dan saran mengenai performance mereka. memberikan penilaian terhadap kemampuan masing-masing peserta dalam menggunakan aplikasi Duolingo. Selain itu, Tim pengabdian masyarakat juga memberikan pendampingan lebih lanjut pada peserta melalui media komunikasi whatsapp atau e-mail. Hal ini dilakukuan untuk memantapkan bahwa para peserta pelatihan benar-benar mampu menggunakan aplikasi Duolingo dengan baik dan benar. Pada kegiatan pelatihan ini, terdapat pendampingan yang dilakukan dengan cara melakukan tatap muka ke sekolah mitra untuk melakukan tatap muka ke sekolah mitra untuk Gambar 2: Kegiatan Pelatihan dan Pendampingan Pemanfaatan aplikasi Duolingo Gambar 2: Kegiatan Pelatihan dan Pend Gambar 2: Kegiatan Pelatihan dan Pendampingan Pemanfaatan aplikasi Duolingo lagi. SIMPULAN DAN SARAN Aplikasi Duolingo sebagai bentuk bagian dari kemajuan teknologi yang digunakan dalam kegiatan masyarakat ini dapat berfungsi sebagai aplikasi edukasi yang bisa mendukung dan memudahkan proses pengajaran dan pembelajaran bahasa Inggris di kelas. Teknologi aplikasi ini juga dapat berfungsi sebagai opsi bagi guru bahasa dalam memberikan pegajaran yang lebih menyenangkan, selain itu dapat mendorong interaksi antara guru dan siswa, dapat meningkatkan pengetahuan siswa, dan dapat meningkatkan motivasi siswa. Babanoğlu, M. P & Reyhan, A. (2015). Explicit and Implicit Types of Corrective Feedback in Turkish Primary Education. International Journal of Languages’ Education and Teaching, 3(2), 126-132. Eggen, P & Kauchak, D. (2012). Strategies and Models for Teachers: Teaching Content and Thinking Skills (6th ed.) Boston: Pearson. Adapun hasil evaluasi dari pelatihan ini menunjukkan bahwa para peserta telah berhasil memanfaatkan aplikasi Duolingo sebagai alat pengajaran dan pembelajaran bahasa Inggris dengan baik dan benar saat mengikuti program pengabdian pada masyarakat selama dua (2) hari. Selain itu, hasil evaluasi juga menunjukkan bahwa para peserta menyatakan bahwa kegiatan pelatihan dan pendampingan ini dapat memberikan banyak manfaat bagi mereka. El Hariry, N. A. (2015). Mobile Phones as Useful Language Learning Tools. European Scientific Journal, 11(16), 298- 317. Gilakjani, A. P. (2017). A Review of the Literature on the Integration of Technology into the Learning and Teaching of English Language Skills. International Journal of English Linguistics, 7(5), 95-106. HASIL DAN PEMBAHASAN Tidak hanya itu saja, ketika melakukan latihan - latihan bahasa Inggris mereka juga dapat mengetahui dan mengevaluasi kesalahan – kesalahan yang mereka lakukan melalui corrective feedback (CF), yaitu suatu korektor yang terdapat dalam aplikasi Duolingo yang dapat menunjukkan, mengidentifkasi kesalahan yang telah dilakukan dan dapat memberikan koreksi. Babanoğlu & Reyhan (2015) menyatakan bahwa CF menjadi hal yang sangat penting dalam pembelajaran dan pengajaran bahasa asing seperti bahasa Inggris, karena perannya yang dapat memberikan kemudahan pada pembelajar dan guru untuk mengetahui kesalahan yang telah dilakukan Di akhir kegiatan ini para peserta diberikan kuesioner (post test) yang isi dan jumlah pertayaannya sama dengan kuesioner di table 1. Dari hasil kuesioner tersebut dapat disimpulkan bahwa semua peserta (100%) berkeinginan untuk memanfaatkan aplikasi Duolingo sebagai alat pengajaran dan pembelajaran bahasa Inggris saat KBM berlangsung di kelas. Mereka juga menyadari bahwasanyya ketika mempratikkan aplikasi Duolingo di kegiatan pelatihan ini mereka mendapatkan banyak pengetahuan bagaimana belajar atau mengajarkan ketrampilan writing, reading, speaking dan listening dari aplikasi tersebut sehingga tanpa mereka sadari hal ini dapat membantu mereka untuk meningkatkan kemampuan bahasa Inggris mereka lebih baik 344 R. Agus Budiharto , Abd Wahab Syahroni. Pendampingan Pemanfaatan Duolingo Gambar 3: Corrective Feedback dalam aplikasi Duolingo Gambar 3: Corrective Feedback dalam aplikasi Duolingo Anshari, M., Almunawar, M.N., Shahrill, M., Wicaksono, D.K., Huda, M. (2017). Smartphones usage in the classrooms: Learning aid or interference?. Education and Information Technologies, 22(6), 3063-3079 DAFTAR RUJUKAN Ahmadi, M. R. (2018). The Use of Technology in English Language Learning: A Literature Review. International Journal of Research in English Education, 3(2), 115-125. Goundar, S. (2011). What is the Potential Impact of Using Mobile Devices in Education?. Proceedings of SIG GlobDev Fourth Annual Workshop 3. 345 Caradde: Jurnal Pengabdian Kepada Masyarakat Vol 2 No 2, Februari 2020 Caradde: Jurnal Pengabdian Kepada Masyarakat Vol 2 No 2, Februari 2020 Kim, H & Kwon, Y. (2012). Exploring Smartphone Applications for Effective Mobile - Assisted Language Learning. Multimedia - Assisted Language Learning, 15(1), 31 - 57. Nalliveettil, G. M & Alenazi, T. H. K. (2016). The Impact of Mobile Phones on English Language Learning: Perceptions of EFL Undergraduates. Journal of Language Teaching and Research, 7(2), 264-272. Larsen-Freeman, D & Anderson, M. (2011). Techniques and Principles in Language Teaching (3rd ed.). Oxford: Oxford University Press. Shyamlee, S. D. & Phil, M. (2012). Use of Technology in English Language Teaching and Learning. Journal of International Conference on Language, Medias and Culture, 33, 150-156. McNeal, T & Van’t Hooft, M. (2006). Anywhere, Anytime: Using Mobile Phones for Learning. Journal of the Research Center for Educational Technology, 2(2), 24-31. Tindell, D. R., & Bohlander, R. W. (2012). The Use and Abuse of Cell Phones and Text Messaging in the Classroom: A Survey of College Students. College Teaching, 60(1). Williams, D., Coles, L., Wilson, K., Richardson, A & Tuson, J. (2000). 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https://openalex.org/W4301397781	https://www.frontiersin.org/articles/10.3389/fneur.2022.985700/pdf	English	null	A non-immersive virtual reality-based intervention to enhance lower-extremity motor function and gait in patients with subacute cerebral infarction: A pilot randomized controlled trial with 1-year follow-up	Frontiers in neurology	2,022	cc-by	8,850	A non-immersive virtual reality-based intervention to enhance lower-extremity motor function and gait in patients with subacute cerebral infarction: A pilot randomized controlled trial with 1-year follow-up OPEN ACCESS EDITED BY Agnieszka Guzik, University of Rzeszow, Poland REVIEWED BY Michela Gofredo, San Rafaele Pisana (IRCCS), Italy Carlos Luque-Moreno, University of Seville, Spain CORRESPONDENCE Dongfeng Huang huangdf@mail.sysu.edu.cn Yurong Mao maoyr@mail.sysu.edu.cn †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Neurorehabilitation, a section of the journal Frontiers in Neurology RECEIVED 04 July 2022 ACCEPTED 15 September 2022 PUBLISHED 04 October 2022 Minjie Bian1, Yuxian Shen1, Yijie Huang1, Lishan Wu1, Yueyan Wang1, Suyue He1, Dongfeng Huang1,2† and Yurong Mao1,2† Minjie Bian1, Yuxian Shen1, Yijie Huang1, Lishan Wu1, Yueyan Wang1, Suyue He1, Dongfeng Huang1,2† and Yurong Mao1,2† 1Department of Rehabilitation Medicine, The Seventh Afliated Hospital, Sun Yat-sen University, Shenzhen, China, 2Guangdong Engineering and Technology Research Center for Rehabilitation Medicine and Translation, Guangzhou, China Introduction: This study was conducted to evaluate whether a non-immersive virtual reality (VR)-based intervention can enhance lower extremity movement in patients with cerebral infarction and whether it has greater short-term and long-term efectiveness than conventional therapies (CTs). CITATION Bian M, Shen Y, Huang Y, Wu L, Wang Y, He S, Huang D and Mao Y (2022) A non-immersive virtual reality-based intervention to enhance lower-extremity motor function and gait in patients with subacute cerebral infarction: A pilot randomized controlled trial with 1-year follow-up. Front. Neurol. 13:985700. doi: 10 3389/fneur 2022 985700 Materials and methods: This was a single-blinded, randomized clinical controlled trial. Forty-four patients with subacute cerebral infarction were randomly allocated to the VR or CT group. All intervention sessions were delivered in the inpatient unit for 3 weeks. Outcomes were measured before (baseline) and after the interventions and at 3-month, 6-month and 1-year follow-ups. The outcomes included clinical assessments of movement and balance function using the Fugl–Meyer Assessment of Lower Extremity (FMA-LE) and Berg Balance Scale (BBS), and gait parameters in the sagittal plane. doi: 10.3389/fneur.2022.985700 COPYRIGHT © 2022 Bian, Shen, Huang, Wu, Wang, He, Huang and Mao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Original Research PUBLISHED 04 October 2022 DOI 10.3389/fneur.2022.985700 TYPE Original Research PUBLISHED 04 October 2022 DOI 10.3389/fneur.2022.985700 A non-immersive virtual reality-based intervention to enhance lower-extremity motor function and gait in patients with subacute cerebral infarction: A pilot randomized controlled trial with 1-year follow-up Introduction study utilized the non-immersive VR techniques to clarify the eﬀectiveness of VR technique. VR is an advanced computer– human interface that provides artiﬁcial sensory feedback for patients while they perform real-time tasks and experience real- time events in virtual environments (9). Training with VR is considered to include the rehabilitation principles of high- intensity, repetitive and task-speciﬁc practice (10). Moreover, VR is well-recognized to improve motivation and enjoyment and consequently decrease the perception of exertion, which promotes adherence to the training activity (5). In addition, VR could reinforce the physiological basis of motor learning and descending neural pathways (11, 12), and its potential cognitive beneﬁts to patients, including improvements in attention or memory, have been demonstrated in situations where they are required to react quickly and deal with busy environments with multiple stimuli. Therefore, thus, VR could play a beneﬁcial role in improving balance and gait capacity among post- stroke patients (13). In clinical, VR is applied independently or in combination with the abovementioned conventional physical therapy techniques for gait rehabilitation and motor function improvement. Stroke is a major health problem with a global incidence of almost 12.2 million cases each year, and has been identiﬁed as the third-leading cause of both death and disability in recent years (1). In China, stroke incidence has almost doubled over the past 30 years, posing a great burden to Chinese society, and the ischemic stroke accounted for more than 80% (2). Approximately 88% of post-stroke patients discharged from hospitals continue to suﬀer from impaired walking ability (3). Moreover, ∼50% of post-stroke patients who regain ambulation capability continue to experience diﬃculties in walking in the community (4). Limited walking ability is a major concern for stroke survivors, both physically and psychologically, as it has a negative impact on their daily function and, ultimately, their quality of life due to limited access to the community (5). The gait pattern among post-stroke survivors usually shows lower walking speed, and an abnormal hip-knee-ankle joint movement (6). Therefore, gait recovery is a major objective of stroke rehabilitation. To improve gait function in post-stroke patients, continued physical therapy at all recovery stages is necessary. Therapeutic techniques such as virtual reality (VR) are being increasingly applied in neurorehabilitation practice, and the beneﬁts of applying VR-based training have been widely recognized in the ﬁeld of stroke rehabilitation. Abbreviations: VR, virtual reality; CT, conventional therapy; FMA, fugl- meyer assessment; BBS, berg balance scale; NHISS national institute of health stroke scale; MMSE, mini mental state examination. A non-immersive virtual reality-based intervention to enhance lower-extremity motor function and gait in patients with subacute cerebral infarction: A pilot randomized controlled trial with 1-year follow-up Results: In the VR group, the walking speed after intervention, at 3-month, 6-month, and 1-year follow-ups were signiﬁcantly greater than baseline (p = 0.01, <0.001, 0.007, and <0.001, respectively). Compared with baseline, BBS scores after intervention, at 3-month, 6-month, and 1-year follow-ups were signiﬁcantly greater in both the VR group (p = 0.006, 0.002, <0.001, and <0.001, respectively) and CT group (p = <0.001, 0.002, 0.001, and <0.001, respectively), while FMA-LE scores after intervention, at 3-month, 6-month, and 1-year follow-ups were signiﬁcant increased in the VR group (p = 0.03, <0.001, 0.003, and <0.001, respectively), and at 3-month, 6-month, and 1-year follow-ups in the CT group (p = 0.02, 0.004 and <0.001, respectively). In the VR group, the maximum knee joint angle in the sagittal plane enhanced signiﬁcantly at 6-month follow-up from that at baseline (p = 0.04). Frontiers in Neurology Frontiers in Neurology 01 frontiersin.org Bian et al. 10.3389/fneur.2022.985700 Conclusion: The efectiveness of the non-immersive VR-based intervention in our study was observed after the intervention and at the follow-ups, but it was not signiﬁcantly diferent from that of CTs. In sum, our results suggest that non-immersive VR-based interventions may thus be a valuable addition to conventional physical therapies to enhance treatment efcacy. Clinical trial registration: http://www.chictr.org.cn/showproj.aspx?proj= 10541, ChiCTR-IOC-15006064. KEYWORDS KEYWORDS virtual reality, ischemic stroke, gait analysis, motor activity, rehabilitation virtual reality, ischemic stroke, gait analysis, motor activity, rehabilitation Introduction VR technique can be divided into non-immersive VR with diﬀerent levels of immersion and immersive VR with a head-mounted display, which is closer to real-life but is easy to leave the adverse eﬀect of dizziness (7). The eﬀectiveness of immersive VR has been demonstrated to train motor patterns of healthy young participants, and the patterns were maintained in real-world settings (8). However, in China, the non-immersive VR is relatively not expensive and user-friendly for both therapists and patients, which is commonly applied in rehabilitation therapy. Hence this Recently, studies on the eﬀectiveness of non-immersive VR among post-stroke patients have reported inconsistent ﬁndings. Some reviews have reported that the current evidence is insuﬃcient to conclude that VR is more eﬀective than conventional therapy (CT) (10, 14–16), while others suggested that VR enhances the lower-limb motor performance including balance and gait function of post-stroke patients more eﬃciently than CT (4, 17–21). Speciﬁcally, several RCTs showed no statistical diﬀerences between the eﬀects of VR treatment and CT treatment on balance or lower extremity motor function for acute stroke patients and subacute patients (22, 23). On the other hand, one study has demonstrated that non-immersive VR as an add on to CT was more eﬀective in balance capability than CT alone among subacute patients without long-term result reported (24). Several studies that adopted clinical measures to assess lower extremity and gait ability have also suggested VR’s potential to promote functional recovery for chronic stroke patients (25), while some have shown the beneﬁts of VR in enhancing balance capability (26, 27). Moreover, an RCT Frontiers in Neurology frontiersin.org 02 10.3389/fneur.2022.985700 Bian et al. and the First Aﬃliated Hospital, Sun Yat-Sen University, China. Adults without neurological pathology were recruited through advertisement. This study was approved by the Human Subjects Ethics Subcommittee of the Aﬃliated Hospitals (NO.2019SYSUSH-019), Sun Yat-Sen University, China. Clinical trial registration number is ChiCTR-IOC-15006064. Written consent was obtained from all participants prior to inclusion in the study. The study was conducted from October 2019 to June 2022. and the First Aﬃliated Hospital, Sun Yat-Sen University, China. Adults without neurological pathology were recruited through advertisement. This study was approved by the Human Subjects Ethics Subcommittee of the Aﬃliated Hospitals (NO.2019SYSUSH-019), Sun Yat-Sen University, China. Clinical trial registration number is ChiCTR-IOC-15006064. Written consent was obtained from all participants prior to inclusion in the study. The study was conducted from October 2019 to June 2022. Introduction showed that cycling training with smartphone VR application led to signiﬁcant improvements in lower extremity function, sitting balance and spatiotemporal gait performance for chronic stroke patients compared with CT (12). The controversial ﬁndings for the motor function after VR intervention possibly result from insuﬃcient VR programs designed for impairments, and the largely varied duration before intervention (16). VR combined with conventional physiotherapy contributed to motor improvement in post-stroke patients in both subacute and chronic stages, but improvement of kinematic outcomes was conﬁrmed for the subacute group, but not for the chronic group (28). Therefore, in our study, diﬀerent VR programs with the same dosage were applied for lower-extremity motor function and gait, and subacute post-stroke patients were recruited. Potential post-stroke inpatients were recruited by physicians at the rehabilitation center using the following inclusion criteria: post-stroke inpatients with (1) Mini-Mental State Examination scores >24 (34); (2) National Institute of Health Stroke Scale scores <20 to exclude heavy ischemic stroke (35); (3) diagnosis of cortical and subcortical schemic stroke (conﬁrmed by magnetic resonance imaging or computer tomography) <6 months before their inclusion in this study; (4) Brunnstrom stage for lower-extremity ≥1; (5) no previous VR-based rehabilitation training experience; (6) Modiﬁed Ashworth Scale scores of the lower-limbs ≤2; (7) the ability to maintain sitting balance for more than 20 min and walking for over 10 meters; and (8) adults under the age of 75. The exclusion criteria were (1) patients who had previously received VR-based training, and (2) patients with other diseases, such as cerebellar and brainstem injuries, severe cognitive impairment, joint stiﬀness, convulsive crisis, congestive heart failure, deep vein thrombosis of a lower extremity, malignant progressive hypertension, respiratory failure, active liver disease, severe hepatic and renal insuﬃciency, history of mental illness and inability to cooperate. Normal age-matched adults were included, if they have no previous and current central nervous system diseases and severe musculoskeletal diseases. Moreover, the majority of published studies followed patients for no longer than 3 months and reported contradictory follow-up outcomes. Wii-based VR intervention was found to increase Berg Balance Scale (BBS) scores both immediately after the intervention and at the 4-week follow-up, and that the scores at both time points were greater than those of the CT group (29). Introduction Nevertheless, another RCT concluded that the eﬀectiveness of VR on balance and gait could not be maintained, and found that the CT group showed greater improvements in weight-bearing symmetry at the 3-month follow-up than the exergame group, among chronic and subacute stroke patients (30). Similarly, the eﬀect of non-immerisve VR was shown not statistically diﬀerent from that of CTs in improving balance performance or gait capability at both post-intervention and the 3-month follow-up among chronic and subacute stroke patients (23, 31, 32). Furthermore, one systematic review summarized that the evidence on the eﬀectiveness of VR analyzed using biomechanical parameters was limited, especially for sustained eﬀectiveness at longer than 4 weeks post-intervention (17). Additionally, the eﬀectiveness of treatment in the follow-ups contribute to the evidence of motor relearning and neural plasticity, which is signiﬁcant for stroke patients (33). Study design Therefore, the current study evaluated the long-term eﬀects of a VR-based intervention, i.e., at 6 months and 1 year post- intervention, using biomechanical analyses of lower extremity motor function, balance function and gait pattern. The study was a single-blinded, randomized controlled trial to explore the eﬀects of VR gait training on the motor function of patients with subacute cerebral infarction. All participants were randomly allocated to either a VR group or a CT group. The baseline and post-intervention assessments and training sessions were conducted in the hospital’s inpatient rehabilitation department, and post-intervention and 3-month, 6-month, and 1-year follow-up assessments were performed in the outpatient rehabilitation center. Sample size estimation was performed by G∗Power software (Düsseldorf, Germany), considering a Minimal Clinically Important Diﬀerence (MCID) change of walking speed equal to 0.13 m/s (36, 37) as the expected eﬀect of the treatment, with a pooled Standard Deviation (SD) of 0.23. Thus, for α = 0.05, β = 0.2, with an f eﬀect size = 0.57, it provides an estimated total sample size of 42 subjects. Moreover, considering a 10% dropout, 47 total subjects were considered suﬃcient for statistical analysis. Given the above-mentioned contradictory results and no reported long-term follow-up result, our study aimed to clarify whether a 3-week course of VR-based lower extremity exercises can eﬀectively improve gait parameters and motor function in post-stroke inpatients in the subacute stage, and whether the eﬀects of these exercises on motor function and gait are sustained at a 1-year follow-up. Frontiers in Neurology Intervention The outcomes of the intervention participants were measured before and immediately after the 3-week intervention and at 3-month, 6-month, and 1-year follow-ups using standard operating procedures. The collected data included demographic data (i.e., age, gender, aﬀected side), clinical assessment outcomes of National Institute of Health stroke scale (NIHSS), Mini Mental State Examination (MMSE), Fugl- Meyer assessment (FMA-LE) and BBS, as well as biomechanical parameters recorded using a 3D gait analysis system, the walking speed parameter of which is the primary outcome of this study. Intention-to-treat analyses were done during the data analysis. The gait performance of twelve age-matched healthy adults were also assessed for comparison. All biomechanical data on gait was obtained and analyzed using a real-time motion tracking/capture system, namely, the standard PlugInGait model with Vicon Nexus software (version 1.7.1; Vicon Motion Systems, UK), as shown in Figure 2. Six infrared 100-Hz cameras recorded the location of 16 markers of pelvic and both lower- extremities during the data collection under the guidance of model sets of PlugInGait model, including the midline sacrum at the level of the posterior superior iliac spines, anterior superior iliac spines, lower lateral 1/3 and 1/2 surface of left and right thigh, lateral epicondyle of knee, lower lateral 1/3 and 1/2 surface of left and right shank, lateral malleolus, the second metatarsal head, and the calcaneus at the same height as the toe marker. The spatiotemporal and lower-extremity joint kinematic data was obtained from the model. During the gait assessment process, each participant was asked to wear ﬂat shoes and close-ﬁtting pants and to walk independently for 10 meters without any crutches or ankle foot orthoses, turn around and return to the starting point at a self-selected walking speed. One researcher collected gait data and calculate the number of successful gait cycles for minimum of six. In addition, during clinical outcome assessments, the post-stroke participants’ FMA-LE (38) and BBS scores for lower limb performance were obtained (39, 40). The interventions in this study mainly focused on the functional ability of the lower extremities, including lower limb movement, balance training and gait exercise. All participants received 5-h rehabilitation programs (either VR-based or CT) on 5 days per week for 3 weeks. Speciﬁcally, the VR group received 15 physical training sessions combined with VR-based training, while the CT group underwent routine CT-based rehabilitation training for the same duration. Study participants Inpatients in the subacute phase after cerebral infarction stroke were recruited from the Seventh Aﬃliated Hospital Frontiers in Neurology frontiersin.org 03 Bian et al. 10.3389/fneur.2022.985700 10.3389/fneur.2022.985700 An independent researcher conducted the random allocation of participants based on a randomization sequence generated by a statistics expert from Sun Yat-sen University. Allocation numbers were sealed in opaque envelopes. The researchers performing the assessments were blinded to treatment allocation, but the participants and the therapists providing the interventions could not be blinded due to the nature of the interventions. and guided the patients at the ﬁrst time of VR intervention, they also supervised the intervention of training of patients during the following sessions. The diﬃculty level of VR training was modiﬁed by experienced therapists based on the abilities and therapeutic goals of each participant, and the system displayed the outcome of each VR training session for the participants and therapists once the training ended. Intervention In each session of the two programs, the training intensity was adjusted by experienced physical therapists in line with the participants’ progress, safety and movement quality. VR techniques adopted in the VR group included the Wii exergame training system, an active and passive trainer with a VR screen, a VR balance training system and a VR gait training system based on the non-immersive VR techniques with feedback including visual, auditory, and numbers. The examples of them are shown in Figure 1 (trajectory tracking, car driving and etc.) and each VR training system is detailly descripted in the Table 1. Therapists introduced, demonstrated FIGURE 1 Examples of VR interventions. (a) Wii exergame training; (b) an active and passive trainer with a VR screen; (c) a VR balance training system; (d) lower extremity training in a VR gait training system. Results Forty-four participants with post-stroke hemiparesis were included in this pilot study and randomly allocated to the VR or CT group. According to the randomization sequence, 23 participants were allocated to the VR group and 21 to the CT group. At the 3-month follow-up, the number of participants in the VR and CT groups has been decreased to 17 and 19, respectively. At the 6-month and 1-year follow-ups, the number of participants in the VR and CT groups was 16 and 18, respectively. The outcome of those 16 and 18 participants in the VR and CT groups were analyzed. The demographic characteristics of the participants, the outcomes of clinical scales and the walking speed are presented in Table 2 and were not signiﬁcantly diﬀerent between the two groups. Additionally, the angles of the hip, knee and ankle joints of the aﬀected lower limbs were measured. Subsequently, from the mean proﬁle of selected strides, data on the following joint kinematic parameters in the sagittal plane were extracted: the maximum angle, minimum angle and range of motion for the hip, knee and ankle joints. For comparison, these joint kinematic parameters from the gait data of 12 adults without neurological pathology were also analyzed. Statistical analysis was performed using GraphPad Prism version 9.0.0 for macOS (GraphPad Software, San Diego, California USA). The normality of data on parameter was checked using the Shapiro–Wilk test and a normal Q–Q plot, and the normality of each outcome was conﬁrmed. Unpaired t- test and X2 test were used to compare between-group diﬀerences at baseline for continuous variables and for categorical variables, respectively. Repeated-measures two-way ANOVA was used for detection of within-group diﬀerences from pre-intervention to post-intervention to follow-ups, while a post-hoc analysis with Catagories FIGURE 2 Three-dimensional data capture. FIGURE 2 Three-dimensional data capture. FIGURE 2 Three-dimensional data capture. Bonferroni’s correction was used for between-group diﬀerences at all time points. Bonferroni’s correction was used for between-group diﬀerences at all time points. events from each walking trial were determined by visual inspection, with reference to the Vicon Nexus software output, and were used to deﬁne the beginning and end of each walking cycle. The following spatiotemporal parameters were collected and analyzed: cadence, stride time, opposite foot oﬀ, opposite foot contact, step time, single support, double support, opposite foot oﬀ, stride length, step length and walking speed. Data processing and analysis FIGURE 1 Examples of VR interventions. (a) Wii exergame training; (b) an active and passive trainer with a VR screen; (c) a VR balance training system; (d) lower extremity training in a VR gait training system. FIGURE 1 Examples of VR interventions. (a) Wii exergame training; (b) an active and passive trainer with a VR screen; (c) a VR balance training system; (d) lower extremity training in a VR gait training system. Gait data from successful gait cycles, speciﬁcally the spatiotemporal and kinematic parameters, were analyzed using Polygon (version 3.5.1; Vicon Motion Systems, UK). Heel strike Frontiers in Neurology 04 frontiersin.org Bian et al. 10.3389/fneur.2022.985700 TABLE 1 VR training systems applied in the study. TABLE 1 VR training systems applied in the study. Catagories Sensor used Movement executed Wii exergame training system Camera Balance training (dynamic) An active and passive trainer with a VR screen Sensors (speed and moment) Cycling A VR balance training system Pressure transducers Balance training (standing) A VR gait training system Sensors (speed and direction) Walking, stepping and lower extremity training FIGURE 2 Three-dimensional data capture. TABLE 1 VR training systems applied in the study. Catagories Sensor used Movement executed Wii exergame training system Camera Balance training (dynamic) An active and passive trainer with a VR screen Sensors (speed and moment) Cycling A VR balance training system Pressure transducers Balance training (standing) A VR gait training system Sensors (speed and direction) Walking, stepping and lower extremity training Movement executed Balance and lower limb motor function Compared with the BBS scores at baseline, those after intervention, at 3-month, 6-month, and 1-year follow-ups were Frontiers in Neurology 05 frontiersin.org Bian et al. 10.3389/fneur.2022.985700 TABLE 2 Demographic characteristics (Mean ± SD) at baseline for three groups. TABLE 2 Demographic characteristics (Mean ± SD) at baseline for three groups. TABLE 2 Demographic characteristics (Mean ± SD) at baseline for three groups. Normal VR group CT group P-value Age 58.17 ± 8.12 53.25 ± 8.72 55.00 ± 10.27 0.599 Gender (male/female) 6/6 14/4 13/3 0.803 Aﬀected side (right/left) 10/8 6/10 0.292 NIHSS 4.69 ± 2.50 4.56 ± 2.854 0.888 MMSE 28.56 ± 1.83 28.06 ± 1.73 0.412 BBS 36.94 ± 10.55 38.28 ± 14.05 0.758 FMA-LE 23.10 ± 5.89 23.50 ± 4.46 0.834 Walking speed 0.42 ± 0.20 0.43 ± 0.21 0.812 MMSE, Mini Mental State Examination; NIHSS, National Institute of Health Stroke Scale; BBS, Berg Balance Scale; FMA-LE, Fugl-Meyer Assessment-Lower Extremity. Mini Mental State Examination; NIHSS, National Institute of Health Stroke Scale; BBS, Berg Balance Scale; FMA-LE, Fugl-Meyer Assessment-Lower Extremity. TABLE 3 Balance and lower motor function results of pre, post and follow-ups between CT and VR group. Variable VR group CT group Baseline Post 3 m follow-up 6 m follow-up 1 yr follow-up Baseline Post 3 m follow-up 6 m follow-up 1 yr follow-up BBS 36.94 ± 10.55 38.38 ± 8.72 48.88 ± 5.74* 52.00 ± 4.66* 53.00 ± 3.74* 38.28 ± 14.05 35.86 ± 13.15* 43.00 ± 10.47* 44.71 ± 11.25* 47.00 ± 10.42* FMA-LE 23.10 ± 5.89 21.63 ± 5.55* 25.88 ± 5.06* 27.13 ± 4.29* 27.50 ± 4.07* 23.50 ± 4.46 23.00 ± 5.35 24.14 ± 5.93* 25.43 ± 5.26* 25.71 ± 4.92* Signiﬁcant diﬀerence between pre and follow-ups of intervention (P < 0.05). TABLE 3 Balance and lower motor function results of pre, post and follow-ups between CT and VR group. Signiﬁcant diﬀerence between pre and follow-ups of intervention (P < 0.05). signiﬁcantly greater in both the VR group (p = 0.006, 0.002, <0.001, and <0.001, respectively) and CT group (p ≥0.001, 0.002, 0.001, and <0.001, respectively) (Table 3). Meanwhile, compared with the FMA-LE scores at baseline, those after intervention, at 3-month, 6-month, and 1-year follow-ups were signiﬁcant increased in the VR group (p = 0.03, <0.001, 0.003, and <0.001, respectively), and at 3-month, 6-month, and 1- year follow-ups in the CT group (p = 0.02, 0.004, and <0.001, respectively). Balance and lower limb motor function Notably, both the BBS and FMA-LE scores before the intervention, immediately post-intervention and at the follow-ups were not signiﬁcantly diﬀerent between the VR and CT groups. 0.002, 0.002, and <0.001, respectively) after intervention, at 3- month, 6-month, and 1-year follow-ups changed signiﬁcantly from baseline, while the decrease of double support (p = 0.006, 0.01, and 0.01, respectively) and the increase of step length (p = 0.009, 0.02, and <0.001, respectively) at 3-month, 6-month and 1-year follow-ups diﬀered signiﬁcantly from baseline. No signiﬁcant change was found in the CT group. The outcomes of all spatiotemporal parameters were not signiﬁcantly diﬀerent between the VR and CT groups at all time points. All spatiotemporal gait data are provided in Table 4. Spatiotemporal gait parameters In the VR group, the maximum knee joint angle in the sagittal plane enhanced signiﬁcantly at 6-month follow-up from that at baseline (p = 0.04) and no signiﬁcant diﬀerence was found in any other parameter. In the CT group, no signiﬁcant diﬀerence was found between baseline and other time points. Moreover, all kinematic parameters were not signiﬁcantly diﬀerent between the VR and CT groups at all time points. All kinematic gait parameters are provided in Table 5. In the VR group, the walking speed after intervention, at 3-month, 6-month, and 1-year follow-ups were signiﬁcantly greater than baseline (p = 0.01, <0.001, 0.007, and <0.001, respectively); additionally, cadence at 3-month, 6-month, and 1-year follow-ups were signiﬁcantly greater than baseline (p = 0.002, 0.02, and <0.001, respectively). Stride time at 3- month and 1-year follow-ups and opposite foot contact at 6- month follow-ups were signiﬁcantly greater than baseline in the VR group (p = 0.04 and 0.002, respectively). In addition, the decrease of step time (p = 0.03, 0.01, 0.007, and 0.008, respectively) and the increase of stride length (p = 0.003, Figure 3 demonstrates the mean kinematic curves for the hip, knee and ankle joints in the sagittal plane for all participants of both groups at baseline, immediately post-intervention and follow-ups. Spatiotemporal gait parameters Frontiers in Neurology frontiersin.org 06 frontiersin.org g p 3 m follow-up 6 m follow-up 1 yr follow-up 24 0.55 ± 0.28 0.57 ± 0.24 0.62 ± 0.23 .67 77.07 ± 22.40 77.78 ± 20.05 82.09 ± 17.52 65 1.70 ± 0.55 1.66 ± 0.49 1.56 ± 0.48 80 16.49 ± 10.53 15.46 ± 9.07 20.34 ± 20.41 72 48.96 ± 6.38 46.62 ± 6.95 45.41 ± 7.78 43 0.86 ± 0.26 0.90 ± 0.35 0.87 ± 0.37 4 0.52 ± 0.10 0.49 ± 0.07 0.37 ± 0.35 50 0.57 ± 0.42 0.55 ± 0.35 0.58 ± 0.42 38 62.78 ± 5.20 62.18 ± 5.04 61.00 ± 5.53 22 0.81 ± 0.28 0.84 ± 0.23 0.88 ± 0.22 1 0.43 ± 0.11 0.43 ± 0.12 0.46 ± 0.11 CT group 3 m follow-up 6 m follow-up 1 yr follow-up 19.55 ± 10.39 21.08 ± 9.81 22.14 ± 9.88 31.85 ± 16.30 31.66 ± 18.29 37.84 ± 18.75 12.74 ± 6.56 9.82 ± 3.91 12.77 ± 4.21 −11.37 ± 8.68 −9.29 ± 9.95 −10.04 ± 7.72 −1.31 ± 1.71 −3.24 ± 7.39 −1.88 ± 7.58 −10.22 ± 5.02 −9.49 ± 5.11 −8.75 ± 6.10 30.92 ± 10.83 30.37 ± 11.61 32.18 ± 10.57 33.15 ± 14.74 34.91 ± 6.98 39.72 ± 16.71 22.96 ± 6.95 19.31 ± 3.13 21.52 ± 4.57 p Pre ± 0.2 ± 24 ± 0.6 ± 12 4 ± 6. ± 0.4 ± 0.0 ± 0.5 3 ± 7. ± 0.2 ± 0.1 8.77 5.61 VR group follow-up 6 m follow-up 1 yr follow-up Pr 5 ± 0.28* 0.65 ± 0.27* 0.73 ± 0.24* 0.43 ± 8 ± 16.34* 88.05 ± 13.01* 93.36 ± 14.53* 72.62 ± 2 ± 0.28* 1.40 ± 0.21 1.32 ± 0.22* 1.84 ± 52 ± 4.97 12.68 ± 3.35 12.61 ± 3.04 18.60 ± 63 ± 3.65 49.28 ± 5.48* 46.70 ± 3.34 45.94 ± 5 ± 0.17* 0.71 ± 0.14* 0.71 ± 0.15* 1.01 ± 49 ± 0.09 0.51 ± 0.10 0.44 ± 0.06 0.50 ± 8 ± 0.19* 0.37 ± 0.12* 0.38 ± 0.16* 0.67 ± 25 ± 4.57 62.78 ± 5.29 62.27 ± 4.27 62.63 ± 5 ± 0.25* 0.87 ± 0.28* 0.93 ± 0.20* 0.69 ± 4 ± 0.11* 0.44 ± 0.13* 0.48 ± 0.10* 0.38 ± up among three groups. foll 65 ± 78 ± 42 ± .52 ± .63 ± 75 ± 49 ± 38 ± .25 ± 85 ± 44 ± Discussion limb motor function after VR-based training (27, 28). One review concluded that VR-based therapies do not contribute to improvements in outcomes, especially when the interventions last for <3 weeks and VR is non-immersive (42). However, non- immersive VR in our study still contributed to the improvement of lower-extrenity motor performance after intervention. Some authors have pointed out that the FMA-LE only indicates abnormal synergistic motor patterns in voluntary and isolated movement tasks and is therefore insuﬃcient to capture all of the necessary information about complicated walking performance, as walking is cyclical and involves considerable sensorimotor integration (43). The hip and knee range of motion increased in our study due to appeared isolated movement, including improvement of hip extention and knee ﬂexion and decreased abnormal joint movement such as knee over-extention. This scale is also not sensitive enough to detect minor changes in physical function, which may have led to our ﬁnding of no diﬀerences between the VR and CT groups. The objective of this study was to determine whether non- immersive VR-based training can improve motor function and gait capability in patients with post-stroke hemiparesis and whether its short- and long-term eﬀectiveness in enhancing lower-limb motion function recovery is greater than that of CT. Overall, our results demonstrated that VR-based training improved lower-limb motion function and gait performance, but non-immersive VR techniques were not more eﬀective than CT techniques. The outcome of balance capacity evaluated based on BBS scores was improved immediately after the intervention and at 3-month, 6-month, and 1-year follow-ups in both the VR and CT groups, with no signiﬁcant changes between two groups at any time point. This result is consistent with previous studies that demonstrated that VR intervention is eﬀective in improving balance performance in post-stroke patients both immediately post-intervention and at follow-ups (28, 29, 41). However, another study that adopted videogame-based training for post- stroke patients showed no signiﬁcant improvement in BBS scores (32). These results indicate the importance of therapists’ assistance and selecting the appropriate type of intervention. According to the FMA-LE results, lower limb motor function was improved only at the 3-month, 6-month, and 1-year follow- ups in the CT group, while VR group’s outcome was improved immediately after the intervention and at 3-month, 6-month, and 1-year follow-ups; in addition, no signiﬁcant diﬀerence between two groups. Spatiotemporal gait parameters group w-up 6 m follow-up 1 yr follow-up Pre .17 24.82 ± 10.43 20.91 ± 10.07 20.94 ± 8.7 4.63 43.87 ± 21.34* 44.43 ± 21.62 32.67 ± 15.6 .67 13.77 ± 2.11 11.36 ± 3.66 11.95 ± 7.8 6.51 −8.53 ± 12.72 −13.51 ± 1.25 −6.75 ± 9.9 09 2.90 ± 5.56 0.75 ± 6.14 −0.71 ± 8.8 .49 −4.63 ± 5.63 −8.84 ± 5.85 −9.67 ± 7.9 .89 33.35 ± 11.28 34.42 ± 7.4 27.69 ± 8.1 6.82 40.97 ± 20.46 43.68 ± 18.82 33.38 ± 12.3 .02 18.40 ± 6.30 20.20 ± 6.75 21.62 ± 9.0 Bian et al. 10.3389/fneur.2022.985700 FIGURE 3 Mean hip, knee, and ankle joint kinematic curve for all participants in the sagittal plane of both groups during pre-, post-training and follow-up (compared with normal gait cycles). FIGURE 3 Mean hip, knee, and ankle joint kinematic curve for all participants in the sagittal plane of both groups during pre-, post-training and follow-up (compared with normal gait cycles). frontiersin.org Discussion This ﬁnding is in accordance with previous studies that have reported immediate improvement in lower At the follow-ups, signiﬁcant improvements compared with the baseline were observed in some gait parameters in the VR group. The parameters of walking speed, cadence, stride time, opposite foot contact, step time, double support, stride length and step length changed signiﬁcantly from baseline to the follow-ups in the VR group, but not in the CT group; in addition, the inter-group diﬀerences were also not signiﬁcant at these time points. This suggests that although the improvements in these parameters may have arisen due to recovery, VR-based training tended to contribute more to the improvements than CT. Our results on walking speed and cadence are consistent with those reported previously. In previous studies, the increases Frontiers in Neurology frontiersin.org 08 Bian et al. 10.3389/fneur.2022.985700 in walking speed, which is regarded as an important indicator of gait performance in post-stroke patients, were signiﬁcantly greater in the VR groups than in the CT groups (15, 44–46). Another study found that walking speed changed signiﬁcantly after VR intervention in participants with mild stroke than in those with moderate and severe stroke (28). The ﬁndings of our study could also be attributable to the participants learning compensatory strategies while getting accustomed to using a VR system. Allen et al. (47) also emphasized this possibility and demonstrated that participants tended to self-select more eﬀective walking speeds by adopting compensatory strategies with the non-paretic limb. Another possible explanation for no signiﬁcant diﬀerences of spatiotemporal gait parameters is that the sample size of the VR group was smaller than that of the control group due to randomized allocation and drop- out of the participants. Studies have shown that improvement in dynamic balance capability is associated with velocity and cadence (48), while lower extremity dysfunction is highly correlated with walking speed (49). In addition, step length is aﬀected by forward propulsion, which is generated by the stance leg to enable the trunk to move forward with dynamic balance control (47). Another possible explanation for the non- signiﬁcant improvements after VR-based training in our study is that the VR environment we adopted was not ecologically valid enough to be more eﬀective than CT. Multiple studies have highlighted the technical limitation that the majority of current VR-based rehabilitation systems do not provide users with a realistic environment or real-life situations (5, 50). Discussion The VR tasks are more like simple games rather than real-world scenarios, which aﬀects rehabilitation eﬀectiveness and creates a potential gap between training and actual daily function. functional recovery, evidence supporting the use of early VR interventions to enhance functional recovery is still lacking (14). The mechanism of performance improvements induced by VR-based training may involve neuroplasticity, motivation and high training intensity. VR is reported to enhance post-stroke experience-dependent neuroplasticity and motor learning by activating related brain regions, inducing cortical reorganization and strengthening the mirror neuron system involved in motor planning, learning and execution (25). VR also increases training motivation and engagement by reducing the perception of exertion, contributing to eﬀortless and sustained exercise (50, 53, 54). The intrinsic and extrinsic feedback on performance and progress given by VR programs can reinforce patients’ correct behavior and help maintain their level of action (25). Meanwhile, user-dependent tasks, objective progression and repetitive training, all of which are part of VR-based training, play important roles in promoting motor learning strategies in clinical practice (11). The augmented feedback from a VR-based rehabilitation system has been shown to beneﬁt participants by enhancing their learning rate and training the mirror neuron system (50, 55). Our study has several limitations. First, the duration of intervention in our study was not suﬃcient, as an intervention of at least 8 weeks is required to observe notable eﬀects of VR-based training due to physical adaptation (18). Second, the calibration of the VR games’ diﬃculty was not accurate in our study and acceptability of VR was not tested among patients, which may have led to poor methodological quality and lack of a clear rationale for the intervention program, particularly in terms of treatment intensity, personalized training and task variation. Third, the sample size in this study is small, so we will conduct a further study with a larger sample size. Lastly, our study analyzed only some kinematic parameters and two clinical scales, but no kinetic parameters, cognitive or physiological changes with clinical scales. Further indexes and instruments such as the hip ﬂexor index and gait deviation index could be used to measure gait patterns (56). Nevertheless, some of the kinematic parameters, especially the maximum knee joint angle in the sagittal plane, were signiﬁcantly improved from baseline to 6-month follow-ups in the VR group. Discussion Though kinematic performance of hip joint had no signiﬁcant diﬀerence from baseline, our study found the hip extention has been increased at follow-ups. The performance of hip and knee joint tends to a more ﬂuent and normal gait pattern instead of stiﬀknee and hip pattern after the intervention in the VR group. These results suggest that VR-based training enhances knee ﬂexion and decreases knee overextension. A study suggested that VR can improve knee strength and performance, and lower limb motor control is highly associated with balance and gait capability (51). Taken together, improvement in knee motion contributes to improved balance capacity, which is reﬂected in the BBS scores. Simonsen (52) pointed out that improved joint performance in the sagittal plane is correlated with enhanced walking speed, which is consistent with the ﬁndings of our study. Similarly, the possible reason for the negative result is that VR training in our study was non-immersive, which may have reduced the participants’ concentration and training eﬀectiveness. Despite some evidence supporting the beneﬁts of an early exercise program on In conclusion, our current study ﬁndings using non- immersive VR to demonstrate that non-immersive VR-based training improves balance and gait performance among subacute stroke patients and contributes to normal gait pattern appearance, but the eﬀectiveness of non-immersive VR techniques is not superior to CT-based training for balance and motor function. Non-immersive VR-based training could be applied as a clinical rehabilitation therapy as well as conventional therapy. Ethics statement The studies involving human participants were reviewed and approved by the Ethics Committee of the Seventh Aﬃliated Hospital of Sun Yat-sen University and the Ethics Committee of the First Aﬃliated Hospital of Sun Yat-sen University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any identiﬁable images or data included in this article. Trial status The trial is still ongoing both in the Seventh Aﬃliated Hospital, Sun Yat-sen University and the First Aﬃliated Hospital, Sun Yat-sen University. The ﬁrst participant was Frontiers in Neurology 09 frontiersin.org Bian et al. 10.3389/fneur.2022.985700 interpreted the data and drafted the manuscript. All authors read and approved the ﬁnal manuscript. included in October 2019 and the patient recruitment in this study was completed in June 2022. interpreted the data and drafted the manuscript. All authors read and approved the ﬁnal manuscript. Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Funding The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. This research is funded by the National Key Research and Development Program of China (2020YFC2004300 and 2020YFC2004304) and 5010 Planning Project of Sun Yat-sen University of China (Grant No. 2014001). Author contributions All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. YM, DH, and MB designed the study. MB and YS recruited the participants. YW provided training instruction. YM, MB, and YS captured the gait data and three-dimensional data. YH and LW assessed the clinical scales. MB, YM, YS, and SH References 1. 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Virtual reality gait training versus non-virtual reality gait training for improving participation in subacute stroke survivors: study protocol of the virtas randomized controlled trial. Trials. (2019) 20:89. doi: 10.1186/s13063-018-3165-7 33. Garay-Sánchez A, Suarez-Serrano C, Ferrando-Margelí M, Jimenez-Rejano JJ, Marcén-Román Y. Eﬀects of immersive and non-immersive virtual reality on the static and dynamic balance of stroke patients: a systematic review and meta-analysis. J Clin Med. (2021) 10:4473. doi: 10.3390/jcm10194473 55. Pichierri G, Wolf P, Murer K, de Bruin ED. Cognitive and cognitive-motor interventions aﬀecting physical functioning: a systematic review. BMC Geriatr. (2011) 11:29. doi: 10.1186/1471-2318-11-29 55. Pichierri G, Wolf P, Murer K, de Bruin ED. Cognitive and cognitive-motor interventions aﬀecting physical functioning: a systematic review. BMC Geriatr. (2011) 11:29. doi: 10.1186/1471-2318-11-29 34. Pendlebury ST, Markwick A, de Jager CA, Zamboni G, Wilcock GK, Rothwell PM. Diﬀerences in cognitive proﬁle between tia, stroke and elderly memory research subjects: a comparison of the mmse and moca. Cerebrovasc Dis. (2012) 34:48–54. doi: 10.1159/000338905 56. Cimolin V, Galli M. Summary measures for clinical gait analysis: a literature review. Gait Posture. (2014) 39:1005–10. doi: 10.1016/j.gaitpost.2014. 02.001 56. Cimolin V, Galli M. Summary measures for clinical gait analysis: a literature review. Gait Posture. (2014) 39:1005–10. doi: 10.1016/j.gaitpost.2014. 02.001 Frontiers in Neurology 11 frontiersin.org
https://openalex.org/W4233726484	https://www.qeios.com/read/WNZUHI/pdf	English	null	SS, Rat Strain	Definitions	2,020	cc-by	114	SS, Rat Strain National Cancer Institute National Cancer Institute Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Open Peer Review on Qeios Qeios ID: WNZUHI · https://doi.org/10.32388/WNZUHI Source National Cancer Institute. SS, Rat Strain. NCI Thesaurus. Code C76186. Derived by Rapp from a colony of Sprague-Dawley rats that were initially derived by LK Dahl at Brookhaven National Laboratories. The SS rat strain has been selected for its acute salt sensitivity, leading to the development of hypertension, diabetes, nephropathy, cardiac hypertrophy, heart failure, cholesterolemia, hyperinsulinemia, hypertriglyceridemia. This rat strain is used as an animal model in the study of human hypertension. Qeios ID: WNZUHI · https://doi.org/10.32388/WNZUHI 1/1
https://openalex.org/W2037215019	https://actavet.vfu.cz/media/pdf/avb_2008077040615.pdf	Breton	null	The Effect of Chronic Otitis Externa-Media on Brainstem Auditory Evoked Potentials in Dogs	Acta veterinaria Brno	2,008	cc-by	5,482	Abstract Besaltı O., Y.S. Sırın, Z. Pekcan: The Effect of Chronic Otitis Externa-Media on Brainstem Auditory Evoked Potentials in Dogs. Acta Vet. Brno 2008, 77: 615-624. Besaltı O., Y.S. Sırın, Z. Pekcan: The Effect of Chronic Otitis Externa-Media on Brainstem Auditory Evoked Potentials in Dogs. Acta Vet. Brno 2008, 77: 615-624. The objective of the study was to present normative Brainstem Auditory Evoked potentials (BAEP) data elicited by both air-conducted clicks (30–100 dBHL) and bone-conducted clicks (100–130 dBHL) for healthy dogs, and dogs with chronic otitis externa-media (COEM). The data were analyzed to acquire reference values in 55 normal dogs and to estimate the degree of hearing impairment associated with the disease. Fifty-four dogs with COEM were divided into two degrees of severity. The first one included dogs with severe (n = 16), and the second with moderate COEM (n = 38). The recognisability wave V was found higher than the others at every stimulation level. Wave VII was not seen by the bone-conducted click at all stimulation levels, and also in cases with severe COEM by the air-conducted click. Recognisability of wave V was seen more than 50% at 60 dBHL in severe COEM. This ratio was seen at 30 dBHL in moderate COEM. In healthy dogs, the latencies of waves (I–VII) were compared between the left and the right side, and there were no significant differences between each side except for a shorter latency of wave VI. In conclusion, COEM caused increasing latency rather than total deafness, and BAEP can be suggested as an ancillary diagnostic tool in ear disorders. Chronic otitis externa is characterized by severe inflammation, with irreversible hyperplasic changes of the ear canal and the surrounding tissues, and mineralization (with subsequent ossification) of the auricular and annular cartilage (Bellah 1997). Chronic inflammation and accumulation of exudates within the canal eventually lead to the disruption of the tympanic membrane. The infection invades the middle ear (otitis media), and may eventually extend to the inner ear (otitis interna), and sometimes the facial nerve is involved (facial paralysis) (Stickney et al. 2004). Although profound hearing loss is most commonly related to neurological causes, chronic otitis externa-media (COEM) can also cause a partial hearing loss (Eger and Lindsay 1997; Krahwinkel et al. 1993). Received October 18, 2007 Accepted June 11, 2008 Received October 18, 2007 Accepted June 11, 2008 Received October 18, 2007 Accepted June 11, 2008 ACTA VET. BRNO 2008, 77: 615-624; doi:10.2754/avb200877040615 ACTA VET. BRNO 2008, 77: 615-624; doi:10.2754/avb200877040615 O. BESALTI1, Y.S. SIRIN2, Z. PEKCAN3 O. BESALTI1, Y.S. SIRIN2, Z. PEKCAN3 Ankara University, Faculty of Veterinary Medicine, Department of Surgery, Diskapi, Ankara, Turkey 2Mayis University, Faculty of Veterinary Medicine, Department of Surgery, Samsun, Turkey 3Kirikkale University, Faculty of Veterinary Medicine, Department of Surgery, Yahsihan, Kirikkale, Turkey Abstract It was demonstrated that total ear canal ablation combined with bulla osteotomy, which is the standard approach to the end stage of COEM, may be the cause of a significant loss of hearing (McAnulty et al. 1995).i Deafness can be classified into conductive or sensory neural deafness according to the involved anatomical structures. Conductive hearing loss occurs with the occlusion of the ear canal, thickening or disruption of the tympanic membrane, replacement of the air within the middle ear with fluid, stiffening or damage to the ossicular chain, and loss of compliance of the vestibular or round windows (Eger and Lindsay 1997). Brainstem auditory evoked potential (BAEP) is an accurate, repeatable and non- invasive method in evaluation of the deafness in dogs and cats. It provides valuable information in the treatment protocol and prognosis, and also differentiates conductive Address for correspondence: O. Besaltı, DVM, PhD Ankara University Faculty of Veterinary Medicine Department of Surgery, Diskapi, Ankara, TURKEY Address for correspondence: O. Besaltı, DVM, PhD Ankara University Faculty of Veterinary Medicine Department of Surgery, Diskapi, Ankara, TURKEY Phone: +90 535 264 0546 Fax: +90 312 316 4472 E-mail: besalti@hotmail.com http://www.vfu.cz/acta-vet/actavet.htm 616 and sensory neural deafness (Sims 1988). This test can be carried out by bone-conducted clicks and air-conducted clicks. By comparing the results of air and bone conduction BAEP tests, an audiologist can determine whether the hearing loss is sensory, neural, conductive or mixed. Brainstem auditory evoked potentials also provide information about which part of the anatomical structures related to hearing is affected (Cox 2002; Sims 1988). ) The aim of this study was to present the effects of COEM on hearing, and provide standard values for BAEP on physiological recordings. Recording Recording g BAER was recorded by Medelec Synergy 5 channel EMG/ EP device (Oxford Instrument-UK). Headset and ear tips for air-conducted clicks (Oxford, S1021) and bone conductor for bone-conducted clicks (Oxford, 017C080) were used for all cases except for dogs with a totally obliterated ear canal in which only bone conductor was used. Ear tips were inserted into both ear canals, and bone conductor was placed against the mastoid process of the temporal bone for delivering the stimulus. Subdermal stainless steel monopolar needle electrodes (Oxford, 017K103) were placed at the vertex (reference) and just rostral (active) to the base of each ear and the ground electrode was placed over the frontal sinus along the midline (Plate I, Fig.1). The impedance of the electrodes was checked to ensure that it was less than 2 kilo ohm. While stimulating one ear, the other was masked by white noise for the purpose of recording each ear individually. Impulses generated in the first 10 milliseconds, and each click was filtered (100 Hz-3 KHz). The tested ear was stimulated at 100 dBHL to 30 dBHL in air-conducted clicks and at 130 dBHL to 100 dBHL in bone-conducted clicks by the decreasing (decrement of) 10 dBHL for each season at a click rate of 11 pps. At each level an average of 1500 clicks/stimuli were used. il g Identification of the waves was carried out as reported before (Sanders and Bagley 2003; Sims 1988). Briefly, wave I is the first and wave V has a large amplitude and characteristic negative trough following the positive peak. Wave III and IV are seen either merged with or separate from each other and their amplitudes are smaller. The other waves were estimated in turn, and latencies were measured from the positive peak point of the wave. Statistical Analysis Latency values were compared with one-way ANOVA for healthy, severe COEM and moderate COEM cases. The latencies were analyzed in all three groups with Tukey test. The differences between right and left sides were compared using paired t-test. Materials and Methods Fifty-five dogs of different age, sex and breed without any history or clinical signs of ear disorders, but anaesthetized for the aim of different elective surgical procedures were subjected to recordings for obtaining physiological values for the study. The cases with COEM that were diagnosed by otoscopic and radiographic examination were included prospectively. Fifty-four dogs met the criteria to be included in the study. Thirty-eight dogs had moderate COEM (stenotic canal and perforated, bulged, cloudy or opaque appearance in colour, and/ or thickened tympanic membrane) and 16 dogs had end stage COEM (ear canal narrowing or total obliteration, unable to visualize tympanic membrane). BAER were recorded under general anaesthesia which was induced using 0.05 mg/kg atropine sulphate SC (Biofarma, Turkey), 2 mg·kg-1 xylazine hydrochlorure IM, (Alfazyne, Izmir, Turkey) and 15 mg·kg-1 ketamine hydrochloride IM (Alfamine, Izmir, Turkey) combination. The study was approved by the Ethics Commitee of the Faculty of Veterinary Medicine, Ankara University. Results Brain stem auditory evoked responses were recorded from 110 normal ears of 55 dogs. Ages ranged from 1 to 11 years (mean 4.22). The breeds included were cross-breed (n = 30), Poodle (n = 6), German shepherd (n = 4), Turkish Anatolian sheep dog (n = 4), Cocker Spaniel (n = 4), Labrador (n = 3), Rottweiler (n = 2), Siberian husky (n = 1) and pointer (n = 1). Moderate COEM was diagnosed in 60 ears of 38 dogs, and ages ranged from 2 to 14 years (mean 7.54). Twenty-two dogs had bilateral and 16 dogs had monolateral moderate COEM. The contralateral ears had mild inflammation or were healthy. The breeds were Cocker Spaniel (n = 11), Poodle (n = 8), German shepherd (n = 4), cross breed (n = 4), Boxer (n = 3), Dalmatian (n = 1), Labrador (n = 3), Golden retriever (n = 1), Turkish Anatolian sheep dog (n = 1), pointer (n = 1) and pit bull Terrier (n = 1). Severe COEM was diagnosed in 28 ears of 16 dogs, and mean ages were 11.6 years old. Twelve dogs had bilateral and 4 had monolateral severe COEM. Breed dispersion was Cocker Spaniel (n = 8), Poodle (n = 6), Labrador (n = 1) and pointer (n = 1). Facial 617 paralysis was diagnosed beside severe COEM in three dogs ipsilaterally and in one Fig. 3. The recognisability of wave I (a, b), V (c, d) and VII (e, f) recorded by air conducted clicks at diffe stimulus intensities in normal (N), moderate (M) and severe (S) COEM. c b a a The recognisability of wave I (a, b), V (c, d) and VII (e, f) recorded by air conducted clicks at dif s intensities in normal (N), moderate (M) and severe (S) COEM. c b a b a he recognisability of wave I (a, b), V (c, d) and VII (e, f) recorded by air conducted clicks at d c b a b Fig. 3. The recognisability of wave I (a, b), V (c, d) and VII (e, f) recorded by air conducted clicks at different stimulus intensities in normal (N), moderate (M) and severe (S) COEM. paralysis was diagnosed beside severe COEM in three dogs ipsilaterally and in one dog bilaterally by neurological examination and needle EMG of the involved facial muscle. Results lysis was diagnosed beside severe COEM in three dogs ipsilaterally and in one do erally by neurological examination and needle EMG of the involved facial muscle. The BAEP appears as a series of I-VII vertex positive peaks (Plate I, Fig. 2). The recognisability ratio of the waves at different stimulation levels is illustrated graphically in Figs 3a-f, 4a-f). 618 y dogs he recognisability of wave I (a, b), V (c, d) and VII (e, f) recorded by air conducted clicks at dif intensities in normal (N), moderate (M) and severe (S) COEM. d e f d e The recognisability of wave I (a, b), V (c, d) and VII (e, f) recorded by air conducted clicks at different us intensities in normal (N) moderate (M) and severe (S) COEM f Fig. 3. The recognisability of wave I (a, b), V (c, d) and VII (e, f) recorded by air conducted clicks at different stimulus intensities in normal (N), moderate (M) and severe (S) COEM. Healthy dogs By increasing the signal intensity, the percentage recognisability of wave I, II and III increased. Wave IV appeared lower than 50% at all stimulation intensity levels. In contrast, wave V recognised at the highest amplitude at all stimulation intensity levels. Waves VI and VII were seen in a higher percentage between 50–90 dBHL. 619 recognisabilit ratio of the a es in moderate COEM: At 30 dBHL a e IV and a b c . The recognisability of waves elicited by bone conducted clicks at different stimulus intensities in he moderate COEM (c, d), severe COEM (e, f) cases. Fig. 4. The recognisability of waves elicited by bone conducted clicks at different stimulus intensities in healthy (a, b), moderate COEM (c, d), severe COEM (e, f) cases. The recognisability ratio of the waves in moderate COEM: At 30 dBHL wave IV and VII, at 40 dBHL wave VII and at 50 dBHL wave IV and VII, and at 60, 70 and 100 dBHL wave VII did not appear. The recognisability ratio of the waves of moderate COEM; wave IV and VII at 50 and 60 dBHL were zero, and at 70–90 dBHL wave VII was not seen. Wave VII was not seen in bone-conducted clicks records at any stimulation intensity level. The recognisability ratio of the waves in moderate COEM: At 30 dBHL wave IV and VII, at 40 dBHL wave VII and at 50 dBHL wave IV and VII, and at 60, 70 and 100 dBHL wave VII did not appear. The recognisability ratio of the waves of moderate COEM; wave IV and VII at 50 and 60 dBHL were zero, and at 70–90 dBHL wave VII was not seen. Wave VII was not seen in bone-conducted clicks records at any stimulation intensity level. The latency values for both air-conducted clicks and bone-conducted clicks are presented in Tables 1 and 2. Significant difference at 70 dBHL of air-conducted click records was seen as: The latency of wave I and II, III in healthy ears was smaller than in moderate The latency values for both air-conducted clicks and bone-conducted clicks are presented in Tables 1 and 2. Significant difference at 70 dBHL of air-conducted click records was seen as: The latency of wave I and II, III in healthy ears was smaller than in moderate 620 d e g. 4. Healthy dogs The recognisability of waves elicited by bone conducted clicks at different stimulus intensities in healt , b), moderate COEM (c, d), severe COEM (e, f) cases. d e f Fig. 4. The recognisability of waves elicited by bone conducted clicks at different stimulus intensities in healthy (a, b), moderate COEM (c, d), severe COEM (e, f) cases. and/or severe COEM. The latency of wave V (on the left side) and VI (on the right side) in healthy ears was smaller than in severe COEM. When the latency values were compared regarding the left or right side, there were no significant differences except for wave VI which was smaller on the left side than on the right. i and/or severe COEM. The latency of wave V (on the left side) and VI (on the right side) in healthy ears was smaller than in severe COEM. When the latency values were compared regarding the left or right side, there were no significant differences except for wave VI which was smaller on the left side than on the right. Healthy dogs i Significant differences of latency at 110 dBHL of bone-conducted click records were seen 621 Healthy 30 left 30 right 40 left 40 right 50 left 50 right 60 left 60 right 70 left 70 right 80 left 80 right 90 left 90 right 100 left 100 right Wave I 1.7 ± 0.24 1.81 ± 0.39 1.66 ± 0.24 1.66 ± 0.3 1.59 ± 0.17 1.55 ± 0.20 1.5 ± 0.12 1.5 ± 0.18 1.48 ± 0.19 1.47 ± 0.16 1.41 ± 0.14 1.43 ± 0.17 1.37 ± 0.14 1.44 ± 0.19 1.48 ± 0.33 1.38 ± 0.05 Wave II 2.49 ± 0.26 2.53 ± 0.39 2.44 ± 0.19 2.54 ± 0.36 2.47 ± 0.28 2.46 ± 0.25 2.37 ± 0.2 2.38 ± 0.21 2.31 ± 0.22 2.33 ± 0.19 2.27 ± 0.16 2.24 ± 0.24 2.25 ± 0.16 2.32 ± 0.22 2.34 ± 0.31 2.26 ± 0.08 Wave III 3.20 ± 0.21 3.24 ± 0.39 3.12 ± 0.23 3.24 ± 0.28 3.13 ± 0.31 3.03 ± 0.53 3.02 ± 0.27 3.08 ± 0.28 3.02 ± 0.28 3.07 ± 0.27 3.06 ± 0.24 3.06 ± 0.29 3.02 ± 0.23 3.02 ± 0.29 3.09 ± 0.39 3.08 ± 0.35 Wave IV 3.83 ± 0.68 4.2 ± 0.55 3.55 ± 0.33 3.85 ± 0.65 3.48 ± 0.49 3.54 ± 0.51 3.28 ± 0.28 3.57 ± 0.38 3.45 ± 0.54 3.56 ± 0.53 3.61 ± 0.59 3.53 ± 0.62 3.84 ± 0.83 3.51 ± 0.61 3.55 ± 0.27 Wave V 4.19 ± 0.58 4.23 ± 0.59 4.12 ± 0.41 4.13 ± 0.4 4.05 ± 0.32 4.11 ± 0.47 4.06 ± 0.37 4.1 ± 0.38 4.14 ± 0.56 4.08 ± 0.52 4.19 ± 0.86 4.12 ± 0.75 4.07 ± 0.58 4.45 ± 1.07 3.95 ± 0.35 4.61 ± 1.09 Wave VI 5.83 ± 0.73 5.56 ± 0.51 5.5 ± 0.43 5.57 ± 0.42 5.56 ± 0.46 5.59 ± 0.44 5.44 ± 0.42 5.58 ± 0.42 5.61 ± 0.91 5.4 ± 0.43 5.35 ± 0.52 5.3 ± 0.37 5.33 ± 0.72 5.23 ± 0.36 5.23 ± 0.4 5.46 ± 0.48 Wave VII 7.05 ± 0.84 6.97 ± 0.9 7.03 ± 0.97 6.95 ± 0.45 6.74 ± 0.8 6.55 ± 0.4 6.57 ± 0.86 6.43 ± 0.64 6.54 ± 0.82 6.24 ± 0.51 6.4 ± 0.82 6.16 ± 0.25 6.06 ± 0.33 6.89 ± 0.96 severe COEM Wave I 2.14 ± 0.59 2.18 ± 0.65 1.75 ± 0.11 1.98 ± 0.37 1.73 ± 0.17 1.67 ± 0.33 1.72 ± 0.39 1.6 ± 0.32 1.58 ± 0.15 1.39 ± 0.35 1.49 ± 0.02 1.42 ± 0.19 1.48 ± 0.04 Wave II 3.2 ± 0.76 3.01 ± 0.64 2.8 ± 0.45 2.78 ± 0.35 2.55 ± 0.17 2.53 ± 0.14 2.65 ± 0.7 2.51 ± 0.06 2.42 ± 0.19 2.39 ± 0.12 2.36 ± 0.06 2.28 ± 0.29 2.54 ± 0.39 Wave III 3.33 ± 0.08 3.62 ± 0.17 3.55 ± 0.27 3.49 ± 0.26 3.58 ± 0.75 3.37 ± 0.11 3.39 ± 0.23 3.16 ± 0.2 3.32 ± 0.2 3.18 ± 0.28 3.13 ± 0.34 Wave IV 3.44 ± 0.25 4.19 ± 0.74 4.49 ± 0.68 4.41 ± 0.21 4.21 ± 0.66 4.1 ± 0.45 Wave V 4.4 ± 0.74 4.2 ± 0.08 4.58 ± 0.43 4.22 ± 0.2 4.43 ± 0.37 4.2 ± 0.33 5.13 ± 1.12 4.64 ± 0.85 4.93 ± 0.93 4.76 ± 0.62 4.56 ± 0.83 4.56 ± 0.55 4.29 ± 0.41 4.23 ± 0.22 Wave VI 6.13 ± 0.44 6.06 ± 0.31 5.99 ± 0.2 5.95 ± 0.12 6.36 ± 0.92 6.12 ± 0.74 6.15 ± 0.98 6.08 ± 1.3 6.1 ± 0.63 5.81 ± 0.66 5.52 ± 0.31 5.4 ± 0.43 Wave VII 7.22 ± 0.68 6.51 ± 0.86 moderate COEM Wave I 1.65 ± 0.01 1.92 ± 0.54 1.84 ± 0.33 1.76 ± 0.33 1.64 ± 0.18 1.68 ± 0.18 1.77 ± 0.39 1.7 ± 0.25 1.7 ± 0.36 1.67 ± 0.24 1.64 ± 0.2 1.73 ± 0.33 1.66 ± 0.3 1.69 ± 0.32 1.65 ± 0.16 1.7 ± 0.29 Wave II 2.43 ± 0.09 2.27 ± 0.1 2.64 ± 0.42 2.57 ± 0.33 2.51 ± 0.22 2.65 ± 0.35 2.66 ± 0.37 2.51 ± 0.29 2.54 ± 0.34 2.47 ± 0.23 2.54 ± 0.3 2.56 ± 0.34 2.55 ± 0.3 2.53 ± 0.31 2.53 ± 0.19 2.57 ± 0.26 Wave III 3.33 ± 0.34 3.4 ± 0.47 3.34 ± 0.47 3.31 ± 0.35 3.25 ± 0.31 3.37 ± 0.57 3.38 ± 0.46 3.35 ± 0.47 3.41 ± 0.42 3.4 ± 0.55 3.31 ± 0.37 3.51 ± 0.52 3.34 ± 0.31 3.29 ± 0.32 3.35 ± 0.34 3.35 ± 0.3 Wave IV 3.84 ± 0.83 3.8 ± 0.47 3.9 ± 0.71 3.56 ± 0.55 3.56 ± 0.11 3.83 ± 0.62 3.45 ± 0.04 3.64 ± 0.53 3.5 ± 0.38 3.97 ± 0.55 3.94 ± 0.45 3.6 ± 0.21 4.26 ± 0.7 Wave V 4.51 ± 0.92 4.66 ± 0.69 4.48 ± 0.86 4.51 ± 0.52 4.46 ± 0.67 4.35 ± 0.35 4.43 ± 0.75 4.39 ± 0.55 4.45 ± 0.68 4.44 ± 0.61 4.28 ± 0.36 4.5 ± 0.56 4.48 ± 0.48 4.46 ± 0.41 4.34 ± 0.27 4.33 ± 0.4 Wave VI 5.64 ± 0.57 6.33 ± 0.55 5.88 ± 0.51 5.78 ± 0.65 5.64 ± 0.21 5.86 ± 0.5 5.73 ± 0.7 5.81 ± 0.59 5.70 ± 0.56 5.51 ± 0.33 5.66 ± 0.55 5.78 ± 0.8 5.68 ± 0.48 5.92 ± 0.52 5.79 ± 0.21 6.16 ± 0.77 Wave VII 7.29 ± 1.2 6.8 ± 1.37 7.52 ± 0.71 6.88 ± 0.81 7.01 ± 0.81 6.63 ± 0.85 7.74 ± 1.1 7.53 ± 0.9 Table 1. Healthy dogs Summary of latency data from air conducted clicks (±SD) 622 Table 2. Healthy dogs Summary of latency data from bone conducted clicks (±SD) Healthy 130 left 130 right 120 left 120 right 110 left 110 right 100 left 100 right Wave I 1.88 ± 0.26 1.76 ± 0.24 1.96 ± 0.25 1.94 ± 0.29 2.07 ± 0.13 2.07 ± 0.14 2.11 ± 0.08 2.18 ± 0.05 Wave II 2.72 ± 0.37 2.67 ± 0.29 2.89 ± 0.2 2.81 ± 0.4 3.04 ± 0.16 3.03 ± 0.19 3.15 ± 0.11 3.11 ± 0.17 Wave III 3.43 ± 0.27 3.45 ± 0.39 3.75 ± 0.16 3.53 ± 0.44 3.68 ± 0.24 3.59 ± 0.34 3.82 ± 0.14 3.67 ± 0.16 Wave IV 4.11 ± 0.22 3.93 ± 0.79 4.13 ± 0.04 Wave V 4.69 ± 0.56 4.51 ± 0.69 5.12 ± 0.22 4.9 ± 0.71 5.09 ± 0.15 4.86 ± 0.35 5.2 ± 0.31 5.02 ± 0.27 Wave VI 5.94 ± 0.44 6.18 ± 0.93 6.33 ± 0.34 6.51 ± 0.27 6.35 ± 0.3 6.74 ± 0.95 6.81 ± 0.33 Wave VII Severe COEM Wave I 1.7 ± 0.45 1.64 ± 0.13 1.76 ± 0.34 1.44 ± 0.03 1.84 ± 0.16 1.81 ± 0.42 2.01 ± 0.38 Wave II 2.44 ± 0.28 2.29 ± 0.14 2.67 ± 0.37 2.54 ± 0.33 2.65 ± 0.15 2.78 ± 0.68 2.81 ± 0.35 Wave III 3.15 ± 0.24 2.86 ± 0.4 3.21 ± 0.38 3.59 ± 0.47 3.35 ± 0.27 2.98 ± 0.79 Wave IV 3.46 ± 0.11 3.4 ± 0.34 3.97 ± 0.31 Wave V 4.01 ± 0.26 3.77 ± 0.43 4.27 ± 0.44 4.28 ± 0.51 4.2 ± 0.59 4.28 ± 0.48 5.53 ± 0.45 4.3 ± 0.44 Wave VI 5.61 ± 0.58 5.38 ± 0.99 5.13 ± 0.55 5.37 ± 0.85 6.01 ± 0.49 5.61 ± 0.07 Wave VII Moderate COEM Wave I 1.77 ± 0.46 1.78 ± 0.3 1.82 ± 0.4 1.83 ± 0.31 1.83 ± 0.29 2.12 ± 0.49 2.09 ± 0.55 2.03 ± 0.11 Wave II 2.54 ± 0.41 2.54 ± 0.31 2.68 ± 0.38 2.59 ± 0.26 2.83 ± 0.47 2.69 ± 0.2 2.79 ± 0.57 2.85 ± 0.35 Wave III 3.38 ± 0.52 3.23 ± 0.63 3.55 ± 0.65 3.26 ± 0.52 3.23 ± 0.58 3.53 ± 0.45 3.4 ± 0.72 3.77 ± 0.2 Wave IV 3.92 ± 0.3 3.47 ± 0.36 4.46 ± 0.63 3.73 ± 0.65 3.55 ± 0.58 4.43 ± 0.61 Wave V 4.22 ± 0.61 4.19 ± 0.71 5.66 ± 0.84 4.48 ± 0.97 4.55 ± 0.71 4.73 ± 1.1 4.62 ± 0.73 4.92 ± 0.89 Wave VI 5.5 ± 0.62 5.61 ± 1.05 7.34 ± 0.98 5.56 ± 0.7 5.72 ± 0.94 6.12 ± 0.91 6.94 ± 0.03 6.04 ± 0.6 Wave VII as follows: Wave I was lower in severe COEM than in healthy ears; however, severe COEM values were lower than moderate COEM on the left side. Healthy dogs Normal values of wave II were higher than in moderate and severe chronic COEM. Wave V was smaller in healthy ears than in severe COEM. Discussion Dogs that only have normal traces at 40 dBHL or above, have a mild hearing loss, whereas those with normal traces at 60 dBHL and above are considered to have a moderate hearing loss. Dogs with normal traces at 75 dBHL or above have a severe or profound hearing loss (Cox 2002). According to this classification there was no hearing problem in 55 normal dogs in which BAER was recognized at 30 dBHL. In addition, r e c o g n i s a b i l i t y of wave V at all stimulus intensities in a higher degree means that it can be used for determining the threshold, as reported before (Cox 2002; Sims 1988). In this study, severe COEM cases had moderate hearing problems. In these 623 cases the recognisability ratio of wave V with more than 50% was seen at 60 dBHL intensity. In moderate COEM there was a non-significant hearing loss, from which it was deducted that the recognisability rate was more than 50% at 30 dBHL. cases the recognisability ratio of wave V with more than 50% was seen at 60 dBHL intensity. In moderate COEM there was a non-significant hearing loss, from which it was deducted that the recognisability rate was more than 50% at 30 dBHL. g y The presence of wave VII whose generator is uncertain in healthy dogs and its absence in severe COEM cases was found interesting. As mentioned before for these cases, having higher hearing thresholds and the absence of wave VII seem to be related. However, before making a clear conclusion, more cases with severe COEM should be investigated. Another interesting finding related to wave VII was its absence at all stimulus intensities in bone- conducted click records. Another conclusion for wave VII can be deducted as conductive structure related to the hearing having a role in the generation of this wave. Since the generator of wave VII is an auditory reflexion (Sims 1988, 2003), in severe COEM cases this reflexion is prevented. l p The latency of all waves increased with the severity of otitis, although the prolongation of times was only significant in advanced cases (Eger and Lindsay 1997). In BAER evoked by air-conducted clicks, latency of wave I increased in both moderate and severe COEM cases compared to healthy dogs in our study. Discussion For wave II on the left side, and for wave III on both sides, for wave V which was recognized at approximately all stimulus intensities, the latency was increased in both moderate and severe COEM compared to healthy dogs. These findings are similar to Eger and Lindsay’s report (1997). The sound waves from the external ear canal are transmitted through the tympanic membrane through the ossicles, which articulate to amplify sound and transmit vibrations to the fluid-filled inner ear. On the surface of the organ of Corti, the inner and outer hair cells lie that connect with the spiral ganglion for transmission to the cochlear nerve (Cook 2004). There is a general agreement that wave I of the BAEP is generated by acoustic nerve activity (Sims 1988). Significant differences of latency, which was lower in severe COEM than normal wave I at 110 dBHL of bone-conducted click can be explained as follows: In severe COEM cases ear canals are mostly obstructed due to chronic inflammation, and sound waves can not be transmitted through the external ear canal. By the bone-conducted click ear canal, tympanic membrane, and perhaps the ossicles vibrations are bypassed, and the receptors in the organ of Corti detect this vibration and the acoustic nerve is stimulated by this impulse. This is the possible cause of the decreased latency of wave I of BAEP in severe COEM. In a study of BAEP in 58 healthy dogs, 4–5 vertex positive waves were explained and latency values at 60 dBHL were I: 1.49 ± 0.13 ms; II: 2.32 ± 0.14 ms; III: 3.01 ± 0.25 ms; IV: 4.22 ± 0.27 ms and V: 5.55 ± 0.37 ms (Bodenhamer et al. 1985). In our study 7 vertex positive waves were identified at different recognisability rates, and also different latency values. The differences are related to the different explanation of wave V. p In conclusion, reference values for both latency and recognisability rate for BAEP in healthy dogs were obtained. Chronic otitis externa-media causes an increase in latency rather than total deafness. Even in severe COEM, dogs have the ability to hear, but to a lesser extent than healthy ones. BAER may be suggested as an ancillary diagnostic tool in ear disorders. Sensory neural deafness associated with COEM can also be differentiated by this test. References BELLAH JR 1997: How and when to perform lateral and vertical ear canal resection. Vet Med 92: 535-543 BODENHAMER RD, HUNTER JF, LUTTGEN PJ 1985: Brain stem auditory-evoked responses in the dog. Am J Vet Res 46: 1787-1792 BELLAH JR 1997: How and when to perform lateral and vertical ear canal resection. Vet Med 92: 535-543 BODENHAMER RD, HUNTER JF, LUTTGEN PJ 1985: Brain stem auditory-evoked responses in the dog. Am J Vet Res 46: 1787-1792 COOK LB 2004: Neurologic evaluation of the ear. Vet Clin North Am-Small Anim Pract 34: 425-435 COX C 2002: Investigation of hearing loss in dogs. In Pract 24: 494-501 g g g EGER CE, LINDSAY P 1997: Effects of otitis on hearing in dogs characterized by brainstem auditor t ti J S ll A i P t 38 380 386 R CE, LINDSAY P 1997: Effects of otitis on hearing in dogs characterized by brainstem auditory evok ponse testing. J Small Anim Pract 38: 380-386 EGER CE, LINDSAY P 1997: Effects of otitis on he response testing. J Small Anim Pract 38: 380-386 response testing. J Small Anim Pract 38: 380-386 p g KRAHWINKEL DJ, PARDO AD, SIMS MH, BUBB WJ 1993: Effect of total ablation of the external acoustic meatus and bulla osteotomy on auditory function in dogs. J Am Vet Med Assoc 202: 949-952 McANULTY JF, HATTEL A, HARVEY CE 1995: Wound healing and brain stem auditory evoked potentials after KRAHWINKEL DJ, PARDO AD, SIMS MH, BUBB WJ 1993: Effect of total ablation of the external acoustic meatus and bulla osteotomy on auditory function in dogs. J Am Vet Med Assoc 202: 949-952 , , , meatus and bulla osteotomy on auditory function in dogs. J Am Vet Med Assoc 202: 949-952 McANULTY JF, HATTEL A, HARVEY CE 1995: Wound healing and brain stem auditory evoked potentials after meatus and bulla osteotomy on auditory function in dogs. J Am Vet Med Assoc 202: 949 952 McANULTY JF, HATTEL A, HARVEY CE 1995: Wound healing and brain stem auditory evoked potentials after i l l i b ll i d V S 24 9 14 McANULTY JF, HATTEL A, HARVEY CE 1995: Wound healing and brain st experimental ventral tympanic bulla osteotomy in dogs. Vet Surg 24: 9-14 experimental ventral tympanic bulla osteotomy in dogs. Acknowledgement This study was supported by TUBİTAK as VHAG 2003. This study was supported by TUBİTAK as VHAG 2003. This study was supported by TUBİTAK as VHAG 2003. Vliv chronické otitis externa-media na akustickou odpověď mozkového kmene u psů Cílem studie bylo prezentovat normativní hodnoty akustické odpovědi mozkového kme- ne (BAEP) na zvukový podnět (kliknutí), šíření zvukové vlny vzduchem (30-100dBHL) a její převod sluchovými kůstkami (100-130 dBHL) u zdravých psů a u psů s chronickou otitis externa-media (COEM). Analýza dat u 55 zdravých psů byla provedena za účelem získání referenčních hodnot pro odhad míry zhoršení sluchu provázející chronický zánět vnějšího a středního ucha u psa. Psi postižení COEM byli rozděleni do dvou skupin dle zá- 624 važnosti onemocnění. Do první skupiny byli zahrnuti jedinci s vážnou formou onemocnění (n = 16), druhá skupina obsahovala jedince s mírným stupněm COEM (n = 38). Ve všech úrovních podráždění byl patrný vzrůst vlny V ve srovnání s ostatními vlnami. U převodu zvukové vlny sluchovými kůstkami chyběla úplně vlna VII ve všech stimulačních hladi- nách a v případě těžkého zánětu chyběla vlna VII i u přenosu akustického signálu vzdu- chem. Vlna V byla patrná ve více než 50% případů na hladině 60 dBHL u jedinců v první skupině. Stejný procentuální podíl byl na hladině 30 dBHL pozorován u mírné COEM. U zdravých psů byla latence jednotlivých vln (I – VII) na pravé i levé straně srovnatelná, hodnoty na pravé i levé straně se statisticky významně nelišily s výjimkou menšího opo- ždění v případě vlny VI. Na závěr je možno konstatovat, že COEM způsobuje spíše vyšší latenci v převodu zvuku než ztrátu sluchu a BAEP lze využít jako jednu z pomocných metod v diagnostice sluchových poruch. Acknowledgement This study was supported by TUBİTAK as VHAG 2003. References Vet Surg 24: 9-14 p y p y g g SANDERS SG, BAGLEY RS 2003: Disorders of hearing and balance: The vestibulocochlear nerve (CN VIII) and associated structures. In: DEWEY CW (Ed.): A practical guide to canine and feline neurology. Iowa State Press, Iowa, pp. 213-240 , , pp SIMS MH 1988: Electrodiagnostic evaluation of auditory function. Vet Clin North Am Small Anim Pract 18: 913-941 SIMS HS 2003: Clinical evaluation of auditory function. In: SLATTER D (Ed.): Textbook of small animal surgery, 3rd ed. WB Saunders, Philadelphia, pp. 1767-1773 g y p pp STICKNEY M, REDDIN-PARKER S, McIAUGHLIN R 2004: Surgical treatment of chronic otitis e dogs. In: Symposium on otitis externa in dogs. Vet Med 99: 274-287 Plate I Besalti O. et al.: The effect of Chronic ... pp. 615-624 Fig. 1. Configuration of recording procedures for BAEP Fig. 2. BAEP at different stimulus intensities elicited by air clicks Plate I Besalti O. et al.: The effect of Chronic ... pp. 615-624 Fig. 1. Configuration of recording procedures for BAEP Plate I Besalti O. et al.: The effect of Chronic ... pp. 615-624 Fig. 1. Configuration of recording procedures for BAEP Fig. 2. BAEP at different stimulus intensities elicited by air clicks Fig. 2. BAEP at different stimulus intensities elicited by air clicks
https://openalex.org/W3132690318	https://bmcmededuc.biomedcentral.com/track/pdf/10.1186/s12909-021-02542-w	English	null	Health management students’ self-regulation and digital concept mapping in online learning environments	BMC medical education	2,021	cc-by	11,754	Alt and Naamati-Schneider BMC Medical Education (2021) 21:110 https://doi.org/10.1186/s12909-021-02542-w Alt and Naamati-Schneider BMC Medical Education (2021) 21:110 https://doi.org/10.1186/s12909-021-02542-w Open Access Health management students’ self- regulation and digital concept mapping in online learning environments Dorit Alt1* and Lior Naamati-Schneider2* * Correspondence: doritalt@014.net.il; liorna@hac.ac.il 1 * Correspondence: doritalt@014.net.il; liorna@hac.ac.il 1Kinneret College on the Sea of Galilee, Tzemach Junction, 15132 Jordan Valley, MP, Israel 2Hadassah Academic College, 37 Hanevi’im St., 9101001 Jerusalem, Israel y, , Hadassah Academic College, 37 Hanevi’im St., 9101001 Jerusalem, Israel * Correspondence: doritalt@014.net.il; liorna@hac.ac.il 1Kinneret College on the Sea of Galilee, Tzemach Junction, 15132 Jordan Valley, MP, Israel 2Hadassah Academic College, 37 Hanevi’im St., 9101001 Jerusalem, Israel Abstract Background: Self-regulation of learning is considered one of the key capabilities deemed essential for the healthcare system and its workers to cope successfully with the current challenges they are facing. Therefore, healthcare curricula are increasingly called upon to support self-regulation as a central learning outcome. With scant relevant publications describing how students of medicine and other healthcare professions regulate their learning, this study set out to design and assess a problem-based learning using digital concept mapping in an online course and to evaluate the set of connections between this intervention and Health Management students’ self-regulation of learning. Method: Students of a Management of Health Service Organizations program (100) were presented with an ill- structured problem, relevant to their course content (accreditation process within hospitals) and were asked to argue for or against the implementation of the accreditation process. The participants were asked to detail five arguments to establish their decision by using Mindomo, a popular digital platform for designing concept maps. The students were given predefined criteria that allowed them to self-assess their maps. Data for the analysis were gathered by two measurements: Concept mapping for problem-based learning scale and the Online self-regulated learning scale and were analyzed by using Partial Least Squares - Structural Equation Modeling. Results: The analyses showed that at the beginning of the process, students’ online self-regulation was found lower than at the end of the intervention, and only two self-regulation sub-factors: Goal setting and Task strategies, were positively linked to students’ perceptions of the intervention. After the intervention, the analyses showed that it increased the levels of four Online self-regulation sub-factors: Goal setting, Task strategies, Environment structuring, and Time management. Conclusions: Teachers need to recognize and account for different types of learners and encourage and scaffold students’ effective use of self-regulation strategies. Low self-regulated learners might fail to see the advantages of concept mapping in problem-solving activities. Combining these teaching and learning tools together with the use of advanced technology in an online course that encourages active learning enables the development and acquisition of abilities of self-directed learning among students in the medical and health management professions. Keywords: Health management education, Self-regulation of learning, Digital concept mapping, Problem-based learning Literature review Self regulation of learning in healthcare curricula SRL is considered a core competence of healthcare pro- fessionals and one that is essential to the safeguarding of patient care [16]. In the health professions, we expect practitioners and trainees to engage in self-regulation of their learning and practice [17]. For example, medical professionals have to ensure high standards in the provision of patient care in the context of a rapidly and constantly changing medical world. Doctors are respon- sible for diagnosing their own learning needs and pursu- ing professional development opportunities; medical residents are expected to identify their knowledge gaps and to seek help from supervisors when they need it [11]. To this end, they have to continuously develop their competencies, define their own learning needs, set personal goals, and engage in the most appropriate learning activities. It means, in essence, that they have to be self-regulated learners [18]. SRL refers to students’ ability to understand and control their learning [19], to the degree to which individuals become metacognitively, motivationally, and behaviorally active participants in their own learning processes [20]. Metacognitive control is the regulation of skills for planning, monitoring, and modifying cognition, which refers to the awareness and control of one’s thought processes. Self-regulated learners use cognitive strategies designed to increase encoding, understanding, and/or retention of learning, to pursue academic goals and regulatory strategies that enable them to self- monitor and control their own learning [21]. Motiv- ational control is the regulation of beliefs and attitudes, self-efficacy, perceptions of task difficulty, task value beliefs, and personal interest in the task [22]. This raises the call to develop curricula that can sup- port healthcare students in developing their SRL, thereby helping them to be proactive, behaviorally, metacogni- tively and motivationally, in their learning process. In view of the importance of this issue and the need to assimilate the development of these abilities among medical professionals and students in the medical field, and in view of the scant research conducted in this field, the present study focuses on promoting SRL. Despite the importance of SRL for students, a system- atic search for relevant publications describing how undergraduate and/or postgraduate medical students regulate their learning points to merely a scant theoret- ical framework [3]. © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 15 Alt and Naamati-Schneider BMC Medical Education (2021) 21:110 Page 2 of 15 Alt and Naamati-Schneider BMC Medical Education (2021) 21:110 Introduction these associations may enhance our understanding of how to effectively use CM for PBL in online courses so as to develop lifelong learning skills for medical personnel and medical managers at an early stage of their training. These abilities will enable medical profes- sionals to better contend with a variety of needs in the digital age and with the changes in, and dynamics of, technological and medical knowledge. The twenty-first century has undoubtedly brought about numerous and profound changes, developments, and ad- vances in complex technologies and in the control they exert over many spheres of our lives, alongside rapid ac- cumulation of knowledge. These widespread changes have also had an impact upon the medical profession. The healthcare system and its professionals must cope with constant changes and new challenges that require adapting the system to the dynamic, ever-changing era. These challenges are linked to the ever-increasing use of advanced technology, artificial intelligence, and alter- ations in the nature and scope of the medical and nurs- ing profession [1]. These trends are occurring alongside changes such as the introduction of many new regula- tions and reforms, the lengthening lifespan, and various demographic changes. The medical field is also facing market conditions characterized by decreasing and lim- ited resources together with increasing competition among multiple entities, including the doctors them- selves [2, 3]. Accordingly, a set of qualifications is cur- rently required of managers and staff in the healthcare system such as flexibility, adaptability, planning ability, and self-regulation of learning (SRL). These capabilities are becoming an essential and inseparable part of the array of tasks that characterize doctors in the twenty- first century [4, 5]. Based on this notion, cultivating these critical lifelong learning thinking skills has become the focus of modern-day medical education [6–10]. The objective is to train medical managers and doctors to adapt themselves to the new era and its corresponding demands. SRL is deemed essential among these learning skills throughout life for doctors and healthcare workers. It enables flexibility and adjustment to dynamic changes in the health market that are similar throughout the world [11, 12]. Literature review CM is a method of learn- ing and an educational tool using diagrammatic interre- lationships between concepts representing subject knowledge. CMs should not simply list information from text randomly but rather should depict the structure of knowledge in propositional statements that illustrate the interrelationships between the concepts in a map [25]. Many studies on CM have focused on health educa- tion, arguing that in today’s challenging and highly complex healthcare settings, students must be able to think critically [43, 44]. The current traditional and rote methods of learning are inefficient in eliciting critical thinking skills among the students; therefore, educators must find a different teaching method to encourage students to make the analytical thinking process part of their daily practice. Educators need to adopt instruc- tional strategies to equip students with knowledge in critical thinking, creative problem solving and collabor- ation [45]. The process enables one to organize and structure information and the interrelationships between them within a particular domain. This may be done in a wholly graphical manner (i.e., using images, photos, colors etc.) to highlight differing concepts and their linkages, or by identifying key concepts by their names or titles and enclosing them in visual boxes [26]. CMs may aid the instructor in assessing what students under- stand and how they relate the material to the overall course goals. CMs are easily taught and can be incorpo- rated in introductory units, midterm reviews and assess- ments, peer assessment, self-assessment, or end-of-course reviews and assessments [27]. Through CM, students should be able to transfer ap- plied didactic objectives from the classroom to their clinical practice, where critical thinking and problem- solving skills are vital for success. This premise was rein- forced by several studies that empirically showed the benefits of CM used with PBL in facilitating students’ twenty-first century skills. For example, Joshi and Vyas [44] maintain that CM should be used in PBL to solve epidemiological problems in community medicine which deals with public health concepts, mathematical calcula- tions, and “applied” interpretations. In their study, they compared two groups of students: the research group was informed on how to make and use CMs out of taught contents, while the control group was taught the same contents in the conventional way. Performance of both groups was assessed by two identical exams. Literature review These reviews mainly indicate that students often struggle to regulate their learning in clinical learning environments as a result of the unpre- dictable, dynamic and disorganized nature of clinical workplace settings [11]. Therefore, healthcare curricula are increasingly called upon to support self-regulation as a central learning outcome. This raises the call to This study sought to design and assess a problem- based learning (PBL) intervention activity with digital concept mapping (CM) in an online course and to evaluate the set of connections between this process and Health Management students’ SRL [13–15]. More specifically, we were interested in evaluating the impact of the decision-making process required in a PBL activ- ity on the manner in which students construe their au- tonomy in an online learning environment. Illuminating Alt and Naamati-Schneider BMC Medical Education (2021) 21:110 Alt and Naamati-Schneider BMC Medical Education (2021) 21:110 Page 3 of 15 Among these multifaceted benefits, critical thinking and problem solving are identified as key skills essential for learners in the twenty-first century [39]. Proficiency in critical thinking is a skill required not only in higher education settings but also in the workplace and in per- sonal, social, and civic life [40]. Students need to know how to frame, analyze, and synthesize information to solve problems and answer questions critically. However, researchers [41, 42] maintain that question posing is challenging for many students, and without appropriate scaffolding it may lead to a heavy cognitive load. Their studies show how concept mapping activities with desig- nated guidance can positively affect learning achieve- ment and question-posing performance and help guide students to learn in a more effective way. develop curricula that might support healthcare students in developing SRL, by designing constructivist-based learning environments, thus helping students to be be- haviorally, metacognitively and motivationally proactive in their learning process. The learning environment sug- gested in the present study is an online PBL supported by CM. The following sections provide a comprehensive definition of this learning environment and show its increased importance as recorded in the literature for advancing SRL in health education. Concept maps for constructivist problem-based learning Concept maps (CMs) [23] have been applied in educa- tion systems for more than 30 years [24]. However, their application to PBL has been less commonly utilized in higher education, and minimal attention has been paid to their use in online learning. Literature review The study group consistently scored higher on the exams and provided largely positive feedback on the utility of CMs in memorizing, confidence-boosting, and under- standing the topic. Similarly, CM’s effectiveness in aca- demic performance in problem-solving as well as in declarative knowledge questions and their perceptions regarding CM was examined among medical students [46]. These researchers found that CM improved academic performance in problem solving but not in declarative knowledge questions. Students’ perception of the effectiveness of CM was positive. Machado and Carvalho [28] have recently reviewed the benefits and challenges of CM in higher education. Based on their findings, CM promotes critical thinking [29]; has a positive effect on exam scores [30]; allows the integra- tion of theory with practice, and links new knowledge with existing knowledge [31]; promotes learning progress and assessment [32]; enhances collaborative learning [33]; in- creases technology inclusion [34]; enables meaningful learning [35]; and is considered an educational tool to facilitate learning and studying [36]. For example, in a re- cent study Chen and Hwang (2020) showed the benefits of concept mapping to English as a foreign language (EFL) learners’ critical thinking awareness and learning perform- ance [37]. In the context of health and clinical education, recent studies showed the advantages of CM to nursing students’ clinical reasoning abilities [38]; motivation for critical thinking skills [6]; critical thinking skills [7, 10]; active learning and connections to clinical concepts [8]. Page 4 of 15 Page 4 of 15 Alt and Naamati-Schneider BMC Medical Education (2021) 21:110 A qualitative study with 20 undergraduate nursing stu- dents sought to identify whether the use of CM can help students extend and revise their expertise in oncology and analyze the abilities they developed in order to con- vert theoretical knowledge into practical knowledge [44]. The resource of graphics and the clinical case data ar- ranged in a mapping form generated an active search and an exercise in self-learning in oncology. Despite the challenges inherent in the use of CM, the results sug- gested an increase in autonomy and clinical reasoning in nursing practice. Another study in nursing education, conducted by Kaddoura et al. [47], explored how stu- dents perceived the effect of a CM on the development of clinical judgment skills. The students created CMs, which were later evaluated both by them and the clinical instructors. Students also completed a clinical evaluation questionnaire at the end of the course. Concept mapping and self-regulation of learning p pp g g g In recent years, researchers have called on higher- education teachers not only to provide students with op- portunities to construct concepts but also to utilize it to foster their ability to self-regulate their learning pro- cesses [14–16]. However, while CMs have been shown to be effective tools for facilitating students’ critical thinking, the benefit of CMs in facilitating their self- regulation abilities in face-to-face or online courses has been underexplored [13]. However, since CM activity requires high mental effort while making meaning, it is equally plausible to posit SRL as an independent variable. Lim et al. [51] suggest that those who possess weak SRL skills are probably less able to direct their own learning, and therefore may gain less benefit from CM strategy. Since learners process in- formation differently, strategies such as CM might not work equally well for all learners [52]. p Most of the research investigating the relationship be- tween CM and SRL skills has considered self-regulation as an outcome variable. As indicated by Novak [49], when students gained skill and experience in construct- ing CMs, they began to report that they were learning how to learn. For example, Chularut and DeBacker [14] maintain that CM is designed to support students in self-regulating their learning. Strategies such as CM can help students attend to tasks, focus on important features, organize material, and maintain a productive psychological climate for learning. While working on CMs, students have very concrete evidence of whether and how well their CM is developing in the direction of their goal. Upon self-observation, they evaluate their CMs with regard to the standards and/or the goals they hold for themselves. Following self-observation and self- judgment, the students experience either satisfaction or dissatisfaction with regard to their progress or their Drawing on the motivational control aspect of SRL, several studies have shown how self-regulated students are more receptive to adopting CM strategies. For example, Schaal [53]argues that self-efficacy might mod- erate the application of a meta-cognitive technique like CM. Consequently, students who believe they are cap- able of using CMs are more likely to participate in this activity. Similarly, Sun and Chen [54] showed how the use of CMs significantly increased elementary school students’ self-efficacy when their initial self-efficacy was already high to begin with. Literature review A descriptive data analysis was performed after the course was completed. The findings revealed that the use of CM provided an interactive way to foster clinical judgment skills in nursing students. In a similar vein, Hsu et al. [48] showed how outcome-based CM can be used as an efficient educational method that encourages nursing students to take a bio-psycho-social approach to medi- cine, which might ultimately lead to improved results. completed map and may react by seeking further infor- mation or assistance. Satisfaction with their learning progress is likely to motivate students to use CM as a learning strategy in other settings. Similarly, Naderifar [15] investigated the effects of CM on Iranian-born English as a Foreign Language (EFL) learners’ self-regulation. One group was given CMs, an- other group received a notebook, the last group served as the control group. The results of the analysis revealed that both CM and notebook keeping significantly en- hanced SRL in vocabulary learning among the learners. Other researchers (e.g., Roy [16]) also revealed that stu- dents in an EFL context gained higher self-regulation as the result of CM strategy teaching. Though limited to EFL studies, these findings have implications for both pedagogy and research. These findings commonly indi- cate that CM increases readers’ autonomy, information comprehension, retention, and recall and additionally promotes uniquely individual performance. Siebene et al. (2020) [9] investigated how concept mapping may support the quality of reflections made by undergraduate medical students. Reflection is viewed as a self-regulatory skill. Learners who are encouraged to evaluate their work through reflection can develop indi- vidual strategies that support their learning [50]. To nur- ture medical students’ reflective writing skills, concept mapping was used by Sieben et al., 2020 [9] as a format for reflection, supporting students to freely shape their thoughts. Concept mapping was detected as a useful tool to teach learners the basics of effective reflection. Alt and Naamati-Schneider BMC Medical Education (2021) 21:110 Participants Data for the analysis were gathered from 100 Israeli undergraduate students of a Management of Health Service Organizations program (covering patient-doctor relations, quality of service in the healthcare system, and ethics and patient rights). The program instructs students regarding the fundamentals in fields such as marketing, finance, organizational behavior, communications, legal issues and strategies. The students were enrolled in a 3rd- year course entitled ‘Assimilation of service quality in health systems’. The mean age of the participants was 25.40 years (SD = 6.76), and 86% were females. The distri- bution regarding ethnicity was: 51% Jewish students; 49% Arab (Muslim and Christian) minority students. This study The literature review indicates that CMs are still under- utilized in health education and that little is known about how higher education students’ SRL might be linked to the way in which they use CMs for effective problem-based online courses. To address this utilization gap, the overarching aims of this study were to design a PBL intervention (see description below) that employs CM with a designated decision-making process (CM for PBL) and to evaluate the set of connections between this intervention and the SRL of Management of Health Service Organizations program students. The intervention was piloted for three months (one semester) in one central Israeli academic college. The following re- search questions and hypotheses were checked: Data were gathered twice, after each phase of the intervention, as described in the following section. Prior to obtaining participants’ consent (informed consent was obtained from all participants involved in the study), it was explained that the questionnaires were anonymous and that it was acceptable should they choose to return a partially completed questionnaire. Finally, participants were assured that no specific identifying information would be processed. The study was preauthorized by the college’s Ethics Committee and in accordance with the relevant guidelines and regulation. Concept mapping and self-regulation of learning They suggest that this type of teaching/learning strategy is more suitable for improving these students’ self-efficacy than that of students with initially low self-efficacy beliefs. For learners who believe in their ability to acquire knowledge, they suggest that teachers adopt dynamic CMs and integrate them in their Alt and Naamati-Schneider BMC Medical Education (2021) 21:110 Page 5 of 15 Page 5 of 15 (Q2) The second research question was: how will CM for PBL impact students’ SRL in an online course? Given the positive effect on SRL attributed to CM [15], it was hypothesized that the activity would enhance students’ SRL (H2). An additional hypothesis was that students’ SRL will be found higher after the intervention (Phase 2) than before the intervention (following Phase 1) (H3). educational materials and questions designed to pro- mote learners’ thinking and enhance their learning achievement. Other researchers, such as Nuuyoma and Fillipus (2020) [55] showed that CM strategies are challenging for students with poor self-regulated learning skills such as time management. The researchers used concept mapping to facilitate learning among nursing students in the human physiology course. Their qualitative study aimed to describe the students’ perceptions and experi- ences while using concept mapping as a learning tool. Focus group discussions were analyzed yielding four themes: concept mapping facilitates deep learning; concept mapping as a group activity; effects of concept mapping on students’ academic performance; and impli- cations of concept mapping for learning resources. Students had positive experiences and perceptions of concept mapping as a learning tool, however, they felt that this tool is time consuming and necessitates many learning resources. Therefore, the authors concluded that students should be guided on time-management strategies, an important aspect of SRL, to encourage the adoption of CM strategies. g Additional variables, such as gender and age, were addressed to examine and control for their potential effect on the research constructs. The accreditation dilemma accreditation. However, the main problem stems from the cost involved in inspections, particularly those that require extra manpower. When the Ministry of Health established accreditation as an obligatory standard, the hospitals did not receive any supplementary budgets and were forced to pay most of the expenditure from their own pockets. Continuing the existing situation undoubt- edly poses a challenging dilemma for the Ministry of Health and the hospitals with regard to the hospitals’ ob- ligation to implement this process and ultimately attain approval. Accreditation is a process by which an independent external entity evaluates organizations that provide healthcare services to determine whether they meet the set of standards and demands that are aimed at improv- ing the quality of treatment. To date, there has been only scant research that supports the benefits of the accreditation process, although various studies do exist that point to the contribution of the accreditation process to promoting quality in healthcare organizations in general, and in hospitals in particular. Yet the process has numerous disadvantages such as a lack of manpower and inadequate funding. The increasingly strict quality control inspections, which have existed in Israel for more than a decade, became an obligatory condition for renewing hospital licenses in July of 2015. There is no doubt that the accreditation has made a huge contribu- tion to hospitals. The competitive spirit that arose and the desire to obtain an international stamp of approval spurred hospitals to implement structural and cultural changes. It additionally served as an incentive for imple- menting basic work processes, most of which occurred in the fields of standardization, order, organization, and management. In practice, accreditation compelled the hospitals to undergo an essential reorganization, from procuring resuscitation equipment and defining jobs to implementing rules and regulations. Additional parame- ters were added to accreditation from year to year, and the cumulative burden forced the nurses to demand that it be eliminated altogether. They claimed that it hin- dered their work to the point at which they could no longer function properly. There is no debate in the pro- fession as to the necessity of quality control and improv- ing medical processes and procedures in the hospitals. The report of quality indexes of the Ministry of Health provides strong evidence of the positive impact of The students in this research were asked to argue for or against the implementation of the accreditation process within hospitals. The intervention G d d (Q1) The first research question was: how might stu- dents’ SRL be related to their perception of CM for PBL in an online course? Based on a literature review [51], the level of SRL skills might be suggested as an inde- pendent variable. High self-regulated students were expected to be more receptive to adopting CM and to demonstrate a predilection to utilize this learning strat- egy, which offers ways to effectively demonstrate their solutions to an ill-structured problem compared to low self-regulated learners. Therefore, it was postulated that health management students’ SRL will be positively linked to their perceptions of CM for PBL in an online course (H1). This hypothesis was checked follow- ing Phase 1 of the intervention. Grounded in a number of theories identified with the constructivist approach to learning such as cognitive schemes, moral development theory, dilemma discus- sion, moral reasoning schemes, radical theory, and social-cultural constructivist theories, the approach to learning known as Values and Knowledge Education (VaKE) [56] was designed and piloted in this study. VaKE is deemed a useful teaching tool that combines morality and values-centered education with knowledge education, with an emphasis on social behavior and the development of critical thinking in a PBL environment. In line with VaKE, the students were presented with the following dilemma, relevant to their course content. Page 6 of 15 Page 6 of 15 Alt and Naamati-Schneider BMC Medical Education (2021) 21:110 The accreditation dilemma The task had two phases. In Phase 1, the participants were asked to detail five argu- ments to establish their decision by using a CM. Group work was allowed, although individual work was prefera- ble. In Phase 2, relying on the materials taught in their courses, the students were asked to obtain the necessary supporting information to substantiate their arguments and to associate the ethical values at stake with at least two of the arguments they had provided. Finally, the participants were instructed to specify and explain the differences or similarities between their respective arguments. Mindomo, a popular platform for designing CMs, was utilized. To facilitate assessment of their maps, well-established criteria were provided to the students, as illustrated in Table 1. This assessment tool was adapted from Panadero et al. [57] to address the current study’s goals. Figure 1 displays a map that illustrates the arguments in favor of assimilating an accreditation process in hos- pitals together with their textual support. An example of a basic argument is improving safety and risk manage- ment; for example, an increase in client satisfaction, a decrease in the number of infections/contaminations, Table 1 Rubric for assessing the concept map (CM) Criteria / Score 4 3 2 1 Arguments and supporting information All five arguments and five justifications with supporting items of information are included. Three-four arguments and justifications with supporting items of information are included. One or two arguments and justifications with supporting items of information are included. Arguments and justifications with supporting items of information are incomplete and/or incorrect. Hierarchy The organization is complete and correct. The supporting information corroborates the arguments. The organization is correct but incomplete. Most of the supporting information corroborates the arguments. The organization is correct but incomplete. Most of the supporting information does not corroborate the arguments. The organization is incomplete and/or incorrect. Relationships among arguments / supporting information Relationships were specified and well-explained. Links to ethical values were added and explained. Relationships were partly specified but explained. Links to ethical values were partly added but explained. Relationships were partly specified but not explained. Links to ethical values were partly added but not explained. Relationships were partly or not specified and poorly/not explained. Links to ethical values were incorrect or missing. Simplicity and easiness of understanding The design is simple and easy to understand. Some relationships are difficult to understand. The accreditation dilemma There is an excessive number of connections. Neither the relationships nor the hierarchy are understandable. Table 1 Rubric for assessing the concept map (CM) Criteria / Score 4 3 2 1 Arguments and supporting information All five arguments and five justifications with supporting items of information are included. Three-four arguments and justifications with supporting items of information are included. One or two arguments and justifications with supporting items of information are included. Arguments and justifications with supporting items of information are incomplete and/or incorrect. Hierarchy The organization is complete and correct. The supporting information corroborates the arguments. The organization is correct but incomplete. Most of the supporting information corroborates the arguments. The organization is correct but incomplete. Most of the supporting information does not corroborate the arguments. The organization is incomplete and/or incorrect. Relationships among arguments / supporting information Relationships were specified and well-explained. Links to ethical values were added and explained. Relationships were partly specified but explained. Links to ethical values were partly added but explained. Relationships were partly specified but not explained. Links to ethical values were partly added but not explained. Relationships were partly or not specified and poorly/not explained. Links to ethical values were incorrect or missing. Simplicity and easiness of understanding The design is simple and easy to understand. Some relationships are difficult to understand. There is an excessive number of connections. Neither the relationships nor the hierarchy are understandable. Table 1 Rubric for assessing the concept map (CM) One or two arguments and justifications with supporting items of information are included. Some relationships are difficult to understand. There is an excessive number of connections. Alt and Naamati-Schneider BMC Medical Education (2021) 21:110 Page 7 of 15 Fig. 1 A concept map detailing the arguments in favor of implementing a hospital accreditation process Fig. 1 A concept map detailing the arguments in favor of implementing a hospital accreditation process data collected. Higher scores on this scale indicate better self-regulation in online learning by students. The items were scored on a six-point Likert scale ranging from 1 = strongly disagree to 6 = strongly agree. Measurements S d h Table 2 Concept Mapping for PBL scale item description 1 Concept mapping helped me learn about the topic. 2 Concept mapping helped me identify the interrelationships among arguments. 3 Concept mapping helped me specify interrelationships among arguments. 4 Concept mapping stimulated me to learn and think independently. 5 Concept mapping helped me to reduce barriers when dealing with decision-making. 6 Concept mapping enhanced my interest in decision-making. 7 I think concept mapping can be easily used in other decision-making discussions. 8 I will consider using concept mapping in other complex decision-making processes. 9 I will consider using concept mapping to make decisions in the future. 10 I was satisfied using concept mapping in making decisions. 11 I liked using concept mapping to assist me in making decisions. 12 I enjoyed using concept mapping during the decision-making process. Table 2 Concept Mapping for PBL scale item description 1 Concept mapping helped me learn about the topic. 2 Concept mapping helped me identify the interrelationships among arguments. 3 Concept mapping helped me specify interrelationships among arguments. 4 Concept mapping stimulated me to learn and think independently. 5 Concept mapping helped me to reduce barriers when dealing with decision-making. 6 Concept mapping enhanced my interest in decision-making. 7 I think concept mapping can be easily used in other decision-making discussions. 8 I will consider using concept mapping in other complex decision-making processes. 9 I will consider using concept mapping to make decisions in the future. 10 I was satisfied using concept mapping in making decisions. 11 I liked using concept mapping to assist me in making decisions. 12 I enjoyed using concept mapping during the decision-making process. The accreditation dilemma The OSLQ con- sists of six subscale constructs including: environment structuring (‘I find a comfortable place to study’); goal setting (‘I set standards for my assignments in online courses’); time management (‘I allocate extra studying time for my online courses because I know they are time-demanding’); help-seeking (‘I find someone who is knowledgeable in course content so that I can consult with him or her when I need help’); task strategies (‘I read aloud instructional materials posted online to cope with distractions’); and self-evaluation (‘I communicate and fewer legal claims. These are accompanied by sup- porting evidence using a variety of formats such as films, figures, tables, excerpts from academic articles, and arti- cles from the local press. The full sources are added as weblinks next to each justification. The connections be- tween the various arguments are marked on the map and accompanied by a written explanation. In the ex- ample shown here, formulating safety improvements and risk management will strengthen the argument that deals with improving the hospital’s reputation. The eth- ical values that were attributed to each argument are also explained; for example, preserving the value of hu- man life by preventing hospital-acquired infection, or preserving human dignity by protecting medical confi- dentiality and, above all, the health of the patients. Student characteristics Data were gathered using a questionnaire aimed at ascertaining the student’s gender, age, and ethnicity. 3 Concept mapping for problem-based learning scale (CM for PBL) A 12-item questionnaire was designed based upon the theoretical framework of CM and VaKE and the authors’ expertise in the field of moral decision-making process in PBL ([58];see Table 2). The questionnaire was aimed at assessing students’ perceptions of the effectiveness of using CMs in the decision-making process required in VaKE. The participants were asked to indicate their level of agreement with each of the statements shown in Table 2. The items were scored on a six-point Likert scale ranging from 1 = strongly disagree to 6 = strongly agree. The questionnaire was given twice, following the first phase (α = .97), and following the second phase (α = .97). The online self-regulated learning questionnaire (OSLQ) The online self-regulated learning questionnaire (OSLQ) This 24-item scale was developed by Barnard et al. [13] from an 86-item pool and then examined for its internal consistency and exploratory factor analysis results for Page 8 of 15 Page 8 of 15 Alt and Naamati-Schneider BMC Medical Education (2021) 21:110 are shown as arrows. In PLS-SEM, single-headed arrows, as shown between the constructs, are considered pre- dictive relationships and, with strong theoretical support, can be construed as causal relationships. Paths were specified from Online SRL the CM for PBL-dependent factor. The PLS-SEM analysis used a path-weighting scheme and a mean value replacement for missing values. It should be noted that based on a previously conducted analysis, background variables were also en- tered into the model to control their effect on the latent variables (age, gender, and ethnicity). The Ethnicity vari- able (majority = 1, minority = 2) was found significantly connected to the model’s constructs and, therefore, was included in the model. with my classmates to find out how I am doing in my online classes’). A principal axis factoring analysis followed by a vari- max rotation was used to corroborate the stability of the scale structure (eigenvalue > 1.00; item loadings > .30). The analysis solution accounted for 58.45% of the vari- ance and yielded five categories: Goal setting (items: 1–5 α = .82); Environment structuring (items: 6–8 α = .81); Task strategies (items: 12, 13 α = .87); Time management (items: 14–16 α = .87); and Peer support (items: 17, 19, 23, 24 α = .80). The last category included items from two original factors, self-evaluation and help seeking. This scale was given to the participants twice, after each phase (1 and 2). Structural validity was established by using the same procedure. The analysis solution accounted for 59.46% of the variance. Five factors were found, with sufficient internal reliability results ranging from α = 0.66 for the 2-item Task strategies factor to α = 0.87 for the 3-item Time management factor. To test the direct effects, we ran the bootstrap routine. Bootstrapping makes no assumptions about the shape of the variables’ distribution or the sampling distribution and can be applied to small sample sizes [59]. As can be learned from Model 1, the Online SRL was found posi- tively connected to the CM for PBL factor (β = .507 p < .001). Data analysis Data were analyzed using Partial Least Squares - Struc- tural Equation Modeling (PLS-SEM) [59],which is rec- ommended to be applied provided that the primary objective of employing structural equation modeling is the prediction of target constructs. SmartPLS 3 software was used. Figure 2 illustrates the research design. The online self-regulated learning questionnaire (OSLQ) Regarding cultural differences, minority students were more reluctant to embrace CM for PBL (β = −.260 p < .001) and had a high level of Online SRL compared to the majority students (β = .269 p < .05). Therefore, the first hypothesis was confirmed. Evaluation of model 1 The coefficient of determination (R2) value was exam- ined, whereby R2 values of 0.75, 0.50, or 0.25 for en- dogenous latent variables can be respectively described as substantial, moderate, or weak [59]. R2 for CM for PBL was found weak (0.254). In addition to measuring the R2 values, the change in the R2 value when omitting a specified exogenous construct from the model was used to evaluate its impact on the endogenous con- structs. This measure is referred to as the f2 effect size whereby values of 0.02, 0.15, and 0.35, respectively, rep- resent small, medium, and large effects [60]. f2 effect size result was 0.320 for Online SRL – CM for PBL, while f2 effect size results for the background variable were very low (0.078 and 0.084). Finally, the blindfolding Second hypothesis In H2 it was expected that the activity of CM for PBL will enhance students’ SRL (H2). To check this hypoth- esis, Model 3 (Fig. 5) was designed to include data gath- ered in Phase 2 (following the intervention). As can be seen in Fig. 5, the model includes the CM for PBL inde- pendent factor and the Online SRL dependent factor with four sub-factors (the Peer support sub-factor was omitted from the analysis due to a low loading result < 0.40; see Hair et al. [59]). Background variables, includ- ing Ethnicity were found non-significantly connected to the model’s constructs and were therefore excluded from the model. According to the bootstrap routine, the CM for PBL factor positively contributed to students’ Online SRL (β = .525 p < .001). Regarding model evaluation re- sults, R2 for Online SRL was found to be moderate (0.276). The Q2 value for Online SRL was 0.162. There- fore, the second hypothesis was confirmed. To evaluate the value of the Online SRL sub-factors in predicting students’ perceptions of the effectiveness of using CM in the decision-making process required in the VaKE, Model 2 (Fig. 4) was designed. The model in- cludes the same latent constructs as those in Model 1; however, this time the Online SRL sub-factors were entered into the model as five independent variables. As shown in Model 2, only two sub-factors were found significantly connected to CM for PBL: Goal setting (β = .325 p < .01) and Task strategies (β = .305 p < .01). Non-significant connections were shown between other Online SRL sub-factors and the dependent variable. First hypothesis In H1 it was postulated that students’ SRL will be posi- tively linked to their perceptions of CM for PBL in an online learning environment. To test this hypothesis, Model 1 (Fig. 3) was designed using data gathered in Phase 1. The model includes two main latent constructs: on the left, the Online SRL factor with its five sub- factors and, on the right, the perception of CM for PBL factor, accompanied by its 12 indicators. The indicators are the directly measured proxy variables, represented as rectangles. Relationships between the constructs as well as between the constructs and their assigned indicators Fig. 2 Research design Alt and Naamati-Schneider BMC Medical Education (2021) 21:110 Page 9 of 15 Fig. 3 Model 1. Analysis results of the examination of H1 by SmartPLS Fig. 3 Model 1. Analysis results of the examination of H1 by SmartPLS shown for the other independent sub-factors. Finally, the Q2 value for CM for PBL was 0.238. procedure was used to assess the predictive relevance (Q2) of the path model. Values larger than 0 suggest that the model has predictive relevance for a certain en- dogenous construct [59]. The Q2 value of CM for PBL in the present study was 0.183. Evaluation of model 2 The model evaluation included several steps. First, col- linearity was examined by the Variance Inflation Factor (VIF) values of all sets of predictive constructs in the structural model. The results showed that the VIF values of all combinations of endogenous and exogenous con- structs are below the threshold of 5 [59], ranging from 1.00 to 2.19. Therefore, collinearity among the predictive constructs is not a critical issue in this structural model. R2 for CM for PBL was found to be moderate (0.329). According to the f2 effect size results, a medium effect (0.071) was exerted by Goal setting, and Task strategies (0.071) on CM for PBL whereas smaller effects were To assess the contribution of CM for PBL to Online SRL sub-factors, Model 4 (Fig. 6) was designed. The model includes the same latent constructs as in Model 3; however, this time the Online SRL sub-factors were entered into the model as four independent variables. As shown in Fig. 6, CM for PBL was found significantly linked to all of the Online SRL sub-factors: Goal setting (β = .438 p < .001), Environment structuring (β = .488 p < .001), Task strategies (β = .366 p < .001), and Time management (β = .450 p < .001). Regarding model evalu- ation results, a collinearity examination yielded sufficient Alt and Naamati-Schneider BMC Medical Education (2021) 21:110 Page 10 of 15 Fig. 4 Model 2. Further analysis results of the examination of H1 by SmartPLS Fig. 4 Model 2. Further analysis results of the examination of H1 by SmartPLS results (i.e., VIF values were equal to 1.00). As indicated in Model 4, the highest R2 result was indicated for Envir- onment structuring (R2 = 0.238), and the lowest for Task strategies (R2 = 0.134). The Q2 values ranged from 0.087 to 0.150. CM. Next, it illustrates the effectiveness of the CM for PBL strategy for students’ SRL in an online course. As postulated, at the beginning of the process, students’ SRL was found positively linked to the CM for PBL strategy. However, only two SRL sub-factors exerted a positive effect on the learning strategy: Goal setting and Task strategies. Evaluation of model 2 It may be inferred that students who were accustomed to setting standards for their assign- ments in online courses, set short-term goals as well as long-term goals, kept a high standard for their learning in online courses and for the quality of their work, and undertook extra problems in their online courses to master the content, were found more receptive to adopt- ing CM for PBL online course and demonstrated a pre- dilection to utilize this learning strategy. This can be corroborated by previous researchers who claim that self-regulated learners are individuals who are already assessing their own performance to varying degrees, while generating their own self-feedback actively and consciously controlling their own learning from cogni- tive, affective, motivational, and behavioral points of view [61]. In view of its findings, the present study adds to the corpus of knowledge by pointing to a specific Third hypothesis Finally, an additional analysis was conducted to detect possible contribution of CM for PBL to Online SRL sub- factors. A repeated-measures analysis yielded a significant result (F(188, 6) = 3.656, p < .01, η2 = .104) in students’ On- line SRL between Phase 1 and Phase 2. SRL Time man- agement sub-factor (F(188, 1) = 4.811, p < .05, η2 = .024), was found significantly higher after the intervention (Phase 2) relative to its beginning (Phase 1). Table 3 presents the mean scores, SD, F values, and Eta-squared statistics (η2) of Online SRL (Phase 1 and 2). Discussion Further analysis results of the examination of H2 by SmartPLS Alt and Naamati-Schneider BMC Medical Education (2021) 21:110 Page 12 of 15 Page 12 of 15 Table 3 Mean scores, SD, F values, and Eta-squared statistics (η2) of Phase 1 and 2 Factors Phase 1 Phase 2 M SD M SD F η2 Online SRL Goal setting 4.54 0.90 4.59 0.88 0.175 .676 Environment structuring 4.58 1.04 4.31 1.06 3.148 .078 Task strategies 3.37 1.34 3.71 1.31 3.310 .070 Time management 4.02 1.18 4.38 1.14 4.811* .029 Communication with classmates 3.60 1.19 3.63 1.23 0.025 .874 p < .05 * Table 3 Mean scores, SD, F values, and Eta-squared statistics (η2) of Phase 1 and 2 for example, the self-determination theory of motivation that aims to explain individuals’ goal-directed behavior [68]. Second, gathering qualitative data alongside quanti- tative measures might be a useful way of assessing and understanding the effectiveness of CM. Combining CM with a reflective journal might be a useful way to gather qualitative data [69] and increase students’ SRL [70]. Third, although entered into the model merely to con- trol its effect on the research constructs, the Ethnicity factor should be further addressed in future studies. It seems that minority students were initially more reluc- tant to embrace CM for PBL despite their higher level of Online SRL, whereas this discrepancy was resolved by the end of the activity. CM facilitated the undertaking of suitable actions to offset the diversity represented by immigrant or minority students [71]; hence, further re- search should be done to investigate how CM can be used as an efficient instructional scaffold to support di- verse students’ learning processes. Finally, this study was conducted in a single course and was limited to Manage- ment of Health Service Organizations program students in Israel; therefore, the results cannot necessarily be gen- eralized to students of other countries and study tracks. Cross-cultural validation of the results is needed to sub- stantiate the findings as well as to validate the factorial structure and the relationship among factors. their time purposefully in order to achieve important educational goals within a given period of time [63]. Time management is characterized as a multidimen- sional process through which students deliberately con- trol the way they engage in academic work; hence it has been portrayed as a manifestation of students’ regulation of their overt behavior [64]. Discussion It may be inferred that the use of such a self-regulatory strategy involves a level of engagement that is often more demanding for students in terms of time and effort than their normal level of en- gagement [65]. In order for them to invest the extra time and effort, the learning environment must be designed in a way that motivates them to allocate time to success- fully accomplish the task and encourages and scaffolds students’ effective use of SRL strategies. Based on this study, the CM for PBL strategy can be suggested as an effective online strategy that motivates students to regu- late their behavior. their time purposefully in order to achieve important educational goals within a given period of time [63]. Time management is characterized as a multidimen- sional process through which students deliberately con- trol the way they engage in academic work; hence it has been portrayed as a manifestation of students’ regulation of their overt behavior [64]. It may be inferred that the use of such a self-regulatory strategy involves a level of engagement that is often more demanding for students in terms of time and effort than their normal level of en- gagement [65]. In order for them to invest the extra time and effort, the learning environment must be designed in a way that motivates them to allocate time to success- fully accomplish the task and encourages and scaffolds students’ effective use of SRL strategies. Based on this study, the CM for PBL strategy can be suggested as an effective online strategy that motivates students to regu- late their behavior. Conclusions and implications This study set out to fill the lacuna of information and practical designated training in the fields of medicine and medical management with respect to the wide var- iety of personal and interpersonal qualifications that are necessary for adaptation to the twenty-first century and its various challenges. The perception that the objective of academia and institutions for medical education is to remain relevant to the demands of the twenty-first cen- tury calls for them to adapt themselves to the changing needs of the labor market and the health system. Conse- quently, their actions and learning must be adapted to the needs of the profession in the present and the future and to the everchanging challenges which are yet un- known. As part of this process, we must place an emphasis on the students in the system and train them by developing the necessary qualifications in early stages of their studies in medical schools, nursing schools, and medical management schools, as well as in the stages of advanced learning and professional training. It is thus important to note that our results might be connected to the way the activity was designed in this research. The students were given predefined criteria that allowed them to self-assess their maps. It is plaus- ible to assume that because the activity included this as- sessment tool, aimed at helping students improve their abilities through progressive goal setting, the students found it valuable and used it as scaffolding to enhance their learning skills. Arguably, strategies of self-evaluation and monitoring are considered vital for students‘SRL [66]. Such strategies, which increase student’s capacity to judge their own performance and results, are considered crucial for cultivating self-regulation [67]. Discussion This study mainly shows how students with different SRL in online courses might tend to differently utilize a learning strategy that offers ways to effectively demon- strate their solutions to an ill-structured problem using Alt and Naamati-Schneider BMC Medical Education (2021) 21:110 Page 11 of 15 Fig. 5 Model 3. Analysis results of the examination of H2 by SmartPLS Fig. 5 Model 3. Analysis results of the examination of H2 by SmartPLS learning activity – CM –revealed by self-regulated learners as being an important and useful tool that helped them take control of their learning and tackle the PBL assignment. the intervention. These findings show that the CM for PBL learning strategy motivated students to choose a quiet study venue to avoid distractions and study opti- mally for the online course, to allocate extra studying time for their task, and distribute their studying time evenly throughout the course. Another interesting finding was shown in the second analysis with data gathered following the intervention (Phase 2). The contribution of CM for PBL to Online SRL sub-factors was found to be significant. The analysis showed that the CM for PBL learning strategy increased the levels of four Online SRL sub-factors: Goal setting, Task strategies, Environment structuring, and Time management. The last two factors failed to predict the perception of the learning strategy at the beginning of Finally, increased levels of Time management were found upon completion of the intervention, as compared to its starting point. Time management represents a strategic process that promotes the accomplishment of important goals and successes within personal, profes- sional, and academic contexts [62]. Academic time man- agement describes students’ efforts to allocate the use of Fig. 6 Model 4. Further analysis results of the examination of H2 by SmartPLS Fig. 6 Model 4. References 1. Jungwirth D, Haluza D. Information and communication technology and the future of healthcare: results of a multi-scenario Delphi survey. Health Informat J. 2019;25:161–73. https://doi.org/10.1177/1460458217704256. 1. Jungwirth D, Haluza D. Information and communication technology and the future of healthcare: results of a multi-scenario Delphi survey. 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The authors declare that they have no competing interests. Received: 23 October 2020 Accepted: 3 February 2021 Received: 23 October 2020 Accepted: 3 February 2021 Availability of data and materials f 14. Chularut P, DeBacker TK. The influence of concept mapping on achievement, self-regulation, and self-efficacy in students of English as a second language. Contemp Educ Psychol. 2004;29:248–63. https://doi.org/1 0.1016/j.cedpsych.2003.09.001. Data are available upon request from Prof. Dorit Alt, email: doritalt@014.net.il Data are available also Mendeley open data repository: https://data. mendeley.com/datasets/ngy6mprc6r/draft?a=1dacd25a-91d2-47f9-83a6-6b5 801683807 15. Naderifar A. The comparative effect of concept mapping and vocabulary notebook keeping on Iranian EFL learners’ self-regulation in vocabulary learning. Cogent Educ. 2018;5(1):1491782. https://doi.org/10.1080/23311 86X.2018.1491782. Other materials can be provided by the authors on request. Consent for publication Not applicable. y y Drawing on the results of the current study, it can be concluded that although the effects of CM interventions might vary with different ability learners, CM for PBL learning strategy can further enhance students’ SRL. This study shows that for a CM-based intervention to be effective in PBL online environments, teachers need to recognize and account for different types of learners and adapt their curricula and learning environment to en- courage and scaffold students’ effective use of SRL strat- egies. For example, they can help straggling students create study schedules, thereby guiding the learning process towards goals that emphasize the successful ac- quisition of skills and knowledge. Low self-regulated learners might fail to see the advantages of CM in problem-solving activities. Well-defined assessment cri- teria that are shared in advance with the students may motivate them to invest adequate time and effort in the task. The students with insufficient SRL skills might need to be further informed of the benefits of CM for their online PBL activities. Authors’ contributions D Al C l 11. Bransen D, Govaerts MJB, Sluijsmans DMA, Driessen EW. Beyond the self: the role of co-regulation in medical students’ self-regulated learning. Med Educ. 2020;54:234–41. https://doi.org/10.1111/medu.14018. Dorit Alt: Conceptualization, data curation, methodology, writing- original draft preparation writing- reviewing and editing. Lior Naamati-Schneider: Conceptualization data curation, writing- original draft preparation writing- reviewing and editing. The authors read and approved the final manuscript. 12. Lajoie SP, Zheng J, Li S, Jarrell A, Gube M. Examining the interplay of affect and self regulation in the context of clinical reasoning. Learn Instruct. 2019. https://doi.org/10.1016/j.learninstruc.2019.101219. Limitations and directions for future studies The present work features several limitations that merit a mention and opens avenues for future research. First, this study proposes a new measurement that captures students’ CM for PBL process. The results seem to con- firm its validity with SRL in online activities used as a criterion variable. However, future studies should exam- ine how it relates to scores from an instrument designed to assess a construct it would theoretically be related to, This study presents the development of SRL among Management of Health Service Organizations program students in the context of online PBL and the use of di- lemmas and constructing a CM using designated pro- grams. Combining these teaching and learning tools together with the use of advanced technology in an on- line course that encourages active learning enables the Page 13 of 15 Page 13 of 15 Alt and Naamati-Schneider BMC Medical Education (2021) 21:110 development and acquisition of abilities of self-directed learning among students in the medical and health man- agement professions. This constitutes an additional step towards adapting the health system and its practitioners to the demands and needs of the twenty-first century. choose to return a partially completed questionnaire. Finally, participants were assured that no specific identifying information would be processed. The study was preauthorized by the Kinneret College on the Sea of Galilee college’s Ethics Committee which approved the research design, and in accordance with the relevant guidelines and regulation. 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https://openalex.org/W4375809583	http://www.cedlas.econo.unlp.edu.ar/wp/wp-content/uploads/doc_cedlas239.pdf	English	null	Educational Upgrading and Returns to Skills in Latin America: Evidence from a Supply-Demand Framework, 1990-2010	World Bank policy research working paper	2,011	cc-by	9,636	Leonardo Gasparini, Sebastián Galiani, Guillermo Cruces y Pablo Acosta Leonardo Gasparini, Sebastián Galiani, Guillermo Cruces y Pablo Acosta * This paper is an updated and modified version of the working paper “Educational Upgrading and Returns to Skills in Latin America: Evidence from a Supply-Demand Framework, 1990–2010”, World Bank Policy Research Paper 5921, 2011. The original document was a background paper for the World Bank report Skills for the 21st Century in LCR. The authors also acknowledge support from the CEDLAS- IDRC LaborAL (www.Labor-AL.org) project. The new version of this document was started as part of G. Cruces’ visiting fellowship in 2013 at Harvard’s David Rockefeller Center for Latin American Studies (DRCLAS), whose support is greatly appreciated. The authors are grateful for comments from (and conversations with) Cristian Aedo, David Autor, Verónica Amarante, Augusto De La Torre, François Bourguignon, Claudia Goldin, Michael Crawford, Francisco Ferreira, Gary Fields, John Gilles, Tim Gindling, Margaret Grosh, Lawrence Katz, Marco Manacorda, Julian Messina, Ricardo Perez Truglia, Jamele Rigolini, Ian Walker, and seminar participants at the World Bank, the OECD Development Center, Harvard’s David Rockefeller Center for Latin American Studies, LaborAL project meeting (Buenos Aires), Academia Nacional de Ciencias Económicas (ANCE-Buenos Aires) and the Banco Central (Argentina). Javier Alejo, Julián Amendolaggine, Santiago Garganta and Emmanuel Vázquez provided outstanding research assistance. ** Affiliations for authors: L. Gasparini, CEDLAS-FCE-UNLP and CONICET; G. Cruces, CEDLAS-FCE- UNLP, CONICET and IZA; S. Galiani, University of Maryland; P. Acosta, World Bank. Corresponding author: gcruces@cedlas.org 1 Introduction Income inequality dynamics in Latin America, one of the most unequal regions in the world, have been far from stable. While income dispersion significantly increased over the 1990s in most Latin American countries, the decade of 2000 was marked by a widespread fall in socioeconomic and labor disparities (López-Calva and Lustig, 2010; Gasparini et al. 2011).1 Recent data for the 2010s suggests a slowing down of the pace of that fall, and even indicates signs of stagnation and reversion in some economies (Gasparini et al. 2016). Understanding the drivers of these changing patterns is a difficult task, plagued by all sorts of methodological and data problems. In this paper, we contribute to that aim by providing evidence on patterns of wage skill premiums and labor supply by educational categories, exploiting a large database of microdata from harmonized national household surveys. Looking at wage premiums is important as the main source of inequality in household incomes (at least in the household incomes captured by surveys) is dispersion in earnings. The analysis is based on a simple but illustrative supply and demand equilibrium framework that has been applied successfully to the study of returns to labor market skills in developed countries (Katz and Murphy, 1992; Goldin and Katz, 2009) as well as in some developing economies (Montes Rojas, 2006; Acosta and Gasparini, 2007; Acosta and Montes Rojas, 2008; Galiani, 2009; Manacorda et al, 2010; Gallego, 2011, García Swartz and Gasparini, 2011). The methodology is based on an examination of changes in wage differentials and the relative supply of workers with different levels of educational attainment, from which consistent patterns in relative demand for skilled and unskilled labor can be derived. This framework is applied to a large database of microdata from harmonized Latin American national household surveys (SEDLAC database). In particular, we make use of more than 10 million individual observations in 250 surveys conducted in almost all countries in the region over the period 1991- 2013. Although there is heterogeneity across countries, we find a pattern of rather constant increase in the relative supply of skilled and semi-skilled workers over the period. Consistently, the returns to secondary education completion fell over time. However, in contrast, the returns to tertiary education display a remarkably changing pattern common to almost all economies: a significant increase in the 1990s, a strong fall in the 2000s, and a deceleration of that fall in the 2010s. Educational Upgrading and Returns to Skills in Latin America: Evidence from a Supply-Demand Framework, 1990-2010* Guillermo Cruces Pablo Acosta** Leonardo Gasparini Sebastián Galiani Abstract: This paper documents the evolution of wage differentials and the supply of workers by educational level for sixteen Latin American countries over the period 1991- 2013. We find a pattern of rather constant rise in the relative supply of skilled and semi- skilled workers over the period. Whereas the returns to secondary education fell over time, in contrast, the returns to tertiary education display a remarkable changing pattern common to almost all economies: significant increase in the 1990s, strong fall in the 2000s and a deceleration of that fall in the 2010s. We conclude that supply-side factors seem to have limited explanatory power relative to demand-side factors in accounting for changes in the wage gap between workers with tertiary education and the rest. 1 Whereas patterns have been similar in most of the developing world the size of the changes were significantly larger in Latin America (Alvaredo and Gasparini, 2015). 1 Introduction We conclude that supply-side factors seem to have limited explanatory power relative to demand-side factors in accounting for changes in the wage skill premium. The rest of the document is organized as follows. The next section explores the motivations for conducting this analysis, documenting briefly the secular expansion in 2 education in the region and trends in income inequality over the last two decades. Section 3 describes the basic supply and demand framework to account for changes in wage skill premium and the relative supply of workers with different skill levels, whereas section 4 presents and discusses the main results. Section 5 examines the role of supply and proxies for demand factors in the evolution of regional aggregates by means of regression analysis. Section 6 ends with some brief concluding remarks. 2.1 Secular trends in education The stock of human capital is the main productive asset for households. As such, formal education constitutes one of the main determinants of an individual’s income, and is arguably a key to a wide set of economic and social opportunities. Latin America has experienced substantial changes in the educational attainment levels of its labor force in the second half of the 20th century, and these have even accelerated over the last decades (see Figure 2.1). This trend can be attributed to the expansion of both secondary and tertiary schooling, which followed an earlier rise in primary school enrollment during the 1960s-1980s. Despite a generalized upward trend in educational attainment of the labor force, there is still substantial heterogeneity between countries (Figure 2.2). Only a minority of countries has an average of more than 9 years of education for adults in the 25-65 age range – this group includes those in the Southern Cone (Argentina, Chile, Uruguay) and Panama – while some countries in Central America (Guatemala, Honduras and Nicaragua) present a much smaller figure, with an average below 6. The Gini coefficient for years of education also fell steadily for all countries in the region during the 1990s and the 2000s. This indicator, however, is not sufficient to signal an unambiguous reduction in inequality of education. While persons from high and low socioeconomic strata have seen their average levels of education increase, Cruces et al. (2014) report substantial differences for adults in the top quintile as compared to those in the bottom quintile; in some countries the disparity is as large as 7.5 years. This quintile gap widened in almost all countries in the 1990s and got smaller in most countries in the 2000s. While the issue of whether or not inequality in education in the region has increased or not depends on which aspect and concept one attempts to capture, there is no ambiguity in the fact that average educational attainment has increased over time for all countries in the region. 2.2 Trends in income inequality In parallel to this educational expansion, there have been considerable changes in the patterns of income inequality in Latin America over the last decades (Figure 2.3). Inequality soared in the 1990s and fell sharply in the 2000s. The contrast between the 3 3 two decades has been widely documented and discussed in other studies (e.g. López- Calva and Lustig, 2010; Gasparini et al., 2011; Gasparini and Lustig, 2011; Azevedo et al., 2012; Cord et al., 2017). Gasparini et al. (2016) report that the mean Gini coefficient for the distribution of household per capita income in Latin America grew at a rate of 0.3 points per year between 1992 and 2002, and then it fell 0.7 points a year between 2002 and 2010. That decline has substantially decelerated in the 2010s: the Gini fell 0.3 points per year between 2010 and 2014, less than half as much as in the previous period. These patterns are statistically significant, generalized within sub-regions of Latin America, and robust to various methodological decisions, including the consideration of various inequality indices (see SEDLAC website). As Figure 2.3 suggests, they are also robust to the consideration of different samples of countries according to the availability and quality of their national household surveys.2 The multitude of policy changes, external shocks and country experiences imply that there is no single satisfying explanation for these trends, which can be attributed to a variety of factors. This paper explores one of the main channels that affect income distribution: changes in labor market returns to education. In principle, these changes are important for a simple reason: earnings are the main source of income for most households. On average, they account for 75% of all income reported in Latin American household surveys. Moreover, several studies that apply different decomposition strategies confirm the leading role of the dispersion in earnings, and in particular in hourly wages, in accounting for inequality in the per capita income distribution (Azevedo et al., 2012; Alejo et al. 2013). Figure 2.4 is illustrative of the close relationship between income inequality at the household level and the dispersion in wages, proxied by the wage gap between skilled and unskilled labor (more details on the construction of this wage skill premium can be found below). 2.2 Trends in income inequality The figure suggests that the wage skill premium is a useful proxy for aggregate levels of inequality: explaining the evolution of these differentials should prove useful for interpreting broader distributional changes in these economies. inequality The sample of “9 countries” includes those with consistent data spanning the whole period 1991-2013. and 4 4 the relative demand for skilled labor, increasing its relative remuneration (and thus overall inequality), whereas educational upgrading would provide a counterbalancing force, reducing this premium. This framework has been formalized, among others, by Katz and Murphy (1992), Card and Lemieux (2001), and Goldin and Katz (2009). As these authors show, it is possible to simplify the analysis by assuming two factors of production, corresponding to two levels of skills among workers – high and low. Inequality is mainly driven by the remuneration differential between these two groups, the wage skill premium, which in turn is determined by an interaction of the relative supply of high skill to low skill workers (which proxies for changes in the human capital composition of the workforce), and the corresponding relative demand for these two factors of production. In this simple supply and demand equilibrium framework, an increase in the relative supply of skilled workers should result in a decline in their relative remuneration, while a positive change in the relative demand for this type of workers would increase the wage skill premium. Katz and Murphy’s (1992) pioneer methodology has been applied to numerous country case studies in Latin America – for instance, for the 1990-2003 period in Mexico (Montes Rojas, 2006), 1960-2000 in Chile (Gallego, 2011), and 1980-2007 in Panama (Galiani, 2009). More recently, Manacorda et al. (2010) develop a full-fledged analysis of the relationship between changes in the wage skill premium and in the relative supply of skilled workers for Argentina, Brazil, Chile, Colombia, and Mexico in the 1980s and the 1990s. They document a substantial rise in the supply of workers with some secondary education, and they find a decline in the relative wage of this group with respect to unskilled workers, consistent with the large increase in their supply. They also find a simultaneous rise in the relative wages of skilled workers, which they attribute to a generalized shift in the demand for workers with tertiary education. 3.2 The model Fram ework and empiri cal impl ementa tion      1 ) 1(        t t t t t t U S A Q (1) (1) Total output Q is a function of the quantities of these factors and the technology parameters ρ and λ. Given this production function, the elasticity of substitution between skilled and unskilled labor is σSU = (1-ρ)-1. This basic framework relies on two broad aggregates of labor. However, the educational structure of the Latin American population discussed in section 2 suggests that the analysis of returns to skills would be enriched by studying the evolution of premiums, supply and demand factors within the unskilled labor group. Following Goldin and Katz (2009), the basic CES production function (1) can be augmented to accommodate 5 5 three skill levels by partitioning the unskilled group. They propose a specification which maintains the previous CES setup, but considers U to be a composite factor – a CES sub-aggregate of the form: three skill levels by partitioning the unskilled group. They propose a specification which maintains the previous CES setup, but considers U to be a composite factor – a CES sub-aggregate of the form:      1 ) 1(          t t t t t D H U (2) (2) where  and η are additional technology parameters, and H and D are units of high school graduate labor and labor with less than a high school degree (referred to as high school dropouts), respectively. The (constant) elasticity of substitution between these two factors is σHD = (1- η)-1. 3.3 Data and empirical implementation Evidence for this paper was obtained by processing microdata from national household surveys included in the Socioeconomic Database for Latin America and the Caribbean (SEDLAC), a project jointly developed by CEDLAS, at the Universidad Nacional de La Plata, and the World Bank. Household surveys are not uniform across Latin American countries, and in several cases, within a country over time. Thus, the issue of comparability is of great concern. Owing to this, we have made all possible efforts to make statistics comparable across countries and over time by using similar definitions of variables in each country/year, and by applying consistent methods of processing the data (see SEDLAC (2015) for details on the harmonization process). The document covers a set of 16 Latin American economies: Argentina, Brazil, Bolivia, Chile, Colombia, Costa Rica, Ecuador, El Salvador, Honduras, Mexico, Nicaragua, Panama, Paraguay, Peru, Uruguay, and Venezuela. Only Guatemala is missing among the continental Latin American countries. The sample represents 97.5 percent of the total Latin American population. We have drawn data from 258 national household surveys in these 16 countries over the period 1991-2013, containing information on wages, hours of work, education, and other relevant variables. We restrict the sample to individuals aged 26-56 that have fully coherent answers about income and education. Following Manacorda et al. (2010), we compute premiums from a sample of male workers only to control for the potential effects of the secular increase in female labor force participation. Even with these restrictions, our sample includes data from more than 10 million workers in Latin America. Adopting an education-based measure of skills, skilled workers (S) are defined as those with some tertiary education (either complete or not complete), while unskilled workers (U) encompasses workers with a high school diploma or lower educational attainment. U is then divided into two groups, which correspond to workers with a high school degree (H) and those with a lower level of education (referred to as high school dropouts) (D), respectively. The two wage skill premiums (left-hand side terms on equations 3 and 4) and the relative supplies of labor (last terms on the right-hand side) are constructed from household survey data for each country. Intuitively, the skill premiums are derived from wage differentials in Mincer-type wage regressions, while the relative supplies are weighted functions of the proportions of each group of workers in the population. 3.2 The model Under perfect competition, with a large number of firms and factors paid at the marginal product value, the wage premium between skilled and unskilled workers (wS /wU) and the differential wages between high school graduates and dropouts (wH /wD) in year t satisfy the following two relationships:                      t t SU t t U S U S w w t t log 1 1 log log    (3)                      t t HD t t D H D H w w t t log 1 1 log log    (4) (3) (4) The skilled-unskilled wage differential in equation (3) captures the traditional Katz and Murphy (1992) college premium, while equation (4) represents the high school premium – the difference in wages within the two groups that constitute the unskilled aggregate. Large values for the elasticities of substitution σ suggest that the two types of labor are close to perfect substitutes, and relative wages would then be uncorrelated with relative quantities. A limitation of the Tinbergen framework and its subsequent applications, however, is that, despite its emphasis on both supply and demand factors, it does not provide for an unambiguous way to approximate changes in demand. This limitation is due to the difficulty of identifying simultaneous demand and supply movements from observed quantities and prices, which are, by definition, equilibrium points resulting from the intersection of the two corresponding curves. While the constructed supply and wage skill premiums also result from market equilibrium, it is plausible that the supply index, based on population stocks, represents the actual relative supply. Katz and Murphy (1992), and subsequent related studies, use observed wage premiums and relative changes in quantities of skilled and unskilled labor as measures of relative prices and supply, respectively; they then compute the changes in the relative demand of skilled labor as a residual – that is, as the figures that would be compatible with the observed changes in prices and supply for a given level of the elasticity of substitution between both factors. 6 3.3 Data and empirical implementation We use the working age population as a basis for the relative labor supply, although the trends described in this paper are qualitatively the same if, instead of using population, we construct our measures of relative supply based on the labor force, employment, or hours of work. The relative supplies represent a weighted sum of 7 individuals in each of the skill categories. Following Katz and Murphy (1992) and Goldin and Katz (2009), we first compute efficiency units dividing the population of each country into 24 groups (cells) according to their gender, educational level, and potential experience.3 For each cell, we compute the average wage for all years with respect to the wage of the largest group in the last year of the sample. These relative wages constitute the factors used to weight the supply for each cell in efficiency units. These efficiency-adjusted labor supplies are then added for all skilled and unskilled workers, and their ratio (in logs) is the measure of relative supply in efficiency units. The relative supply of high school graduate workers with respect to dropouts is derived analogously. The two terms on the right-hand side of equations (3) and (4) correspond to the log of the relative supply aggregates. The skill premiums, in turn, correspond to weighted averages of the coefficient for each educational category (primary, secondary, and tertiary, each then divided into complete and incomplete) in a Mincer regression for each country and year in the sample. The weights are given by the share of employment of individuals with a given educational level during a base period. See the Appendix for more details. A residual relative demand can be computed from these relative supplies and premiums. As shown by Katz and Murphy (1992), under perfect competition and considering a CES technology with two factors (skilled and unskilled workers), we can obtain a time series of relative demand between skilled and unskilled workers for a given value of the elasticity of substitution between factors σSU:               t t U S U S w w D t t SU t ln ln  (5) (5) (5) Estimates of the elasticities of substitution are needed to empirically compute equation (5). 3.3 Data and empirical implementation Most of the literature arrives at those estimates from the inverse of the coefficient of the relative supply of skilled labor in a wage gap regression, controlling by proxies of the relative demand – usually a time trend, linear or of higher order. The mean value of σ in our regression analysis indicates values fluctuating around 4, while Manacorda et al. (2010) find values around 3 for Latin America. Goldin and Katz (2009), in the context of the United States, report values of around 1.6. The tables in the document will show results, alternatively, for σ equal to 2, 3, and 4. 3 Four educational levels (complete college, incomplete college, complete secondary and less than complete secondary) and three groups of potential experience (0 to 15 years, 16 to 30 years, and more than 30 years) are considered. 4 Empirical Results Table 4.1 presents the results for the skilled-unskilled (tertiary educated workers vs. the rest) wage premium and relative supplies, as well as the residual estimates of 8 relative demand for different values of the elasticity of substitution. The period under analysis is divided into three “episodes” that roughly correspond to the 1990s, the 2000s, and the 2010s. Figure 4.1 presents aggregate trends, net of country effects (i.e., as deviations of each country’s means). Although there is substantial heterogeneity across the countries in our sample, some patterns are common to most economies in the region. The relative supply of skilled workers increased substantially and continuously over the period under analysis, on average at approximately the same rate over the three episodes. This sustained and remarkable education upgrading of the region’s workforce is consistent with the trends in the population outlined briefly in Section 2 above. The pattern for the wage premium in Table 4.1 exhibits heterogeneity both across countries and in particular over time. The wage skill premium increased for all countries in the region during the 1990s, modestly in some countries such as Brazil, Chile, Costa Rica, Honduras and Panama, and substantially more in the remaining countries in the sample, especially Argentina, Colombia, Nicaragua, and Uruguay. On the contrary, during the decade of 2000, the wage skill premium fell in all countries; the average rate of reduction was about 3.2 percent per year, twice the size of the average increase in the previous decade. In most countries, the fall in the wage skill premium in the 2000s was enough to offset the increase of the 1990s. The decline began in the late 1990s in some economies (e.g., Mexico) and later in other South American economies that experienced serious macroeconomic crises (e.g. Argentina, Paraguay, and Uruguay). The reduction in the wage gap continued throughout the 2000s until signs of deceleration showed up in several economies around the turn of the decade. According to our estimates, while the wage premium fell at 3.2 percent on average in the 2000s, the reduction slowed down to 1.4 percent per year in the 2010s. On average, then, the wage gap between skilled and unskilled workers widened in the 1990s and shrunk considerably in the 2000s and more slowly in the 2010s, in the context of a seemingly secular increase in the relative supply of skilled workers (Figure 4.1). 4 Empirical Results Interestingly, this pattern is remarkably similar to the one for the Gini coefficient of the distribution of household per capita income (Figure 2.3). This similarity is partly due to the relevance of wage gaps in driving earnings and household inequality, given that labor income is the main income source for households in Latin America, but it could also be the consequence of factors affecting wage skill premiums and other income sources (e.g. public transfers, capital incomes, pensions) or demographic variables in the same direction. The patterns in wage skill premiums and relative labor supply are consistent with a positive and strong increase in the relative demand for skilled labor in the 1990s in all countries, as witnessed by the estimates of residual demand presented in the last three panels of Table 4.1 and in the aggregate estimates in Figure 4.3. The estimates of relative demand for the 2000s, consistent with the observed changes in relative supply and wage gaps, indicate a reversal in the trends of the previous decade. The indicators 9 signal a negative shift in the relative demand for skilled labor; the decline was stronger in the 2000s, and mild in the 2010s. Besides changes in relative remuneration between workers with some tertiary education and those with lower levels of education, the analysis also allows for differentiating within the group of unskilled workers. The estimates that divide the group of the unskilled into high school graduates and high school dropouts or less educated, are presented in Table 4.2. As described in Section 2, the supply of workers with secondary education increased as compared to that of workers with lower education levels for most countries and periods under study, with only a few exceptions. Table 4.2 indicates a somewhat faster rate of expansion in the 2000s than in the 1990s, and an acceleration in the 2010s. These mostly uniform trends in the relative supply of secondary school graduates, however, did not translate into common trends in the wage gaps. During the 1990s, the wage differential between high school graduates and dropouts significantly increased in Argentina and Peru and substantially fell in Brazil, Colombia, Mexico, and El Salvador. 4 Empirical Results The increase in the relative supply of high-school graduates in the 2000s was concurrent with a decline in the relative remuneration of this factor in most countries, with a few exceptions, including Uruguay, where the relative supply of high school graduates fell over the period. The reduction in the skill premium between high school graduates and dropouts continued in the 2010s, although it was less generalized and intense. The results in Table 4.2 and Figure 4.2 indicate that, on average and concurrent with a sharp rise in the supply of high school graduates with respect to dropouts, the average wage differential between the two groups fell modestly over time, and more strongly in the 2000s. These patterns are consistent with an increase in the relative demand for high school graduates with respect to dropouts, although at a slower pace than the increase in their relative supply (Figure 4.3). 5 Wage skill premium and relative skill supply This section further explores some of the correlations that emerged from the descriptive analysis of the previous section. The methodology follows Katz and Murphy’s (1992) study of the United States, which was based on time series regressions of the wage skill premium as a function of the relative supply of skilled labor and other factors. Here, and following Manacorda et al. (2010), pooled data from all the countries in the sample are used. The advantage of pooling the data for the region is that it allows for the inclusion of countries for which the limited number of years is not suitable for a country-specific regression. The results, thus, should be interpreted as reduced form 10 estimates of averages for the whole region, with the caveat that the aggregate patterns do not necessarily correspond to the experiences of all 16 countries.4 The wage skill premium regressions a la Katz and Murphy (1992) attempt to establish the relevance of supply and demand factors. In this framework, however, there is no unambiguous indicator of relative demand. The literature for the United States has thus proxied these factors by adopting different specification of time trends (Goldin and Katz, 2009; Acemoglu and Autor, 2011, among many others). The conjecture that time trends might adequately capture the shifts in relative demand seems to obey to the implicit assumption that the driving force behind these shifts is a relatively steady trend determined by cumulative factors, such as technological change. This assumption, however, does not seem to suit the nature of changes in Latin American economies. While technical change is probably a relevant force behind the demand for skilled labor in the region, a multitude of other factors can be expected to play a role, either directly mediating the forces of technological change (for instance, through relative prices of technology embedded in capital) or indirectly – for instance, external shocks, macroeconomic crises and the ensuing devaluations (which affect the relative price of capital with respect to labor), structural reforms (such as trade liberalization and privatizations, with direct effects on employment and demand for skills), and policy reversals. These types of events, more common in Latin American economies than in the advanced countries traditionally studied in Katz and Murphy-type of analysis, imply that time trends might be unable to capture the potential changes, swings, and reversals in the pattern of relative demand for skilled labor. 5 We present the results of the weighted regressions, where weights are the inverse of the standard error of the skill premium squared. Results for the unweighted regressions, available upon request, are similar. and relati ve s kill s upply unweighted country averages. 5 Wage skill premium and relative skill supply With these limitations in mind, the analysis here follows Manacorda et al.’s (2010) approach, where no explicit proxy for relative demand is included. Instead, country and year fixed effects capture the demand shifters in the right-hand side of equations (3) and (4) above. The regressions for the college wage premium have the following form: ct t c ct ct U S T C U S w w                       log log (6) (6) where the left-hand side variable is the wage skill premium in year t for country c, and the right-hand side variables are a constant, the relative supply of skilled labor, and country (C) and year (T) fixed effects. The regressions for the high school premium are defined analogously. Wage skill premi um 11 can be expected given the aggregate patterns in Figure 5.1 (and from the country- specific scatter plots not presented in the paper), the simple correlations between the levels of the wage differentials and the respective relative supplies are negative and strongly significant, even when including country fixed effects, year effects, and when clustering standard errors by country. This evidence is compatible with the Tinbergen framework and the Katz and Murphy (1992) results for the United States: the educational upgrading of the labor force, manifested through an increase in the relative supply of workers with some college education or with a high school degree, implied on its own a decline in relative remunerations. The effect seems to have been stronger for the skilled-unskilled wage premium than for the high school-dropout differential. 6 Concluding remarks This paper studies the evolution of wage differentials and trends in the supply and demand of workers by skill level (as proxied by educational attainment) for 16 Latin American countries, during the period 1991-2013. Although there is considerable heterogeneity across countries, we find a rather consistent rise in the relative supply of skilled and semi-skilled workers over the period. Consistent with this pattern, the returns to secondary education completion fell over time. By contrast, the returns to tertiary education display a remarkable changing pattern common to nearly all countries. The wage premium to that educational level significantly increased in the 1990s and fell strongly in the 2000s and more slowly in the early 2010s. Given this, we conclude that supply-side factors seem to have limited explanatory power relative to demand-side factors in accounting for changes in the wage skill premium, especially between workers with tertiary education and the rest. This finding stresses the need to further our understanding of the changes in the relative demands for skilled and unskilled labor. 12 References Acemoglu, D., and Autor, D. (2011). “Skills, tasks and technologies: Implications for employment and earnings”. Handbook of Labor Economics, 4, 1043-1171. Acosta, P. and Gasparini, L. (2007). “Capital Accumulation, Trade Liberalization, and Rising Wage Inequality: The Case of Argentina”. Economic Development and Cultural Change 55. Acosta, P. and Montes Rojas, G. (2008). “Trade Reform and Inequality: The Case of Mexico and Argentina in the 1990s”. World Economy 31, 763-780. Conclu ding remar ks Barro, R. and Lee, J (2010). “A New Data Set of Educational Attainment in the World, 1950–2010”, NBER Working Paper 15902. Card, D. and Lemieux, T. (2001). “Can Falling Supply Explain the Rising Return to College for Young Men?” Quarterly Journal of Economics 116, 705-746. Cord, L., Barriga‐Cabanillas, O., Lucchetti, L., Rodríguez‐Castelán, C., Sousa, L. D., and Valderrama, D. (2017). “Inequality stagnation in Latin America in the aftermath of the global financial crisis”. Review of Development Economics, 21(1), 157-181. Cruces, G., García Domench, C. and Gasparini, L. (2014). "Inequality in Education: Evidence for Latin America". En Cornea, G. (ed.). Falling Inequality in Latin America. Policy Changes and Lessons. Oxford University Press, 2014. pp. 318- 339. (ISBN 978-0-19-870180-4) i, S. (2009). “Salarios y Educación en el Mercado Laboral Panameño”. Mimeo Gallego, F. A. (2012). “Skill premium in Chile: studying skill upgrading in the South”. World Development, 40(3), 594-609. García Swartz, D. and Gasparini, L. (2011). “General-Equilibrium Perspectives on Relative Wage Changes in a Highly Volatile Macroeconomic Environment: Argentina between 1974 and 1995”. CEDLAS working paper. Gasparini, L., Cruces, G. and Tornarolli, L. (2011). “Recent Trends in Income Inequality in Latin America”. Economia 10, 147-201. Gasparini, L., Cruces, G. y Tornarolli, L. (2016). “Chronicle of a Deceleration Foretold: Income Inequality in Latin America in the 2010s”. Revista de Economía Mundial 43, 25-46. 13 Gasparini, L., and Lustig, N. (2011). “The rise and fall of income inequality in Latin America” (No. 118). Documento de Trabajo. Goldin, C. and Katz, L. (2007). “The Race Between Education and Technology: The Evolution of U.S. Educational Wage Differentials, 1890 to 2005”. NBER Working Paper No 12984. Goldin, C. D., and Katz, L. F. (2009). “The race between education and technology”. Harvard University Press. Katz, L. and Murphy, K. (1992). “Changes in Relative Wages, 1963-1987: Supply and Demand Factors”. Quarterly Journal of Economics 107, 35-78. López-Calva, L. and Lustig, N. (2010). The New Dynamics of Income Inequality in Latin America. Tinbergen, J. (1975). Income Distribution: Analysis and Policies. North-Holland: Amsterdam. References Brookings Institution and UNDP, Washington DC. Manacorda, M., Sánchez-Páramo, C. and Schady, N. (2010). “Changes in Returns to Education in Latin America: The Role of Demand and Supply of Skills”. Industrial and Labor Relations Review 63, 307-326. Montes Rojas, G. (2006). “Skill Premia in Mexico: Demand and Supply factors.” Applied Economics Letters 13, 917–924. Tinbergen, J. (1975). Income Distribution: Analysis and Policies. North-Holland: Amsterdam. 14 Figure 2.1: Percentage of the labor force by highest educational level achieved, Latin America, 1950-2010 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 No educucation Primary Secondary Tertiary Source: Barro and Lee (2010). Figure 2.2: Distribution of the working age population by education level Source: own computations based on SEDLAC (CEDLAS and World Bank). Figure 2.1: Percentage of the labor force by highest educational level achieved, Latin America, 1950-2010 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 No educucation Primary Secondary Tertiary S B d L (2010) Figure 2.1: Percentage of the labor force by highest educational level achieved, Latin America, 1950-2010 Tertiary Source: Barro and Lee (2010). Source: Barro and Lee (2010). Figure 2.2: Distribution of the working age population by education level Source: own computations based on SEDLAC (CEDLAS and World Bank). Figure 2.2: Distribution of the working age population by education level Source: own computations based on SEDLAC (CEDLAS and World Bank). 15 Figure 2.3: Gini coefficient for the distribution of household per capita income Unweighted average for Latin America, 1991-2013. 0.48 0.50 0.52 0.54 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 9 countries 12 countries 16 countries Source: own computations based on SEDLAC (CEDLAS and World Bank). Note: “9 countries” refers to: Argentina, Brazil, Chile, Costa Rica, Honduras, Mexico, Panama, Uruguay and Venezuela. “12 countries” also includes: Colombia, Paraguay and El Salvador. “16 countries” also includes: Bolivia, Ecuador, Peru and Nicaragua. Figure 2.3: Gini coefficient for the distribution of household per capita income Unweighted average for Latin America, 1991-2013. 0.48 0.50 0.52 0.54 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 9 countries 12 countries 16 countries 16 countries wn computations based on SEDLAC (CEDLAS and World Bank). Source: own computations based on SEDLAC (CEDLAS and World Bank). References p ( ) Note: “9 countries” refers to: Argentina, Brazil, Chile, Costa Rica, Honduras, Mexico, Panama, Uruguay and Venezuela. “12 countries” also includes: Colombia, Paraguay and El Salvador. “16 countries” also includes: Bolivia, Ecuador, Peru and Nicaragua. Figure 2.4: Gini coefficient for the distribution of household per capita income and wage premium skill-unskilled. Latin America, 1991-2013. Figure 2.4: Gini coefficient for the distribution of household per capita income and wage premium skill-unskilled. Latin America, 1991-2013. Figure 2.4: Gini coefficient for the distribution of household per cap wage premium skill-unskilled. Latin America, 1991-2013. 0.4 0.5 0.5 0.6 0.6 0.7 0.5 1.0 1.5 Wage Premium, Skilled-Unskilled Scatter Binned Scatter OLS Fit R²= 0.56 Source: own computations based on SEDLAC (CEDLAS and World Bank). 0.4 0.5 0.5 0.6 0.6 0.7 0.5 1.0 1.5 Wage Premium, Skilled-Unskilled Scatter Binned Scatter OLS Fit R²= 0.56 Wage Premium, Skilled-Unskilled Source: own computations based on SEDLAC (CEDLAS and World Bank). 16 Figure 4.1: Wage premium and relative supply over time, net of country effects. Skilled-unskilled labor. 0.80 0.90 1.00 1.10 1.20 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 9 countries 12 countries 16 countries -1.00 -0.80 -0.60 -0.40 -0.20 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 9 countries 12 countries 16 countries Source: own computations based on SEDLAC (CEDLAS and World Bank). Note: “9 countries” refers to: Argentina, Brazil, Chile, Costa Rica, Honduras, Mexico, Panama, Uruguay and Venezuela. “12 countries” also includes: Colombia, Paraguay and El Salvador. “16 countries” also includes: Bolivia, Ecuador, Peru and Nicaragua. 0.80 0.90 1.00 1.10 1.20 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 9 countries 12 countries 16 countries -1.00 -0.80 -0.60 -0.40 -0.20 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 9 countries 12 countries 16 countries -1.00 -0.80 -0.60 -0.40 -0.20 Source: own computations based on SEDLAC (CEDLAS and World Bank). Source: own computations based on SEDLAC (CEDLAS and World Bank). Note: “9 countries” refers to: Argentina, Brazil, Chile, Costa Rica, Honduras, Mexico, Panama, Uruguay and Venezuela. “12 countries” also includes: Colombia, Paraguay and El Salvador. “16 countries” also includes: Bolivia, Ecuador, Peru and Nicaragua. Source: own computations based on SEDLAC (CEDLAS and World Bank). Note: “9 countries” refers to: Argentina, Brazil, Chile, Costa Rica, Honduras, Mexico, Panama, Uruguay and Venezuela. “12 countries” also includes: Colombia, Paraguay and El Salvador. References “16 countries” also includes: Bolivia, Ecuador, Peru and Nicaragua. 17 Figure 4.2: Wage premium and relative supply over tim High school-dropouts 0.35 0.40 0.45 0.50 0.55 0.60 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 9 countries 12 countries 16 countries -1.40 -1.20 -1.00 -0.80 -0.60 -0.40 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 9 countries 12 countries 16 countries Source: own computations based on SEDLAC (CEDLAS and Note: “9 countries” refers to: Argentina, Brazil, Chile, Costa Uruguay and Venezuela. “12 countries” also includes: Colomb countries” also includes: Bolivia, Ecuador, Peru and Nicaragu Figure 4.2: Wage premium and relative supply over time, net of country effects. High school-dropouts Figure 4.2: Wage premium and relative supply over time, net of country effects. High school-dropouts Figure 4.2: Wage premium and relative supply over time, net of country effects. High school-dropouts 0.35 0.40 0.45 0.50 0.55 0.60 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 9 countries 12 countries 16 countries -1.40 -1.20 -1.00 -0.80 -0.60 -0.40 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 9 countries 12 countries 16 countries Source: own computations based on SEDLAC (CEDLAS and World Bank). Note: “9 countries” refers to: Argentina, Brazil, Chile, Costa Rica, Honduras, Mexico, Panama, Uruguay and Venezuela. “12 countries” also includes: Colombia, Paraguay and El Salvador. “16 countries” also includes: Bolivia, Ecuador, Peru and Nicaragua. High school-dropouts 0.35 0.40 0.45 0.50 0.55 0.60 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 9 countries 12 countries 16 countries 0.35 0.40 0.45 0.50 0.55 0.60 -1.40 -1.20 -1.00 -0.80 -0.60 -0.40 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 9 countries 12 countries 16 countries -1.40 -1.20 -1.00 -0.80 -0.60 -0.40 Source: own computations based on SEDLAC (CEDLAS and World Bank). Source: own computations based on SEDLAC (CEDLAS and World Bank). Note: “9 countries” refers to: Argentina, Brazil, Chile, Costa Rica, Honduras, Mexico, Panama, Uruguay and Venezuela. “12 countries” also includes: Colombia, Paraguay and El Salvador. “16 countries” also includes: Bolivia, Ecuador, Peru and Nicaragua. Source: own computations based on SEDLAC (CEDLAS and World Bank). Note: “9 countries” refers to: Argentina, Brazil, Chile, Costa Rica, Honduras, Mexico, Panama, Uruguay and Venezuela. “12 countries” also includes: Colombia, Paraguay and El Salvador. “16 countries” also includes: Bolivia, Ecuador, Peru and Nicaragua. 18 Figure 4.3: Relative demands over time (residual assuming σ=3 for both elasticities) Figure 4.3: Relative demands over time (residual assuming σ=3 for both elasticities) Source: own computations based on SEDLAC (CEDLAS and World Bank). Source: own computations based on SEDLAC (CEDLAS and World Bank). Source: own computations based on SEDLAC (CEDLAS and World Bank). Figure 5.1: Wage premiums and relative supply Figure 5.1: Wage premiums and relative supply 0.6 0.8 1.0 1.2 1.4 -1.0 -0.5 0.0 Relative Supply, Skilled-Unskilled Scatter Binned Scatter OLS Fit R²= 0.31 0.2 0.4 0.6 0.8 -1.5 -1.0 -0.5 0.0 Relative Supply, High School-Dropouts Scatter Binned Scatter OLS Fit R²= 0.54 Source: own computations based on SEDLAC (CEDLAS and World Bank). 0.6 0.8 1.0 1.2 1.4 -1.0 -0.5 0.0 Relative Supply, Skilled-Unskilled Scatter Binned Scatter OLS Fit R²= 0.31 Relative Supply, Skilled-Unskilled 0.2 0.4 0.6 0.8 -1.5 -1.0 -0.5 0.0 Relative Supply, High School-Dropouts Scatter Binned Scatter OLS Fit R²= 0.54 Relative Supply, High School-Dropouts Source: own computations based on SEDLAC (CEDLAS and World Bank). 19 Table 4.1: Changes in the wage premium and the relative supply and demand for skilled-unskilled workers assuming alternative values for elasticity SU 1990s 2000s 2010s 1990s 2000s 2010s 1990s 2000s 2010s 1990s 2000s 2010s 1990s 2000s 2010s Argentina 2.5 -3.5 -1.3 4.8 2.9 2.1 9.9 -4.2 -0.4 12.4 -7.7 -1.7 15.0 -11.2 -3.0 Bolivia . -6.9 -1.1 . 3.7 5.3 -10.1 3.1 -17.0 2.0 -23.8 0.9 Brazil 0.7 -5.0 -0.6 1.9 5.5 1.1 3.2 -4.4 0.0 3.9 -9.4 -0.6 4.6 -14.4 -1.1 Chile 0.5 -2.5 -2.1 3.2 1.3 3.8 4.2 -3.7 -0.5 4.6 -6.2 -2.6 5.1 -8.7 -4.8 Colombia 2.5 -1.1 -1.6 4.4 4.6 5.4 9.4 2.5 2.3 11.8 1.5 0.7 14.3 0.4 -0.8 Costa Rica 0.7 -0.5 -2.3 4.7 3.3 3.8 6.2 2.4 -0.7 6.9 1.9 -3.0 7.7 1.5 -5.2 Ecuador . -4.4 -3.0 . 3.1 2.0 -5.6 -3.9 -9.9 -6.9 -14.3 -9.8 Honduras 0.0 -1.9 0.6 2.5 2.4 7.9 2.6 -1.4 9.2 2.7 -3.3 9.8 2.7 -5.2 10.5 Mexico 1.8 -2.8 0.4 3.7 2.2 0.7 7.3 -3.5 1.6 9.2 -6.3 2.0 11.0 -9.1 2.4 Nicaragua 3.5 -6.7 . 4.5 5.4 . 11.4 -8.0 . 14.9 -14.7 . 18.4 -21.4 . Figure 4.3: Relative demands over time (residual assuming σ=3 for both elasticities) Household surveys used for each country: Argentina: 1992, 2000, 2009, 2013; Bolivia: 2001, 2009, 2012; Brazil: 1993, 2002, 2009, 2013; Chile: 1990, 2000, 2006, 2013; Colombia: 1996, 2001, 2008, 2013; Costa Rica: 1992, 2002, 2008, 2013; Ecuador: 2003, 2009, 2012; Honduras: 1993, 2003, 2009, 2013; Mexico: 1989, 2000, 2008, 2012; Nicaragua: 1993, 2001, 2009; Panama: 1992, 2002, 2009, 2013; Paraguay: 1995, 2003, 2009, 2013; Peru: 1997, 2002, 2009, 2013; El Salvador: 1995, 2002, 2009, 2013; 1992, 2002, 2009, 2013. Venezuela: 1995, 2002, 2008, 2011. Note: annual log changes multiplied by 100. Household surveys used for each country: Argentina: 1992, 2000, 2009, 2013; Bolivia: 2001, 2009, 2012; Brazil: 1993, 2002, 2009, 2013; Chile: 1990, 2000, 2006, 2013; Colombia: 1996, 2001, 2008, 2013; Costa Rica: 1992, 2002, 2008, 2013; Ecuador: 2003, 2009, 2012; Honduras: 1993, 2003, 2009, 2013; Mexico: 1989, 2000, 2008, 2012; Nicaragua: 1993, 2001, 2009; Panama: 1992, 2002, 2009, 2013; Paraguay: 1995, 2003, 2009, 2013; Peru: 1997, 2002, 2009, 2013; El Salvador: 1995, 2002, 2009, 2013; 1992, 2002, 2009, 2013. Venezuela: 1995, 2002, 2008, 2011. Table 4.2: Changes in the wage premium and the relative supply and demand for unskilled workers (high school-dropouts) assuming alternative values for elasticity HD 1990s 2000s 2010s 1990s 2000s 2010s 1990s 2000s 2010s 1990s 2000s 2010s 1990s 2000s 2010s Argentina 1.1 -1.7 -1.7 1.6 5.6 3.3 3.8 2.2 -0.1 4.9 0.5 -1.8 6.0 -1.2 -3.4 Bolivia . -4.0 1.8 . 7.3 4.8 . -0.7 8.4 . -4.8 10.3 . -8.8 12.1 Brazil -1.4 -2.3 -2.9 6.1 7.1 6.8 3.4 2.5 1.0 2.0 0.2 -1.9 0.6 -2.1 -4.8 Chile 0.5 -2.1 -1.5 6.9 4.3 4.5 8.0 0.2 1.4 8.5 -1.9 -0.1 9.0 -3.9 -1.6 Colombia -1.7 0.0 -1.2 6.7 5.3 4.3 3.2 5.2 2.0 1.5 5.1 0.8 -0.3 5.1 -0.4 Costa Rica -0.5 -0.5 1.3 1.2 2.5 5.0 0.2 1.5 7.6 -0.3 0.9 8.9 -0.8 0.4 10.1 Ecuador . -1.2 -0.4 . 2.5 10.1 0.0 9.3 . -1.2 8.9 . -2.4 8.5 Honduras 0.0 -1.9 0.6 2.5 2.4 7.9 2.6 -1.4 9.2 2.7 -3.3 9.8 2.7 -5.2 10.5 Mexico -1.4 -1.2 0.2 4.6 -0.3 5.1 1.7 -2.6 5.4 0.3 -3.8 5.6 -1.2 -5.0 5.7 Nicaragua -0.2 -0.4 . 2.1 4.9 . 1.7 4.0 . 1.4 3.5 . 1.2 3.1 . Figure 4.3: Relative demands over time (residual assuming σ=3 for both elasticities) Panama 0.7 -3.3 -0.9 2.6 2.0 4.1 4.1 -4.5 2.4 4.8 -7.8 1.5 5.6 -11.0 0.6 Paraguay 1.2 -5.6 -1.1 5.4 6.1 2.9 7.7 -5.1 0.6 8.9 -10.8 -0.5 10.0 -16.4 -1.6 Peru 1.8 -2.3 -1.9 1.1 3.6 3.9 4.6 -1.0 0.1 6.3 -3.4 -1.8 8.1 -5.7 -3.7 El Salvador 1.7 0.6 -1.1 3.5 0.3 1.7 7.0 1.5 -0.5 8.7 2.1 -1.5 10.5 2.7 -2.6 Uruguay 3.4 -0.2 -3.1 0.6 2.2 0.5 7.5 1.8 -5.7 10.9 1.6 -8.7 14.3 1.4 -11.8 Venezuela 1.1 -4.8 -2.2 3.9 4.7 8.4 6.2 -5.0 4.1 7.3 -9.8 2.0 8.5 -14.7 -0.2 Mean 1.6 -3.2 -1.4 3.3 3.3 3.6 6.5 -3.0 0.8 8.1 -6.2 -0.6 9.7 -9.4 -2.0 Mean 9 countries 1.3 -2.7 -1.3 3.1 2.9 3.6 5.7 -2.5 1.1 7.0 -5.2 -0.1 8.3 -7.9 -1.4 Mean 12 countries 1.4 -2.5 -1.3 3.4 3.1 3.5 6.3 -2.0 1.0 7.7 -4.5 -0.2 9.1 -7.1 -1.5 Skilled - Unskilled Country Relative supply Wage premium Relative demand (σsu=2) Relative demand (σsu=3) Relative demand (σsu=4) Source: own computations based on SEDLAC (CEDLAS and World Bank). Note: annual log changes multiplied by 100. Household surveys used for each country: Argentina: 1992, 2000, 2009, 2013; Bolivia: 2001, 2009, 2012; Brazil: 1993, 2002, 2009, 2013; Chile: 1990, 2000, 2006, 2013; Colombia: 1996, 2001, 2008, 2013; Costa Rica: 1992, 2002, 2008, 2013; Ecuador: 2003, 2009, 2012; Honduras: 1993, 2003, 2009, 2013; Mexico: 1989, 2000, 2008, 2012; Nicaragua: 1993, 2001, 2009; Panama: 1992, 2002, 2009, 2013; Paraguay: 1995, 2003, 2009, 2013; Peru: 1997, 2002, 2009, 2013; El Salvador: 1995, 2002, 2009, 2013; 1992, 2002, 2009, 2013. Venezuela: 1995, 2002, 2008, 2011. p ( ) Note: annual log changes multiplied by 100. Household surveys used for each country: Argentina: 1992, 2000, 2009, 2013; Bolivia: 2001, 2009, 2012; Brazil: 1993, 2002, 2009, 2013; Chile: 1990, 2000, 2006, 2013; Colombia: 1996, 2001, 2008, 2013; Costa Rica: 1992, 2002, 2008, 2013; Ecuador: 2003, 2009, 2012; Honduras: 1993, 2003, 2009, 2013; Mexico: 1989, 2000, 2008, 2012; Nicaragua: 1993, 2001, 2009; Panama: 1992, 2002, 2009, 2013; Paraguay: 1995, 2003, 2009, 2013; Peru: 1997, 2002, 2009, 2013; El Salvador: 1995, 2002, 2009, 2013; 1992, 2002, 2009, 2013. Venezuela: 1995, 2002, 2008, 2011. p ( ) Note: annual log changes multiplied by 100. Figure 4.3: Relative demands over time (residual assuming σ=3 for both elasticities) Panama -0.3 -2.0 0.6 2.9 5.3 1.6 2.2 1.3 2.7 1.9 -0.6 3.3 1.6 -2.6 3.9 Paraguay 0.3 -4.1 -4.1 2.7 6.0 4.6 3.4 -2.1 -3.7 3.7 -6.2 -7.8 4.1 -10.3 -11.9 Peru 1.7 -2.0 0.3 1.9 2.1 5.9 5.3 -1.9 6.5 7.1 -4.0 6.7 8.8 -6.0 7.0 El Salvador -3.3 0.5 -1.6 7.0 2.0 2.3 0.4 3.1 -0.8 -3.0 3.6 -2.4 -6.3 4.1 -4.0 Uruguay -0.3 0.1 -3.4 -1.4 -2.2 11.1 -2.0 -2.0 4.3 -2.3 -1.9 1.0 -2.5 -1.8 -2.4 Venezuela 0.0 -2.6 -0.9 2.9 7.5 6.5 3.0 2.2 4.6 3.0 -0.5 3.7 3.0 -3.1 2.8 Mean -0.4 -1.6 -0.9 3.4 3.9 5.6 2.6 0.7 3.9 2.2 -0.9 3.0 1.9 -2.5 2.1 Mean 9 countries -0.2 -1.6 -0.9 3.0 3.6 5.8 2.5 0.4 4.0 2.3 -1.2 3.2 2.0 -2.7 2.3 Mean 12 countries -0.6 -1.5 -1.2 3.7 3.8 5.3 2.5 0.8 2.8 1.9 -0.7 1.6 1.3 -2.1 0.4 Country Highschool - Dropouts Wage premium Relative supply Relative demand (σHD=2) Relative demand (σHD=3) Relative demand (σHD=4) Source: own computations based on SEDLAC (CEDLAS and World Bank). Note: annual log changes multiplied by 100. Household surveys used for each country: Argentina: 1992, 2000, 2009, 2013; Bolivia: 2001, 2009, 2012; Brazil: 1993, 2002, 2009, 2013; Chile: 1990, 2000, 2006, 2013; Colombia: 1996, 2001, 2008, 2013; Costa Rica: 1992, 2002, 2008, 2013; Ecuador: 2003, 2009, 2012; Honduras: 1993, 2003, 2009, 2013; Mexico: 1989, 2000, 2008, 2012; Nicaragua: 1993, 2001, 2009; Panama: 1992, 2002, 2009, 2013; Paraguay: 1995, 2003, 2009, 2013; Peru: 1997, 2002, 2009, 2013; El Salvador: 1995, 2002, 2009, 2013; 1992, 2002, 2009, 2013. Venezuela: 1995, 2002, 2008, 2011. Table 4.2: Changes in the wage premium and the relative supply and demand for unskilled workers (high school-dropouts) assuming alternative values for elasticity HD Table 4.2: Changes in the wage premium and the relative supply and demand for unskilled workers (high school-dropouts) assuming alternative values for elasticity HD urce: own computations based on SEDLAC (CEDLAS and World Bank). Note: annual log changes multiplied by 100. Figure 4.3: Relative demands over time (residual assuming σ=3 for both elasticities) Household surveys used for each country: Argentina: 1992, 2000, 2009, 2013; Bolivia: 2001, 2009, 2012; Brazil: 1993, 2002, 2009, 2013; Chile: 1990, 2000, 2006, 2013; Colombia: 1996, 2001, 2008, 2013; Costa Rica: 1992, 2002, 2008, 2013; Ecuador: 2003, 2009, 2012; Honduras: 1993, 2003, 2009, 2013; Mexico: 1989, 2000, 2008, 2012; Nicaragua: 1993, 2001, 2009; Panama: 1992, 2002, 2009, 2013; Paraguay: 1995, 2003, 2009, 2013; Peru: 1997, 2002, 2009, 2013; El Salvador: 1995, 2002, 2009, 2013; 1992, 2002, 2009, 2013. Venezuela: 1995, 2002, 2008, 2011. Note: annual log changes multiplied by 100. Household surveys used for each country: Argentina: 1992, 2000, 2009, 2013; Bolivia: 2001, 2009, 2012; Brazil: 1993, 2002, 2009, 2013; Chile: 1990, 2000, 2006, 2013; Colombia: 1996, 2001, 2008, 2013; Costa Rica: 1992, 2002, 2008, 2013; Ecuador: 2003, 2009, 2012; Honduras: 1993, 2003, 2009, 2013; Mexico: 1989, 2000, 2008, 2012; Nicaragua: 1993, 2001, 2009; Panama: 1992, 2002, 2009, 2013; Paraguay: 1995, 2003, 2009, 2013; Peru: 1997, 2002, 2009, 2013; El Salvador: 1995, 2002, 2009, 2013; 1992, 2002, 2009, 2013. Venezuela: 1995, 2002, 2008, 2011. 20 Table 5.1: Correlates of the skilled-unskilled wage premium Source: own computations based on SEDLAC (CEDLAS and World Bank) and WDI (World Bank). Notes: Robust standard errors in brackets, clustered at the country level. * significant at 10%; significant at 5%; * significant at 1%. Weights: inverse of the standard error of the skill premium squared (1) (2) (3) (4) (5) Relative Supply S-U -0.2111 -0.4651* -0.4928* -0.4792 -0.3705 [0.166] [0.082] [0.145] [0.145] [0.129] Constant 0.9220 0.6936 0.6333 0.1461 0.3451 [0.086] [0.021] [0.098] [1.189] [1.025] Country fixed effects No Yes Yes Yes Yes Year fixed effects No No Yes Yes Yes Countries 16 16 16 9 12 Observations 269 269 269 174 225 R-squared 0.058 0.928 0.952 0.966 0.960 Table 5.1: Correlates of the skilled-unskilled wage premium Source: own computations based on SEDLAC (CEDLAS and World Bank) and WDI (World Bank). Notes: Robust standard errors in brackets, clustered at the country level. * significant at 10%; significant at 5%; * significant at 1%. W i ht i f th t d d f th kill i d g g Weights: inverse of the standard error of the skill premium squared. Table 5.2: Correlates of the high school-dropout wage premium Source: own computations based on SEDLAC (CEDLAS and World Bank) and WDI (World Bank). %; s g ca t at %. se of the standard error of the skill premium squared. g ; g Weights: inverse of the standard error of the skill premium squared. own computations based on SEDLAC (CEDLAS and World Bank) and WDI (World Bank). Figure 4.3: Relative demands over time (residual assuming σ=3 for both elasticities) Notes: Robust standard errors in brackets, clustered at the country level. * significant at 10%; significant at 5%; * significant at 1%. Weights: inverse of the standard error of the skill premium squared. (1) (2) (3) (4) (5) Relative Supply H-D -0.0354 -0.2829* -0.2822* -0.3203* -0.2897* [0.057] [0.030] [0.053] [0.064] [0.060] Constant 0.4601 0.2378 0.2422 -0.4892 -0.5420 [0.085] [0.015] [0.054] [1.266] [1.177] Country fixed effects No Yes Yes Yes Yes Year fixed effects No No Yes Yes Yes Countries 16 16 16 9 12 Observations 269 269 269 174 225 R-squared 0.007 0.959 0.964 0.970 0.966 Table 5.2: Correlates of the high school-dropout wage premium 6 As in Katz and Murphy (1992), the base period is defined here as the average of all years for each country. Appendix: Empirical calculations of wage premium Wage premiums are estimated by means of a Mincer wage regression, based on individual worker’s microdata. The Mincer equation consists of a regression of the logarithm of the hourly wages on dummies for educational levels, a potential experience variable (constructed as age minus years of education minus 6) and its powers up to the order of 4, and a series of regional and urban-rural controls. The remuneration for each input (ln wkt) is computed from this regression as a weighted average of the returns to education of each of the educational levels belonging to the corresponding input. The wage premiums are then obtained as the difference of these remunerations. The regressions have the following form: (A1) it it it X D D D D D w t t t t t t t t t t                 c.pri c.pri i.sec i.sec c.sec c.sec i.coll i.coll c.coll c.coll ln (A1) it it X D D D D D w t t t t t t t t t t               c.pri c.pri i.sec i.sec c.sec c.sec i.coll i.coll c.coll c.coll ln where w is the wage for worker i at time t, the D variables are indicators for the level of educational attainment (college complete and incomplete, secondary complete and incomplete, and primary complete – primary incomplete is the omitted category) with their corresponding β coefficients, and the X variables represent a set of individual characteristics, which include years of experience, region of residence and urban/rural status (when available). Appendix: Empirical calculations of wage premium Defining skilled workers as those with some college (complete or incomplete) and the unskilled as those without any college education, the corresponding skilled-unskilled wage premium is given by:    t t t t t t t U S w w c.pri U c.pri i.sec U i.sec c.sec U c.sec i.coll S i.coll c.coll S c.coll γ γ γ γ γ ln                 (A2) (A2) where βit is the coefficient associated to educational level i in the Mincer equation at time t, and γik=Ei/Ek is the share of employment of the individuals with the educational level i in the employment of input k in a base period.6 Analogously, the high school-dropout wage premium or differential can be o Analogously, the high school-dropout wage premium or differential can be obtained as:   t t t Dt Ht w w c.pri D c.pri i.sec D i.sec c.sec γ γ ln             (A3)   t t t Dt Ht w w c.pri D c.pri i.sec D i.sec c.sec γ γ ln             (A3) (A3) 6 As in Katz and Murphy (1992), the base period is defined here as the average of all years for each country.
https://openalex.org/W3090617035	https://www.frontiersin.org/articles/10.3389/fchem.2020.589560/pdf	English	null	An Electrochemical Sandwich Immunosensor Based on Signal Amplification Technique for the Determination of Alpha-Fetoprotein	Frontiers in chemistry	2,020	cc-by	4,483	ORIGINAL RESEARCH published: 16 September 2020 doi: 10.3389/fchem.2020.589560 Edited by: Hassan Karimi-maleh, University of Electronic Science and Technology of China, China Reviewed by: Li Fu, Hangzhou Dianzi University, China Sadegh Salmanpour, Islamic Azad University Sari Branch, Iran Edited by: Hassan Karimi-maleh, University of Electronic Science and Technology of China, China Keywords: sandwich immunosensor, alpha-fetoprotein, electrochemical analysis, cancer biomarker, glassy carbon electrode Correspondence: Jianwei Jiang swpru5v2@21cn.com An Electrochemical Sandwich Immunosensor Based on Signal Ampliﬁcation Technique for the Determination of Alpha-Fetoprotein Changming Shen 1,2, Lin Wang 3, Hongyan Zhang 1,2, Shaojuan Liu 4 and Jianwei Jiang 1,2 1 Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Hangzhou, China, 2 Institute of Cancer and Basic Medicine (IBMC), Chinese Academy of Sciences, Hangzhou, China, 3 The Third Afﬁliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China, 4 The Third People’s Hospital of Hangzhou, Hangzhou, China The synthesis of Au nanocubes is used to label alpha-fetoprotein antibody (anti-AFP) and horseradish peroxidase (HRP) to form an immune complex for antibody detection. Graphene oxide-methylene blue-gold nanoparticles (GO-MB-AuNPs) nanocomposites were used as the immunosensing platform. This proposed sandwich-type immunoassay shows good performance. This method establishes a feasible amperometric immunoassay method for sensitive analysis of AFP in serum samples. Under the optimal experimental conditions, the DPV current response of the immunosensor is proportional to the logarithmic value of the AFP concentration. The linear detection range can achieve to 0.005–20 ng/mL with a detection limit of 1.5 pg/mL. The proposed immunosensor has good precision, selectivity and stability, and can be used for AFP determination in clinical tests. INTRODUCTION Alpha-fetoprotein (AFP) is a glycoprotein, mainly produced by embryonic liver cells. About 2 weeks after birth, AFP disappears from the blood. Under normal circumstances, the content of AFP in adult serum is <25 ng/mL. However, it is elevated in the blood of about 80% of liver cancer patients. When liver cells become cancerous, they restore their ability to produce AFP. When the condition gradually worsens, the content will increase substantially (Li et al., 2017, 2020; Yang et al., 2018; Ma L. et al., 2019). Therefore, AFP can be used as a speciﬁc clinical detection index for the diagnosis of primary liver cancer. AFP can be detected in multiple channels, generally including ﬂuorescence analysis, enzyme-linked immunoassay, chemiluminescence analysis, chromatography, radioimmunoassay, enzyme-labeled electrophoresis, and electrochemical analysis. From the analysis of markers, it can be divided into labeled AFP sensors and label-free AFP sensors (Fang et al., 2017; Wang et al., 2017; Liu et al., 2018; Fu et al., 2019a; Shamsadin-Azad et al., 2019). Specialty section: This article was submitted to Electrochemistry, a section of the journal Frontiers in Chemistry Received: 31 July 2020 Accepted: 17 August 2020 Published: 16 September 2020 MATERIALS AND METHODS an immune response dominated by antibodies and lymphocytes (Alizadeh et al., 2018; Karimi-Maleh et al., 2020a,b). Immunosensor is a detection device designed using the principle of speciﬁc recognition and binding of antigen and antibody. Antigen and antibody molecules are ﬁxed on the surface of the electrode in some form, and form a stable complex with the corresponding antibody or antigen to be detected (Wang et al., 2018; Fu et al., 2020a,b; Karimi-Maleh and Arotiba, 2020). These compounds can cause changes in sensor electrical signals, such as electrode potential or current and capacitance. Graphene oxide (GO) was purchased from Xianfeng Nano Co., Ltd. HAuCl4·4H2O was purchased from Shanghai Yuanye Biological Co., Ltd. Methylene blue (MB), cetyltrimethylammonium bromide (CTAB), NaBH4, and trisodium citrate were purchased from Sinopharm Chemical Co., Ltd. The AFP standard solution and mouse monoclonal antibody (anti-AFP) were purchased from Zhengzhou Bosai Biotechnology Co., Ltd. All reagents were analytical grade. The electrochemical experiment was carried out on the CHI 760C electrochemical analyzer (Shanghai Chenhua Instrument Co., Ltd., China). Three electrode system was adopted: modiﬁed electrode as working electrode, platinum wire electrode as counter electrode, saturated calomel electrode as reference electrode (SCE). The solution used in the electrochemical impedance analysis was 0.1 M KCl containing 10 mM K4[Fe(CN)6] and 10 mm K3[Fe(CN)6]. Both cyclic voltammetry (CV) and diﬀerential pulse voltammetry (DPV) experiments use 0.1 M PBS as the test solution (containing 0.1 M KCl, pH 6.4). Immunolabeling mainly refers to labeling antibodies or antigens in speciﬁc reactions to catalyze reactions and improve sensor sensitivity. Common markers include luciferin, enzymes, radioisotopes, nanomaterials, and electronic dense substances. The label sensor is highly sensitive and speciﬁc, which has been widely used (Peng et al., 2018; Fu et al., 2019b; Ge et al., 2019; Ma N. et al., 2019; Hu et al., 2020; Ying et al., 2020). When technology enters the nano-era, a large number of nanomaterials were used in biosensors (Baghayeri et al., 2018; Feng et al., 2020; Hojjati-Najafabadi et al., 2020; Hou et al., 2020; Karimi-Maleh et al., 2020c). Therefore, in addition to the above immunolabels, many researchers use nanomaterials as labels to expand the antigen and antibody immune response signals. The preparation of Au cubes was according to the literature. The formed Au cubes were then dispersed into 0.1 M PBS with the addition of HRP and anti-AFP. The mixed solution was stirred under 4◦C overnight to form AuC-HRP-anti-AFP. Citation: Shen C, Wang L, Zhang H, Liu S and Jiang J (2020) An Electrochemical Sandwich Immunosensor Based on Signal Ampliﬁcation Technique for the Determination of Alpha-Fetoprotein. Front. Chem. 8:589560. doi: 10.3389/fchem.2020.589560 Immunosensor is a combination of sensing technology and speciﬁc immune response to detect the reaction between antigen and antibody. A healthy body can defend against the invasion of microorganisms, viruses, and other harmful substances through a variety of mechanisms, including natural immunity and acquired immunity. Antigens are exogenous substances that can induce speciﬁc immunity, which can stimulate the immune system of animals and plants and produce September 2020 \| Volume 8 \| Article 589560 Frontiers in Chemistry \| www.frontiersin.org Immunosensor for Alpha-Fetoprotein Determination Shen et al. Frontiers in Chemistry \| www.frontiersin.org RESULTS AND DISCUSSION FIGURE 2 \| UV-vis spectra of GO, MB AuNPs, and GO-MB-AuNPs nanocomposite. FIGURE 2 \| UV-vis spectra of GO, MB AuNPs, and GO-MB-AuNPs nanocomposite. Figure 1A shows the SEM image of the formed Au cubes. The diameter of the Au cubes was at an average of 80 nm, in which the structure facilitates the immobilization of anti-AFP antibody and HRP enzyme (Yu et al., 2017). Diﬀerent substances can produce diﬀerent UV-visible absorption peaks due to their diﬀerent structures. It can be seen from Figure 1B that there are two obvious absorption peaks at 460 nm and 532 nm in the UV absorption peak of Au cubes (Zhang et al., 2020). As shown in Figure 2, GO has an adsorption peak around 230 nm, while MB has two absorption peaks at around 290 and 650 nm. AuNPs have speciﬁc ultraviolet absorption peaks between 500 and 600 nm due to their diﬀerent particle sizes. The preparation of GO-MB-AuNPs nanocomposites was investigated by UV-vis spectrometer as well (Yang et al., 2017). There are four absorption peaks of GO-MB-AuNPs nanocomposites, which are located at 232, 290,527, and 644 nm respectively, indicating the formation of GO-MB-AuNPs nanocomposites. FIGURE 2 \| UV-vis spectra of GO, MB AuNPs, and GO-MB-AuNPs nanocomposite. FIGURE 2 \| UV-vis spectra of GO, MB AuNPs, and GO-MB-AuNPs nanocomposite. p Cyclic voltammetry (CV) was used to investigate the electrochemical behavior of modiﬁed electrodes at diﬀerent preparation stages in pH 7.0 PBS (containing 4 mM H2O2). As shown in Figure 3, the bare GCE has no redox peak in the PBS solution. When the GO-MB-AuNPs nanocomposite material was modiﬁed on the electrode surface, a pair of stable redox peaks appeared on the electrode (Zhang et al., 2019). This is the redox of MB immobilized on graphene oxide. After GO-MB-AuNPs capture the anti-AFP antibody and AFP antigen, the redox peak current on the electrode decreases successively, which proves that the antibody and the generated antigen-antibody immune complex have poor conductivity and hinder the electrons transfer on the electrode surface. FIGURE 3 \| CVs of bare GCE, GO-MB-AuNPs/GCE, anti-AFP-GO-MB-AuNPs/GCE, AFP-anti-AFP-O-MB-AuNPs/GCE, and AuC-HRP-anti-AFP/AFP-anti-AFP-O-MB-AuNPs/GCE. EIS can give information about the impedance change on the interface of the electrode during the modiﬁcation process (Naderi Asrami et al., 2020). MATERIALS AND METHODS In this work, we synthesized Au nanocubes, which are used to eﬀectively label biomacromolecules in electrochemical immunosensors and immunoassays. At the same time, they can also have a signiﬁcant catalytic eﬀect on the response of the sensor, thereby signiﬁcantly improving the electrochemical response of the sensor. We then use Au nanocubes to immobilize both AFP antibody and horseradish peroxidase (HRP) as the detection antibody labeling material. In the presence of AFP antigen, a sandwich immunosensor analysis mode is used to perform sensitive and quantitative detection of AFP. The immunosensor has few consumables, high sensitivity and high selectivity, and can be applied to clinical detection. GO-MB-AuNPs were prepared by mixing of three substances into a 0.1 M PBS overnight. The composite was obtained using a centrifugation process. Then, a certain amount of GO-MB- AuNPs was dip coated on the GCE surface and dried naturally. Ten microliter of anti-AFP was dip coated on the modiﬁed electrode and incubated overnight. Then, the electrode was immersed into a BSA solution for removing the excess of anti- AFP. During the AFP sensing, the standard AFP solution was dip-coated on the above-mentioned electrode and incubated for 30 min. Then, the AuC-HRP-anti-AFP was dip-coated on the electrode to forming the sandwich sensor. The DPV scan was recorded in a 0.1 M PBS (containing 4 mM H2O2). FIGURE 1 \| (A) SEM and (B) UV-vis spectrum of Au cubes. FIGURE 1 \| (A) SEM and (B) UV-vis spectrum of Au cubes. September 2020 \| Volume 8 \| Article 589560 Frontiers in Chemistry \| www.frontiersin.org 2 Immunosensor for Alpha-Fetoprotein Determination Shen et al. FIGURE 2 \| UV-vis spectra of GO, MB AuNPs, and GO-MB-AuNPs nanocomposite. FIGURE 3 \| CVs of bare GCE, GO-MB-AuNPs/GCE, anti-AFP-GO-MB-AuNPs/GCE, AFP-anti-AFP-O-MB-AuNPs/GCE, and AuC-HRP-anti-AFP/AFP-anti-AFP-O-MB-AuNPs/GCE. Frontiers in Chemistry \| www.frontiersin.org RESULTS AND DISCUSSION The semicircle diameter of EIS is equal to the electron transfer resistance (Ret), which controls the electron transfer kinetics of the redox probe on the electrode surface (Campuzano et al., 2017; Ganbat et al., 2020). The EIS results were consistent with the CV results, as shown in Figure 4. These results fully indicate that the electrode preparation was successful and the sandwich-type immunosensor can be formed in turn. The synthesized Au nanocubes have high conductivity and electron transfer eﬃciency, which facilitates the exchange of electrons between the solution and the substrate electrode. FIGURE 3 \| CVs of bare GCE, GO-MB-AuNPs/GCE, anti-AFP-GO-MB-AuNPs/GCE, AFP-anti-AFP-O-MB-AuNPs/GCE, and AuC-HRP-anti-AFP/AFP-anti-AFP-O-MB-AuNPs/GCE. FIGURE 3 \| CVs of bare GCE, GO-MB-AuNPs/GCE, anti-AFP-GO-MB-AuNPs/GCE, AFP-anti-AFP-O-MB-AuNPs/GCE, and AuC-HRP-anti-AFP/AFP-anti-AFP-O-MB-AuNPs/GCE. We used the CV method to optimize the amount of modiﬁer, incubation time, pH, and H2O2 concentration of the GO- MB-AuNPs nanocomposite. The experimental results show that as the amount of GO-MBAuNPs nanocomposite gradually increases, the peak current gradually increases and the peak current reaches the maximum at 5 µL. When it continues to increase, the peak current decreases instead. Therefore, 5 µL is the best amount of modiﬁer in this work. The incubation time is the time for the antigen-antibody immune reaction on the electrode surface. The results show that the maximum current can be reached when the antigen and antibody reacted for 30 min. There is almost no change in peak current over 30 min. Therefore, we selected 30 min as the incubation time. At the same time, we tested the peak current response of the electrolyte immunosensor at diﬀerent pH. At pH 5.5–7.0, the peak current of the sensor gradually increases and then gradually decreases. The results prove that pH 7.0 is the optimal pH for the antigen-antibody reaction in this environment, which can ensure the good activity of antigen and antibody. HRP catalyzes H2O2 as an important part of signal ampliﬁcation. The peak current of the sensor increases as the concentration of H2O2 increases, and reaches the maximum value at 4 mM. Therefore, 4 mM is used as the optimal concentration of H2O2 in subsequent experiments. At the same time, we tested the peak current response of the electrolyte immunosensor at diﬀerent pH. At pH 5.5–7.0, the peak current of the sensor gradually increases and then gradually decreases. RESULTS AND DISCUSSION The results prove that pH 7.0 is the optimal pH for the antigen-antibody reaction in this environment, which can ensure the good activity of antigen and antibody. HRP catalyzes H2O2 as an important part of signal ampliﬁcation. The peak current of the sensor increases as the concentration of H2O2 increases, and reaches the maximum value at 4 mM. Therefore, 4 mM is used as the optimal concentration of H2O2 in subsequent experiments. September 2020 \| Volume 8 \| Article 589560 3 Immunosensor for Alpha-Fetoprotein Determination Shen et al. Shen et al. Immunosensor for Alpha Fetoprotein Determination FIGURE 4 \| EIS of bare GCE, GO-MB-AuNPs/GCE, anti-AFP-O-MB-AuNPs/GCE, AFP-anti-AFP-O-MB-AuNPs/GCE, and AuC-HRP-anti-AFP/AFP-anti-AFP-O-MB-AuNPs/GCE. FIGURE 5 \| (A) DPV curves of the proposed immunosensor for different concentrations of AFP detection from 0.005 to 20 ng/mL. (B) Corresponding linear relationship between the AFP concentrations and peak currents. Under the optimum conditions, we used the proposed immunosensor to detect AFP standard solutions of diﬀerent concentrations. Figure 5A shows the DPV graph of the sensor to diﬀerent concentrations of AFP. When the concentration between the AFP concentrations and the peak current. The limit of detection was calculated to be 1.5 pg/mL. Compared with other related reports (Jiao et al., 2016; Wei et al., 2016; Yuan et al., 2017; Li et al., 2018), this experimental result has FIGURE 4 \| EIS of bare GCE, GO-MB-AuNPs/GCE, anti-AFP-O-MB-AuNPs/GCE, AFP-anti-AFP-O-MB-AuNPs/GCE, and AuC-HRP-anti-AFP/AFP-anti-AFP-O-MB-AuNPs/GCE. FIGURE 5 \| (A) DPV curves of the proposed immunosensor for different concentrations of AFP detection from 0.005 to 20 ng/mL. (B) Corresponding linear relationship between the AFP concentrations and peak currents. FIGURE 5 \| (A) DPV curves of the proposed immunosensor for different concentrations of AFP detection from 0.005 to 20 ng/mL. (B) Corresponding linear relationship between the AFP concentrations and peak currents. FIGURE 5 \| (A) DPV curves of the proposed immunosensor for different concentrations of AFP detection from 0.005 to 20 ng/mL. (B) Corresponding linear relationship between the AFP concentrations and peak currents. between the AFP concentrations and the peak current. The limit of detection was calculated to be 1.5 pg/mL. Compared with other related reports (Jiao et al., 2016; Wei et al., 2016; Yuan et al., 2017; Li et al., 2018), this experimental result has a wider linear range and lower detection. REFERENCES based on the enhanced electrochemical ﬁngerprint recorded from plant tissue. Biosens. Bioelectron. 159:112212. doi: 10.1016/j.bios.2020.112212 based on the enhanced electrochemical ﬁngerprint recorded from plant tissue. Biosens. Bioelectron. 159:112212. doi: 10.1016/j.bios.2020.112212 Alizadeh, N., Salimi, A., and Hallaj, R. (2018). Magnetoimmunosensor for simultaneous electrochemical detection of carcinoembryonic antigen and α- fetoprotein using multifunctionalized Au nanotags. J. Electroanal. Chem. 811, 8–15. doi: 10.1016/j.jelechem.2017.12.080 Ganbat, K., Pan, D., Chen, K., Ning, Z., Xing, L., Zhang, Y., et al. (2020). One-pot electrografting preparation of bifunctionalized carbon nanotubes for sensitive electrochemical immunosensing. J. 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Layer-by-layer self-assembly of MoS2/PDDA hybrid ﬁlm in microﬂuidic chips for ultrasensitive electrochemical immunosensing of alpha-fetoprotein. Microchem. J. 158:105209. doi: 10.1016/j.microc.2020.105209 Feng, Y., Wei, Z., and Zhang, J. (2020). Determination of ursolic acid in extracts from ligustri lucidum fruit using an electrochemical method. Front. Chem. 8:444. doi: 10.3389/fchem.2020.00444 Fu, L., Liu, Z., Ge, J., Guo, M., Zhang, H., Chen, F., et al. (2019a). (001) plan manipulation of α-Fe2O3 nanostructures for enhanced electrochemical Cr(VI) sensing. J. DATA AVAILABILITY STATEMENT The proposed electrochemical immunosensor was used to detect the AFP in the serum samples by the standard addition method. The results were shown in Table 1. The recovery rate obtained from the experimental results was 97.83–101.75%, indicating that the immunosensor has the potential for clinic AFP determination. The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s. CONCLUSION Experimental results show that Au cubes were ideal material for immobilizing antigen-antibody, which can greatly improve the response signal of the electrode. The proposed sandwich-type immunosensor has higher sensitivity. The detection of AFP also has a wide linear range and a low detection limit, and a good recovery rate is obtained when the actual sample is detected. improves the sensitivity and signal response of the electrode, so that the electrode has a wider linear range and lower detection limit. RESULTS AND DISCUSSION The results show that Au nanostructures can be used as a labeling material to immobilize more antibodies and enzymes, which greatly Under the optimum conditions, we used the proposed immunosensor to detect AFP standard solutions of diﬀerent concentrations. Figure 5A shows the DPV graph of the sensor to diﬀerent concentrations of AFP. When the concentration of AFP is between 0.005 and 20 ng/mL, the peak current of DPV increases with the increase of AFP concentration with a good linear relationship. Figure 5B shows the linear relationship September 2020 \| Volume 8 \| Article 589560 Frontiers in Chemistry \| www.frontiersin.org 4 Immunosensor for Alpha-Fetoprotein Determination Shen et al. TABLE 1 \| Detection of AFP in serum samples with recovery rate. Concentration (ng/mL) Add (ng/mL) Found (ng/mL) Recover (%) RSD (%) 2 2 4.07 101.75 3.37 5 2 6.87 98.14 5.20 10 2 11.74 97.83 2.66 TABLE 1 \| Detection of AFP in serum samples with recovery rate. successive measurements for each electrode was ∼2.61%, suggesting acceptable repeatability. \| p y Concentration (ng/mL) Add (ng/mL) Found (ng/mL) Recover (%) RSD (%) Concentration (ng/mL) Add (ng/mL) Found (ng/mL) Recover (%) RSD (%) AUTHOR CONTRIBUTIONS The reproducibility is a key issue of the electrochemical sensor. For this purpose, the current response of the four sensors prepared at the same conditions was measured. The results displayed that the relative standard deviation (RSD) of electrochemical response for four sensors was about 3.31%, indicating that the immunosensor had a good reproducibility. Meanwhile, the RSD obtained from six JJ contributed the conception and design of the study. CS and LW conducted the electrochemical experiments. 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Development of an electrochemical biosensor for phylogenetic analysis of Amaryllidaceae September 2020 \| Volume 8 \| Article 589560 Frontiers in Chemistry \| www.frontiersin.org 5 Immunosensor for Alpha-Fetoprotein Determination Shen et al. Wang, Y., Zhao, G., Wang, H., Cao, W., Du, B., and Wei, Q. (2018). Sandwich- type electrochemical immunoassay based on Co3O4@ MnO2-thionine and pseudo-ELISA method toward sensitive detection of alpha fetoprotein. Biosens. Bioelectron. 106, 179–185. doi: 10.1016/j.bios.2018.02.002 Karimi-Maleh, H., Karimi, F., Malekmohammadi, S., Zakariae, N., Esmaeili, R., Rostamnia, S., et al. (2020b). An ampliﬁed voltammetric sensor based on platinum nanoparticle/polyoxometalate/two-dimensional hexagonal boron nitride nanosheets composite and ionic liquid for determination of N-hydroxysuccinimide in water samples. J. Mol. Liq. 310:113185. doi: 10.1016/j.molliq.2020.113185 Wei, Y., Li, Y., Li, N., Zhang, Y., Yan, T., Ma, H., et al. (2016). 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https://openalex.org/W2301875775	https://figshare.com/articles/journal_contribution/p73_promotes_glioblastoma_cell_invasion_by_directly_activating_POSTN_periostin_expression/10175966/1/files/18339101.pdf	English	null	p73 promotes glioblastoma cell invasion by directly activating POSTN (periostin) expression	Oncotarget	2,016	cc-by	11,999	p73 promotes glioblastoma cell invasion by directly activating POSTN (periostin) expression Vivien Landré1, Alexey Antonov1, Richard Knight1 and Gerry Melino1,2 1 Medical Research Council Toxicology Unit, Leicester LE1 9HN, UK 2 University of Rome Tor Vergata, 00133-Rome, Italy Correspondence to: Gerry Melino, email: gm89@le.ac.uk Keywords: periostin, p53 family, cell death, temozolomide, metastasis Received: September 29, 2015 Accepted: January 18, 2016 Published: February 22, 2016 , y o o , 1 Medical Research Council Toxicology Unit, Leicester LE1 9HN, UK 2 University of Rome Tor Vergata, 00133-Rome, Italy Correspondence to: Gerry Melino, email: gm89@le.ac.uk Keywords: periostin, p53 family, cell death, temozolomide, metastasis Received: September 29, 2015 Accepted: January 18, 2016 Abstract Glioblastoma Multiforme is one of the most highly metastatic cancers and constitutes 70% of all gliomas. Despite aggressive treatments these tumours have an exceptionally bad prognosis, mainly due to therapy resistance and tumour recurrence. Here we show that the transcription factor p73 confers an invasive phenotype by directly activating expression of POSTN (periostin, HGNC:16953) in glioblastoma cells. Knock down of endogenous p73 reduces invasiveness and chemo-resistance, and promotes differentiation in vitro. Using chromatin immunoprecipitation and reporter assays we demonstrate that POSTN, an integrin binding protein that has recently been shown to play a major role in metastasis, is a transcriptional target of TAp73. We further show that POSTN overexpression is sufficient to rescue the invasive phenotype of glioblastoma cells after p73 knock down. Additionally, bioinformatics analysis revealed that an intact p73/ POSTN axis, where POSTN and p73 expression is correlated, predicts bad prognosis in several cancer types. Taken together, our results support a novel role of TAp73 in controlling glioblastoma cell invasion by regulating the expression of the matricellular protein POSTN. Oncotarget, Vol. 7, No. 11 Oncotarget, Vol. 7, No. 11 www.impactjournals.com/oncotarget/ Introduction to target GBM [11-13]. While differentiation therapy is successfully used to treat acute promyeloctic leukaemia, where it leads to a dramatic increase of patient survival [14], no drug that induces differentiation in solid tumours has yet been developed. This is mainly due to a lack of development-based classification of these tumours, which often have considerably more varied genetic abnormalities compared to acute promyeloctic leukaemia, as well as our limited understanding of the pathways and components involved in the differentiation of different cancer types [14]. Glioblastoma Multiforme (GBM) is the most common malignant brain tumour in adult patients with an extremely poor prognosis of only 15 month median survival after diagnosis [1]. Despite aggressive treatment, usually consisting of tumour resection followed by radiation- and chemotherapy, tumour recurrence and therapeutic resistance is exceptionally common, making these tumours de facto incurable [2-6]. Several studies over the last decade have suggested that GBM develops from a subsection of tumour cells called stem-like glioblastoma cells [7-9]. These cells, characterised by a self-renewing, often chemo- resistant and immature differentiation phenotype, have tumour-initiating properties and are largely believed to be responsible for tumour recurrence and therapeutic resistance. Even single cells that survive therapy have the ability to initiate growth of a new tumour, and are therefore also called tumour-seeding cells [2, 10]. This suggests that differentiation therapies that induce cancer stem-cell differentiation would be a promising approach The transcription factor p73 is a member of the p53 family, which comprises three members p53, p63 and p73 that share similarities in their structure and function [15-19]. Having been discovered already in 1979, p53 is the most studied component of this family of transcription factors, showing a very complex gene activation program spanning from autophagy [20, 21], ROS, metabolism and mitochondria regulation [22-24], DNA damage response [25-29] to stemness and lineage determination [30-32]. Despite 35 years of high quality research, several crucial issues remain unanswered www.impactjournals.com/oncotarget Oncotarget 11785 originating in the brain. Several studies in GBM [86- 88] revealed a correlation between mRNA levels of the TAp73 and ΔNp73 isoforms and tumour grade as well as patient survival, leading to the suggestion that p73 may be a potential biomarker of GBM tumour grade. Furthermore, analysis of datasets of glioma patients showed a correlation between p73 gene deletion and longer survival (Figure 1A). Introduction Therefore, as a first step to understand the potential mechanism for this correlation, we knocked down total p73 mRNA using siRNA in the U251 and U87 glioblastoma cell lines (Figures 1B, S1, S2A). Strikingly, p73 knock down resulted in a dramatic change of cell morphology, from a flat polygonal shape to a small, round cell body with very long and fine processes, an appearance, which resembles that of mature astrocytes. To further demonstrate that the change in morphology is due to cell differentiation, glioblastoma cells, transfected with control siRNA or siRNA specific for total p73, were stained using a GFAP (glial fibrillary acidic protein) antibody (Figures 1C, S2B). GFAP is an intermediate filament that is highly expressed in differentiated astrocytes. The GFAP signal in the cell processes was quantified, and the results showed that GFAP expression is higher in the cells in which p73 had been knocked down (Figure 1C, right panel). This observation suggests that glioblastoma cells may differentiate into astrocytes in the absence of p73. This is also in line with previous studies published by our group showing a decrease in stemness markers in neurons isolated from p73 KO compared to wt mice [89] and reduced Nestin expression, a neuronal stem cell marker, in the dentate gyrus of p73 KO mice [90]. in order to fully understand the biological role and function of this tumour suppressor. In fact p53, as well as its family members, shows an extreme complexity, including, for example, its stability and degradation [33- 37], its connection and regulation to micro-RNA [38- 44] and its splicing isoforms [45, 46]. In parallel to the advances in p53 biology, crucial progress is also under way for its therapeutic exploitation [47-56]. Although having been identified much later, p63 and p73 already show their complexity and interaction with p53 [57-63]; where p63 function is highly relevant in skin formation and homeostasis [64] as well as in cancer [63, 65, 66]. p73 exists in a variety of isoforms which can be divided into transactivation (TA) domain containing isoforms (TAp73) and those lacking the TA domain (ΔNp73), and each of these can be expressed as a number of C-terminal isoforms to exert distinct functions [67-74]. Depending on cell type and p73 isoform, the protein has been shown to exhibit both tumour suppressive and oncogenic functions [75- 77]. Results The fact that stem-like glioblastoma cells are generally associated with a mesenchymal phenotype, and that p73 affects the differentiation status of glioblastoma cells, suggests that p73 can affect epithelial to mesenchymal transition (EMT). We therefore performed western blot analysis of different EMT markers after total p73 knock down. As shown in Figure 2A (and S3A), transient knock down of p73 led to a remarkable reduction of SNAIL, an important factor in the delamination process in neuronal tissue development, together with up-regulation of E-cadherin [92, 93]. We observed no Introduction Unlike p53, p73 is rarely mutated in human cancers and both the TA and ΔN isoforms have been shown to be overexpressed in several tumour types [78, 79]. p73 deficient mice have striking developmental deficiencies in the CNS, mainly characterised by cortical loss leading to ex vacuo hydrocephalus as well as dysgenesis of the hippocampus and caudal cortex [80, 81]. Interestingly, these severe neurological deficiencies are phenocopied neither by the ΔNp73 nor the TAp73 KO mice, suggesting redundancy of the two isoforms during neurogenesis [82]. TAp73 KO mice display several abnormalities in the hippocampus, while ΔNp73 KO mice are healthy and show a relatively mild neurological phenotype with loss of the hydrocephalus, but no cortical loss [83-85]. As these results suggest a role of p73 in regulating the differentiation status of glioblastoma cells, we were interested in the effect of induced differentiation on p73 protein levels. To study this, differentiation of glioblastoma cells was induced by serum withdrawal (Figure 1E, 1F) and p73 levels were determined by western blotting (Figure 1D). Serum withdrawal led to almost complete loss of p73 protein expression, while no change in p73 protein levels was seen when serum was replaced by growth factors and supplements promoting stem-cell-like properties [91]. Here we demonstrate that p73 plays a role in glioblastoma cell differentiation, since loss of p73 in glioblastoma cells leads to a more differentiated phenotype with reduced migration and invasion abilities. Furthermore, we show that the extracellular protein POSTN (periostin, osteoblast specific factor, HGNC:16953), that has been shown to play a role in glioblastoma carcinogenesis and is an important factor in metastasis, is transcriptionally activated by p73, and that this activation leads to increased cell invasion of glioblastoma cells in vitro. Loss of p73 leads to glioblastoma cell differentiation Although the role of p73 in neuronal development and differentiation has been studied extensively in recent years, relatively little attention has been paid to its role in the development and metastasis of malignancies www.impactjournals.com/oncotarget Oncotarget 11786 Figure 1: P73 knock down induces morphological transformation of glioblastoma cells. A. p73 gene deletion is correlated with prolonged survival of glioblastoma patients. Data is from the REMBRANT database/NIH (https://caintegrator.nci.nih.gov/rembrandt/). The p-value corresponds to the gene deletion group in comparison to all other patients. B. Morphological changes of U251 cells after 72 h of p73 knock down using siRNA transfection. C. Cells as in B but fixed and stained with an anti-GFAP antibody. Pictures were taken using the Cellomics, GFAP signal was quantified and is expressed as arbitrary units (right panel). D. Cells were grown in media that was serum rich (10%), serum free or media complemented with growth factors (GF) (20 ng/ml EGF and FGF) and p27 supplement. Total protein was extracted and blotted with antibodies against p73 and GAPDH. E. Cells were incubated in serum free medium or full medium for 72 h and visualised and quantified (F) using a GFAP antibody as in C. Scale Bars in B, C and E represent 100 µm. p < 0.0001. Error bars represent SEM. Figure 1: P73 knock down induces morphological transformation of glioblastoma cells. A. p73 gene deletion is correlated with prolonged survival of glioblastoma patients. Data is from the REMBRANT database/NIH (https://caintegrator.nci.nih.gov/rembrandt/). The p-value corresponds to the gene deletion group in comparison to all other patients. B. Morphological changes of U251 cells after 72 h of p73 knock down using siRNA transfection. C. Cells as in B but fixed and stained with an anti-GFAP antibody. Pictures were taken using the Cellomics, GFAP signal was quantified and is expressed as arbitrary units (right panel). D. Cells were grown in media that was serum rich (10%), serum free or media complemented with growth factors (GF) (20 ng/ml EGF and FGF) and p27 supplement. Total protein was extracted and blotted with antibodies against p73 and GAPDH. E. Cells were incubated in serum free medium or full medium for 72 h and visualised and quantified (F) using a GFAP antibody as in C. Scale Bars in B, C and E represent 100 µm. p < 0.0001. Error bars represent SEM. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 11787 of glioblastoma cells. Cell migration was assayed using the xCELLigence system, whereby cells that migrated through a membrane, from serum free towards serum containing media, were quantified. The assay showed a change in other EMT marker i.e. Twist and Vimentin (data not shown). EMT is generally associated with an invasive phenotype, and we therefore wanted to establish whether p73 affects the migration and/or invasion ability Oncotarget 11788 www.impactjournals.com/oncotarget whether p73 affects the migration and/or invasion ability containing media, were quantified. The assay showed a Figure 2: P73 knock down reduces migration and invasion of glioblastoma cells. A. Whole protein extract of U251 cells 72 h post-transfection with scr or p73 siRNA was analysed by immunoblotting with antibodies against p73, SNAIL, E-cadherin and GAPDH. Line indicates where 2 lanes that are not next to each other on the gel were moved side by side (see Figure S3A for full scan). B. The migration ability of U251 cells after siRNA transfection (scr or p73) was determined using the xCelligence. C. Invasion into matrigel of U251 cells that were transfected with siRNAs targeting p73 or a non-targeting control is shown. D. As in C but cells were transiently transfected with plasmids encoding TAp73α, ΔNp73α or empty vector control. P-values shown are for EV compared to TAp73 and EV compared ΔNp73. p < 0.1, p < 0.05. Error bars represent SEM. nd invasion of glioblastoma cells. A. Whole protein extract of U251 cells 72 by immunoblotting with antibodies against p73, SNAIL, E-cadherin and GAPDH. other on the gel were moved side by side (see Figure S3A for full scan). B. The n (scr or p73) was determined using the xCelligence. C. Invasion into matrigel ng p73 or a non-targeting control is shown. D. As in C but cells were transiently r empty vector control. P-values shown are for EV compared to TAp73 and EV Figure 2: P73 knock down reduces migration and invasion of glioblastoma cells. A. Whole protein extract of U251 cells 72 h post-transfection with scr or p73 siRNA was analysed by immunoblotting with antibodies against p73, SNAIL, E-cadherin and GAPDH. Line indicates where 2 lanes that are not next to each other on the gel were moved side by side (see Figure S3A for full scan). B. The migration ability of U251 cells after siRNA transfection (scr or p73) was determined using the xCelligence. C. www.impactjournals.com/oncotarget Invasion into matrigel of U251 cells that were transfected with siRNAs targeting p73 or a non-targeting control is shown. D. As in C but cells were transiently transfected with plasmids encoding TAp73α, ΔNp73α or empty vector control. P-values shown are for EV compared to TAp73 and EV compared ΔNp73. p < 0.1, *p < 0.05. Error bars represent SEM. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 11788 the control cells (Figure 3A). Using a pathway analysis tool [94], we identified the pathways most enriched in differentially expressed genes to be: Integrin binding, fibronectin binding, blood coagulation and cell adhesion (Figure 3A-3C). This molecular data is in agreement with the biological effect of p73 in cell migration, as integrin and fibronectin as well as cell adhesion pathways are all involved in cell mobility, and alterations of these pathways have been shown to be involved in metastasis of cancer cells [95]. To verify the results of the microarray we tested the expression of the top ten down- and seven up-regulated genes and found that we could validate ~ 73 % of the genes in the array (Figure S4). striking reduction in cell migration ability after knock down of p73 (Figure 2B). Similarly, in an invasion assay, where the ability of the cells to enter and invade a matrigel matrix was assessed, knock down of total p73 resulted in consistent reduction of invasion (Figure 2C), whereas overexpression of p73 isoforms led to an increase in matrigel invasion (Figures 2D, S3B). Together these results demonstrate a role of p73 in glioblastoma cell morphology, associated with a more invasive phenotype. To gain insight into the molecular mechanisms underlying these changes of U251 morphology and invasion, we performed a gene microarray analysis of U251 cells transfected with siRNA for total p73 or a scrambled sequence for 72 h. We found 632 genes differentially expressed in the knock down compared to Figure 3: Analysis of mRNA expression after p73 knock down suggests a role for p73 in cell migration and invasion. A. Results of the microarray revealed 632 genes that were differentially expressed in control cells compared to knock down of endogenous p73 (Schematic illustration of gene array results). Pathway analysis of microarray results comparing U251 transfected with scr siRNA and p73 siRNA indicated changes in the pathways shown. Odds ratios for pathways are Integrin binding = 4.4, Fibronectin binding 9.05 and Cell adhesion = 2.04. p- values refer to the significance of enrichment of genes of the pathways shown. Heat maps of B. “Integrin and Fibronectin binding” (combined), 10 genes for integrin binding and 5 genes for fibronectin binding were identified, while CTGF is involved in both pathways. C. “Cell Adhesion” genes identified by GO term analysis, as differentially expressed between si scr and si p73. www.impactjournals.com/oncotarget Expression of POSTN mRNA 72 h after transfection with p73 or scr siRNA was determined using RT- qPCR in U251 (left panel) and U87 (right panel) cells. C. U251 protein extracts of cells transfected with siRNA for p73 or a scrambled control and analysed by immunoblotting using p73, POSTN and β-tubulin antibodies. Asterisk indicates an unspecific band of the p73 antibody. D. Expression of POSTN mRNA 24 h after transfection with plasmids encoding TAp73α, ΔNp73α or an empty vector control was determined using RT-qPCR in U251 (left panel) and U87 (right panel) cells. E. Analysis of the POSTN promoter revealed one potential binding site for transcription factors of the p53 family 600 bp upstream of the transcriptional start site. F. ChIP assay demonstrating binding of TAp73α to the POSTN promoter in U251 cells. G. U251 (left panel) or U87 (right panel) cells were transfected with p73 constructs (TAp73α or ΔNp73α) plus POSTN-Luc wt or mutant and control Renilla-Luc. Post transfection (24 h), cells were harvested and dual luciferase reporter assays performed. Results were normalised by expressing firefly/renilla luciferase activity in relative light units (RLU) as the mean +/- S.D.H. H. U251 (left panel) or U87 (right panel) cells were transfected with siRNA (p73 or scrambled control), 48 h post transfection cells were treated with 5 ng/ml TGFβ for another 24 h. Cells were harvested and mRNA expression of p73 and POSTN was analysed using RT-qPCR. p < 0.05, *p < 0.001. Error bars represent SEM. Figure 4: POSTN is a direct target of p73. A. High POSTN mRNA expression is strongly correlated with reduced survival of glioblastoma patients. Data is from the REMBRANT database/NIH (https://caintegrator.nci.nih.gov/rembrandt/). p-value corresponds to comparison between all three conditions with each other. B. Expression of POSTN mRNA 72 h after transfection with p73 or scr siRNA was determined using RT- qPCR in U251 (left panel) and U87 (right panel) cells. C. U251 protein extracts of cells transfected with siRNA for p73 or a scrambled control and analysed by immunoblotting using p73, POSTN and β-tubulin antibodies. Asterisk indicates an unspecific band of the p73 antibody. D. Expression of POSTN mRNA 24 h after transfection with plasmids encoding TAp73α, ΔNp73α or an empty vector control was determined using RT-qPCR in U251 (left panel) and U87 (right panel) cells. E. www.impactjournals.com/oncotarget Figure 3: Analysis of mRNA expression after p73 knock down suggests a role for p73 in cell migration and invasion. A. Results of the microarray revealed 632 genes that were differentially expressed in control cells compared to knock down of endogenous p73 (Schematic illustration of gene array results). Pathway analysis of microarray results comparing U251 transfected with scr siRNA and p73 siRNA indicated changes in the pathways shown. Odds ratios for pathways are Integrin binding = 4.4, Fibronectin binding 9.05 and Cell adhesion = 2.04. p- values refer to the significance of enrichment of genes of the pathways shown. Heat maps of B. “Integrin and Fibronectin binding” (combined), 10 genes for integrin binding and 5 genes for fibronectin binding were identified, while CTGF is involved in both pathways. C. “Cell Adhesion” genes identified by GO term analysis, as differentially expressed between si scr and si p73. www.impactjournals.com/oncotarget Oncotarget 11789 STN is a direct target of p73. A. High POSTN mRNA expression is strongly correlated with reduced s tients. Data is from the REMBRANT database/NIH (https://caintegrator.nci.nih.gov/rembrandt/). p-value corre ween all three conditions with each other. B. Expression of POSTN mRNA 72 h after transfection with p73 or s using RT- qPCR in U251 (left panel) and U87 (right panel) cells. C. U251 protein extracts of cells transfe or a scrambled control and analysed by immunoblotting using p73, POSTN and β-tubulin antibodies. Asterisk in of the p73 antibody. D. Expression of POSTN mRNA 24 h after transfection with plasmids encoding TAp73α tor control was determined using RT-qPCR in U251 (left panel) and U87 (right panel) cells. E. Analysis of th ed one potential binding site for transcription factors of the p53 family 600 bp upstream of the transcriptional emonstrating binding of TAp73α to the POSTN promoter in U251 cells. G. U251 (left panel) or U87 (right p d with p73 constructs (TAp73α or ΔNp73α) plus POSTN-Luc wt or mutant and control Renilla-Luc. Post transf arvested and dual luciferase reporter assays performed. Results were normalised by expressing firefly/renilla ve light units (RLU) as the mean +/- S.D.H. H. U251 (left panel) or U87 (right panel) cells were transfected w Figure 4: POSTN is a direct target of p73. A. High POSTN mRNA expression is strongly correlated with reduced survival of glioblastoma patients. Data is from the REMBRANT database/NIH (https://caintegrator.nci.nih.gov/rembrandt/). p-value corresponds to comparison between all three conditions with each other. B. www.impactjournals.com/oncotarget Analysis of the POSTN promoter revealed one potential binding site for transcription factors of the p53 family 600 bp upstream of the transcriptional start site. F. ChIP assay demonstrating binding of TAp73α to the POSTN promoter in U251 cells. G. U251 (left panel) or U87 (right panel) cells were transfected with p73 constructs (TAp73α or ΔNp73α) plus POSTN-Luc wt or mutant and control Renilla-Luc. Post transfection (24 h), cells were harvested and dual luciferase reporter assays performed. Results were normalised by expressing firefly/renilla luciferase activity in relative light units (RLU) as the mean +/- S.D.H. H. U251 (left panel) or U87 (right panel) cells were transfected with siRNA (p73 or scrambled control), 48 h post transfection cells were treated with 5 ng/ml TGFβ for another 24 h. Cells were harvested and mRNA expression of p73 and POSTN was analysed using RT-qPCR. p < 0.05, *p < 0.001. Error bars represent SEM. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 11790 POSTN is a direct target of p73 database to examine the effect of POSTN mRNA up- regulation on patient survival and found that POSTN overexpression correlates strongly with poor prognosis in glioblastoma patients (Figure 4A). To study the regulation of POSTN by p73, we initially used knock down experiments in U251 and U87 glioblastoma cell lines, where both full p73 and specifically the transcriptionally active TAp73 isoform expression were abolished. As shown in Figure 4B, both the isoform specific and total knock down resulted in a striking decrease of POSTN mRNA levels in both cell lines. Furthermore, knock down of p73 also led to a marked decrease of POSTN protein levels (Figure 4C). Correspondingly, when TAp73 is overexpressed there is a small, but significant increase in POSTN mRNA levels, while ΔNp73 has no effect on its expression (Figure 4D). To gain insight into the mechanism of how p73 regulates cell migration, we decided to study the regulation of one of the genes identified in the microarray in more detail. We chose POSTN (Periostin), as it has previously been implicated in glioblastoma malignancy and to be important for EMT and cancer metastasis [96-98]. The matricellular protein POSTN was initially identified as a cell adhesion protein in a mouse osteoblastic cell line [99, 100]. More recently a role of POSTN in carcinogenesis and metastasis has been discovered [101, 102]. Numerous studies showed an up-regulation of both POSTN protein and mRNA levels in a plethora of different tumours, including glioblastoma, neuroblastoma, breast, colon and pancreatic cancer [96, 101]. We used the Rembrandt Figure 5: POSTN overexpression rescues invasion ability of glioblastoma cells with p73 knock down. A. Invasion into matrigel of U251 cells that were transfected with siRNA targeting p73, POSTN, both or a nontargeting control is shown. B. As in A, but cells were transfected with siRNA (scr or p73) and plasmids encoding POSTN or empty vector as indicated. Figure 5: POSTN overexpression rescues invasion ability of glioblastoma cells with p73 knock down. A. Invasion into matrigel of U251 cells that were transfected with siRNA targeting p73, POSTN, both or a nontargeting control is shown. B. As in A, but cells were transfected with siRNA (scr or p73) and plasmids encoding POSTN or empty vector as indicated. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 11791 To investigate whether POSTN is a direct transcriptional target of TAp73 we performed a promoter analysis of the POSTN gene and found one potential p73 binding site ~600 bp upstream of the POSTN transcriptional start site (Figure 4E). We employed chromatin immunoprecipitation (ChIP) assays to investigate binding of TAp73 to this potential binding site using overexpression of TAp73α-HA in U251 cells followed by DNA-protein crosslinking and isolation of TAp73α using an HA antibody. The results showed that TAp73 bound to the POSTN promoter, as well as to the MDM2 promoter used as a positive control (Figure 4F). To confirm that TAp73 activates the POSTN promoter, reporter assays were carried out using the POSTN promoter, either in its wild type form or with a three- nucleotide deletion in the previously identified p73 binding site (Figure 4G). The results showed that TAp73, and in U251 cells also ΔNp73, transcriptionally activates reporter activity in both U251 and U87 cells, and that this is abrogated when the p73 binding site is mutated. Taken together the data from the ChIP and reporter assays demonstrated direct binding of TAp73 to the POSTN promoter leading to POSTN transcription. Expression of POSTN is induced via the TGFβ pathway, and we next asked whether p73 is also required to activate POSTN expression when induced by TGFβ. To ure 6: Glioblastoma are more chemo-sensitive after p73 knock down. A. U251 cells were treated with 50 µM Temozo 8 h. Early and late apoptosis was detected using Annexin V/PI-double staining followed by flow cytometry analysis. B. As in additional time points of Temozolomide treatment of 24 and 72 h, total apoptosis was quantified and is shown. C. Colony for y after p73 knock down. p = 0.05, *p < 0.0001. Error bars represent SEM. Figure 6: Glioblastoma are more chemo-sensitive after p73 knock down. A. U251 cells were treated with 50 µM Temozolomide for 48 h. Early and late apoptosis was detected using Annexin V/PI-double staining followed by flow cytometry analysis. B. As in A, but with additional time points of Temozolomide treatment of 24 and 72 h, total apoptosis was quantified and is shown. C. Colony formation assay after p73 knock down. p = 0.05, **p < 0.0001. Error bars represent SEM. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 11792 To test this we performed an invasion assay with cells transfected with siRNA for total p73, POSTN or both. As expected loss of either p73 or POSTN decreased invasion of glioblastoma cells; however, a double knock down of both did not lead to further reduction in invasion, suggesting that indeed both proteins are acting in the same pathway with p73 regulating POSTN expression (Figures 5A, S5A). To further support this hypothesis, we overexpressed POSTN in cells (Figure S5B) transfected with either siRNA for total p73 or control cells to see if POSTN expression can rescue the invasive phenotype of these cells. As shown in Figure 5B, overexpression of POSTN led to a strong increase in invasion that is not reduced after p73 knock down. Moreover, the reduced invasion produced by p73 knock down is rescued by overexpression of POSTN. test this, cells were transfected with either siRNA targeting total p73 or a scrambled control, treated with TGFβ and mRNA levels of POSTN and p73 were measured using RT-qPCR. As expected, TGFβ treatment led to a strong increase in POSTN mRNA and knock down of p73 reduced this by around 3 fold, further demonstrating that p73 plays a role in POSTN activation (Figure 4H). The fact that p73 knock down reduced the activation of POSTN in response to TGFβ treatment suggests that p73 acts downstream of TGFβ in this pathway. It remains to be investigated how/if p73 is directly activated by TGFβ leading to an increase of POSTN expression. p73 mRNA levels do not change after TGFβ treatment, suggesting that p73 activity could be modified by post translational modifications. Since POSTN is known to promote invasion of glioblastoma cells we speculated that the reduction of POSTN after p73 knock down is, at least in part, responsible for the loss of invasive ability of the cells. Taken together this data demonstrates that p73 regulates POSTN levels and thereby invasion of glioblastoma cells. Figure 7: Correlation of p73 and POSTN expression predicts bad patient prognosis. Positive total p73/POSTN correlation represents a negative prognostic factor for patient survival in astrocytic gliomas (dataset: GSE18166, N = 51), lung (GSE4573, N = 128), breast cancer (GSE19783, N = 106) and colon cancer (GSE17538, N = 566). Panels represent patient survival estimation of p73/POSTN- positive correlation groups compared with negative or absent correlation groups. Loss of p73 leads to a decreased chemo-resistance In the current study we demonstrate that p73 promotes invasion and migration in glioblastoma cells through directly activating expression of POSTN. Previous studies have proposed a role for p73 in cell migration and invasion. Sablina et al (2003) showed that TAp73 leads to increased colon cancer cell migration [109], while Zhang et al (2012) observed a reduction of cell migration in non-cancerous breast cells (MCF10a) after TAp73 overexpression [110], suggesting that the effect is cell type and condition dependent. The ΔNp73 isoform has also been implicated with increased invasion and metastasis in a study by Steder et al, [111], that showed that ΔNp73 can initiate metastasis by inhibiting IGF1R-AKT/STAT3 signalling. As an undifferentiated phenotype of glioblastoma cells is usually associated with increased invasiveness and chemoresistance, we investigated the effect of total p73 knock down on glioblastoma cell sensitivity to Temozolomide. We measured apoptosis in cells after drug treatment using Annexin V/PI staining. p73 knock down led to a modest, but significant, increase of apoptosis after treatment with the Temozolomide (Figure 6A, 6B). Additionally, we examined the effect of POSTN overexpression on apoptosis after Temozolomide treatment with and without p73 knock down. However, no change in cell death after POSTN overexpression was observed, irrespectively of p73 knock down (Figure S6). This suggests that the effect on chemo-resistance after p73 knock down is mediated by a different axis compared to the effect on cell invasion and is POSTN independent. Here we show that, strikingly, loss of p73 leads to a differentiated phenotype that is less invasive and more susceptible to chemotherapeutic treatment in glioblastoma cells. Using a gene microarray, we identified POSTN as a direct transcriptional target of p73 that is strongly down regulated in glioblastoma cells upon p73 loss. A previous study has shown regulation of POSTN mRNA by p73 in a thyroid cancer cell line [109]. Here, however, we show for the first time that p73 directly binds to the POSTN promoter to activate its transcription and that p73 directly regulates POSTN protein levels. The study of POSTN regulation by p73 in thyroid cancer detected a strong upregulation of POSTN expression in a reporter assay in response to ΔNp73 overexpression, while we did not observe an upregulation of POSTN mRNA in response to ΔNp73 and only a mild effect in a reporter assay. Loss of p73 leads to a decreased chemo-resistance This could be due to cell type specific non-transcriptional activation of POSTN by ΔNp73, as this isoforms does not have intrinsic transcriptional activity. p As we observed cell differentiation after p73 knock down and this is often associated with a decrease in proliferation we, furthermore, probed the effect of p73 on cell proliferation using a colony formation assay. The results showed a dramatic decrease of cell proliferation after total p73 knock down (Figure 6C). www.impactjournals.com/oncotarget Figure 7: Correlation of p73 and POSTN expression predicts bad patient prognosis. Positive total p73/POSTN correlation represents a negative prognostic factor for patient survival in astrocytic gliomas (dataset: GSE18166, N = 51), lung (GSE4573, N = 128), breast cancer (GSE19783, N = 106) and colon cancer (GSE17538, N = 566). Panels represent patient survival estimation of p73/POSTN- positive correlation groups compared with negative or absent correlation groups. www.impactjournals.com/oncotarget Oncotarget 11793 Correlation between p73 and POSTN levels predicts bad prognosis for patient survival Cells were lysed in Triton X-100 lysis buffer (50 mM HEPES (pH 8), 0.4 % Triton X-100, 150 mM NaCl, 10 mM NaF, 2 mM DTT, 0.1 mM EDTA, 1x protease inhibitor mix (Roche)). Western Blotting was performed as described previously [115]. survival that might drive carcinogenesis. Further studies, especially in vivo, are necessary to establish whether p73 or its target POSTN could provide a drug target for development of new drugs that reduce glioblastoma invasion. The POSTN gene was subcloned into the pcDNA 3.1 (-) vector (Life Technologies) after PCR amplification of the gene from U251 cell cDNA using the following primers including the restriction sites for NheI and Hind III, Fw: 5’GATGTGCTGCATAGATTCAACATCAC3’ , Rv: 5’GTGATGTTGAATCTATGCAGCACATC3’. Restriction enzymes and T4 DNA Ligase were purchased from NEB and restriction digest, ligation and transformation into competent cells (DH5α, Life Technologies) were carried out following the supplier’s instructions. Correlation between p73 and POSTN levels predicts bad prognosis for patient survival If up-regulation of POSTN by p73 leads to increased invasion, we would expect that the tumours in which this regulation is active and thus POSTN and p73 levels correlate, are more aggressive and have a worse prognosis than tumours where this axis is not functional. To assess this bioinformatically, glioblastoma datasets from patients were divided into two groups; those where p73 and POSTN mRNA correlate and those where there is no correlation. We then compared the survival probability of the two groups (Figure 7). While the survival of the group where POSTN and p73 levels correlate is decreased, the difference between this group and that in which there is no interaction does not reach significance, possibly because the sample size is too small. We therefore decided to probe other cancers, specifically lung, breast cancer and colon cancer, where larger datasets are available. And importantly, POSTN was implicated to be a negative prognostic marker in breast [103, 104], lung cancer [105, 106] and colon cancer [107, 108]. All three datasets show a dramatic, and statistically significant, reduction in survival of patients where levels of p73 and POSTN correlate (Figure 7). Taken together we have shown that p73 ablation leads to morphological changes associated with a differentiated phenotype in glioblastoma cells, that leads to a decrease in their invasion ability through inactivation of the p73/POSTN axis. Several studies have demonstrated that POSTN is up regulated in a variety of tumours, including glioblastoma [96, 101], and acts as a metastasis-promoting factor [102]. Likewise, POSTN has been shown to promote epithelial to mesenchymal transition leading to increased cell invasion and proliferation [98, 112, 113]. In addition to being overexpressed by tumour cells, POSTN is also expressed in the stroma of normal stem cell niches and aids the metastatic success of circulating cancer cells [102]. Taken together, these data suggests that POSTN plays an important role in tumour formation and metastasis, being either expressed by the tumour cells or the surrounding stroma. Here we have demonstrated that TAp73 directly activates POSTN expression leading to an invasive phenotype in glioblastoma cells. Moreover, total p73 knock down renders glioblastoma cells more sensitive to Temozolomide treatment and bioinformatic analysis revealed that correlation of p73 and POSTN expression predicts poor patient survival. This suggests that the p73/ POSTN axis is a negative predictive factor of patient www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 11794 anti-rabbit (Molecular Probes). Reporter assay Reporter activity was tested using the Dual Luciferase Reporter System from Promega. Briefly, cells were seeded in 24 well plates and transfected with Renilla (50 ng/well), POSTN reporter construct wt or mutant (120 ng/well) and 100 ng of the indicated p73 isoforms. After 24 h, cells were washed and lysed on the plate using the Promega lysis buffer. Firefly luciferase and Renilla activities were measured as indicated by Promega. Results shown are firefly luciferase expression normalised by Renilla and are expressed as relative light units (RLU). Results are shown as the median of 3 independent experiments, all carried out in duplicate and measured twice. Inverted invasion assay The inverted invasion assay was carried out as described previously [116]. In brief, geltrex (Gibco, A1413302) was mixed 1:1 with PBS and supplemented with 25 μg/ml fibronectin (FN, Sigma), 60 μl of the mix was added to each transwell dish (8 μm polycarbonate membrane, Costar) and allowed to set at 37°C for 45 min. Next 30,000 cells were added to the top of the membrane and transwells were incubated upside down for 4 h to allow cells to settle on the membrane. The wells were then washed twice by dipping into serum free media and placed in 1 ml serum free media in a 24 well plate. On top of the geltrex layer 100 μl serum-rich media was added supplemented with 10 ng/ml EGF and 10 ng/ml HGF. Cells were incubated for 72 h and stained using 4 μM Calcein-AM (Santa Cruz Biotechnology) in media for 1 h. Invading cells were quantified using confocal microscopy by taking pictures every 10 μm throughout the geltrex matrix. Fw: 5’GCTACGGCTAGCATGATTCCCTTTTTACCCATG’3, Rv: 5’ GCTACGAAGCTTTCACTGAGAACGACCTTCCC’3. Fw: 5’GCTACGGCTAGCATGATTCCCTTTTTACCCATG’3, Rv: 5’ GCTACGAAGCTTTCACTGAGAACGACCTTCCC’3. Glioblastoma cell lines U251 and U87 were maintained in DMEM (Life Technologies) and EMEM (ATCC) respectively; all media were supplemented with 10 % FBS (v/v) (Labtech) and 1 % (v/v) penicillin/ streptomycin (Invitrogen), and grown with 5 % CO2 at 37°C. To maintain cells, U251 and U87 cells were grown to 90 and 70% confluence respectively and then split 1:8 using trypsin:EDTA (Gibco). For the Luciferase reporter assay a reporter construct with 1000bp of the POSTN promoter upstream of the transcription start site were cloned into the pGL3-Basic Vector (Promega). The region was amplified using PCR from genomic U251 cell DNA using the following primers including restriction sites for SacI and BgIII: Transfection of cells with siRNA was carried out using Hyperfect (Qiagen) at 60% (U251) and 50% (U87) confluence following the supplier’s instructions using 30 nM siRNA, siRNA Silencer Select was from Ambion (IDs: TAp73: 115665, total p73: 2671, POSTN: s20889). DNA transfections were carried out using Effectene (Qiagen) at 80% confluence and Lipofectamine 2000 (Life Technologies) at 70% confluence for U251 and U87 respectively. 5’CATGGAGCTCTGGCTAGGGATTGCATAGTGT’3, 5’CATGGAGCTCTGGCTAGGGATTGCATAGTGT’3, A deletion mutant of the plasmid was created using the Quick Change II XL Site directed mutagenesis Kit (Agilent Technologies) and the following primers Fw: 5’GATGTGCTGCATAGATTCAACATCAC3’ , Rv: 5’GTGATGTTGAATCTATGCAGCACATC3’. g g Fw: 5’CATGGAGCTCTGGCTAGGGATTGCATAGTGT’3, Rv: 5’CATGAGATCTAGAACTGGCAGTGGGCTTTG’3. Fw: 5’CATGGAGCTCTGGCTAGGGATTGCATAGTGT’3, Rv: 5’CATGAGATCTAGAACTGGCAGTGGGCTTTG’3. A d l i f h l id d i Microarray analysis Cells were transfected and incubated as indicated in the figure legends and then fixed using 3 % paraformaldehyde in PBS. After blocking and permeabilisation for 1 h (0.4 % Triton X-100, 1 % BSA in PBS), the 96 well plates were incubated with α-GFAP antibody (1:500) in 1 % BSA in PBS overnight at 4°C. After 3 washes (10 min each) in PBST the slides were incubated in secondary antibody (1:1000) for 30 min at 37°C, washed again twice with PBST and then incubated for 10 min in PBS containing Hoechst Staining (1:10000). Cells were visualised using the Cellomics (Thermo Fisher). RNA was extracted from U251 cells that had been transfected with siRNA (scr or p73) 72 h prior to RNA isolation using TRIZOL (Invitrogen). Next, RNA was reverse transcribed, converted to cRNA, amplified, and labeled with a cyanine-3 dye using a Low Input Quick Amp labeling kit (Agilent). Then cRNAs were hybridized to human gene expression microarrays (Agilent, catalog number G4851B) containing 50,599 different probes. Slides were washed with the Agilent wash buffer reagents and scanned using the G2505C Agilent Microarray Scanner (scan control version A.8.4.1). Using the Agilent Feature Extraction software (version 10.7.3.1) data was extracted and analyzed using Agilent GeneSpring GX software (version 12.1). To analyze significant differential expression, an unpaired Student’s t test with Benjamini-Hochberg multiple testing correction was applied. Hits with a p-value ≤ 0.05 and a fold change >2 were considered to be both statistically and biologically significant. Plasmids, antibodies and western blot Antibodies were anti-p73 (Melino lab [114]), anti- SNAIL (Cell Signalling L70G2, mAb), anti-E-Cadherin (Cell Signalling 4A2, mAb), anti-POSTN, anti-GFAP (DAKO, pAb). Anti-GAPDH (SIGMA, G8795, mAb) and anti-β-tubulin (Santa Cruz H-235, pAb). Secondary antibodies were HRP conjugated goat-anti-rabbit and goat-anti-mouse (Bio Rad), and Alexa Flour 488 donkey www.impactjournals.com/oncotarget Oncotarget 11795 generated using the Revert Aid cDNA synthesis kit (Life Technologies). Actual qPCR Primer for RT-qPCR were: generated using the Revert Aid cDNA synthesis kit (Life Technologies). Actual qPCR Primer for RT-qPCR were: Colony formation assay For the colony formation assay U251 cells were transfected with si scr or si p73. 24 h post transfection 500 cells were seeded per well of a 6 well plate in duplicate. After 2 weeks cells colonies were stained using crystal violet. The experiment was carried in 3 independent biological replicates. Annexin V/ PI To analyse potential p73 binding sites within the POSTN promoter an analysis was carried out using Math-Inspector Professional software and the TRANSFAC database on a region of 3000 kb upstream of the transcription-start site and within the first intron of human POSTN. The analysis highlighted a 25 bp region containing a p53-like RE site roughly 600 bp upstream of the transcription start site [118]. U251 cells were seeded in 6 well plates and transfected with 30 nM siRNA targeting p73 or a scrambled control, 24 h post transfection cells were treated with 50 μM Temozolomide for the times indicated. Apoptosis was measured using the Annexin V-FITC Apoptosis Detection Kit (eBioscience) following the supplier’s instructions. Staining was analysed using the FACS Calibur (Becton Dickinson) with CellQuestPro Software, per sample 10,000 cells were analysed. Chromatin immunoprecipitation (ChIP) assay ChIP assays were carried out using the MAGnify™ Chromatin Immunoprecipitation System (Life Technologies) following the manufacturer’s instructions. Briefly, U251 cells were transfected with HA-TAp73α DNA, harvested 24 h post transfection and fixed using formaldehyde. After sonication an immunoprecipitation for TAp73 was performed using an HA antibody g ) q q L32 (internal control) Fw: TTCCTGGTCCACAACGTCAAG Rv: TGTGAGCGATCTCGGCAC, total p73: Fw: GCCTGGAGCTGATGGAGTT, Rv:ACGGGGGCTGTAGGTGAC, POSTN: Fw:CTCATAGTCGTATCAGGGGTCG Rv: ACACAGTCGTTTTCTGTCCAC. RT-PCR and PCR were carried out as described previously [117]. Migration assays were carried out using the xCELLigence system (ACEA Bioscience) following the supplier’s instructions. Briefly, cells were harvested and washed in serum free media, and 20,000 cells were added to the upper chamber of the CIM plate, with the lower chamber filled with serum rich media. Cells were allowed to migrate for up to 60 h and the numbers of migrated cells read every 15 min. Bioinformatics analyses There is no conflict of interest. Gene expression data sets GSE18166, GSE17538, GSE4573 and GSE19783 were downloaded from the GEO omnibus repository. The gene expression rank that reflects relative mRNA expression levels is more consistent as it does not require normalization and thus no normalization bias is introduced [94, 119-121]. Gene expression values were transformed into rank expression values on a scale from 100 to 0, where a rank value of 55 shows that 55% of probes in the sample have a lower expression value for a given gene. Pearson coefficient was used as a measure of correlation between expression profiles. g p POSTN Fw:5’CAATATTGGCTGCTTTTCACCA’3, Rv: 5’AAGGTTTGAAATGAAGCAGAAAGG’3, MDM2 Fw: 5’GGTTGACTCAGCTTTTCCTCTTG’3, Rv: 5’GGAAAATGCATGGTTTAAATAGCC’3. g p POSTN Fw:5’CAATATTGGCTGCTTTTCACCA’3, Rv: 5’AAGGTTTGAAATGAAGCAGAAAGG’3, MDM2 Fw: 5’GGTTGACTCAGCTTTTCCTCTTG’3, Rv: 5’GGAAAATGCATGGTTTAAATAGCC’3. References 1. Jackson M, Hassiotou F and Nowak A. Glioblastoma stem- like cells: at the root of tumor recurrence and a therapeutic target. Carcinogenesis. 2015; 36:177-185. 2. Chen J, McKay RM and Parada LF. 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This procedure is repeated until there is no sample left in cohort 1 which increases the positive correlation between p73 and POSTN, if it was removed. Next the statistical difference in survival of the two cohorts was calculated and graphs were blotted using R (statistical package). 4. Dickinson A, Yeung KY, Donoghue J, Baker MJ, Kelly RD, McKenzie M and Johns TG. The regulation of mitochondrial DNA copy number in glioblastoma cells. Cell death and differentiation. 2013; 20:1644-1653. 5. Lugli N, Kamileri I and Halazonetis TD. PARP inhibitors and IR join forces to strike glioblastoma-initiating cells. Cell death and differentiation. 2014; 21:192-193. 6. Zhang Y, Kim J, Mueller AC, Dey B, Yang Y, Lee DH, Hachmann J, Finderle S, Park DM, Christensen J, Schiff D, Purow B, Dutta A and Abounader R. Multiple receptor tyrosine kinases converge on microRNA-134 to control KRAS, STAT5B, and glioblastoma. Cell death and differentiation. 2014; 21:720-734. RT-qPCR and PCR RNA was isolated from cells using the TRIZOL reagent (Life Technologies). For RT-qPCR first 3 μg of isolated RNA was digested using DNase (SIGMA) to eliminate any DNA contamination and then cDNA was www.impactjournals.com/oncotarget Oncotarget 11796 Abbreviations 7. Bageritz J, Puccio L, Piro RM, Hovestadt V, Phillips E, Pankert T, Lohr J, Herold-Mende C, Lichter P and Goidts V. Stem cell characteristics in glioblastoma are maintained by the ecto-nucleotidase E-NPP1. Cell death and differentiation. 2014; 21:929-940. POSTN, periostin; GBM, glioblastoma multiforme; TAp73, transactivation domain-containing p73 isoform; ΔNp73, amino-truncated p73 isoform; GFAP, glial fibrillary acidic protein; EMT, epithelial to mesenchymal transition; ChIP, chromatin immunoprecipitation. 8. Liu JK, Lubelski D, Schonberg DL, Wu Q, Hale JS, Flavahan WA, Mulkearns-Hubert EE, Man J, Hjelmeland AB, Yu J, Lathia JD and Rich JN. Phage display discovery of novel molecular targets in glioblastoma-initiating cells. Cell death and differentiation. 2014; 21:1325-1339. www.impactjournals.com/oncotarget Grant Support (Covance) or an IgG control, cross-linking was reversed and DNA eluted using the components of the kit. An PCR was performed on the input and diluted DNA using the following primers: This work has been supported by the Medical Research Council, UK; grants from Associazione Italiana per la Ricerca contro il Cancro (AIRC; 2011-IG11955; 5xmille MCO #9979) and Fondazione Roma (NCD), Min. Salute to Istituto Dermopatico dell’Immacolata/Istituto di Ricovero e Cura a Carattere Scientifico (IDI-IRCCS RF08 c.15, RF07 c.57) to GM. Acknowledgments We thank K. Dudek for assistance in gene microarray analysis, L. Pinon for assistance with imaging using the Cellomics, P. Muller for help setting up the inverted invasion assay and M. Agostini, I. Amelio and B. Rotblat for scientific discussion. 9. 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https://openalex.org/W2798571499	http://www.e-revistes.uji.es/index.php/recerca/article/download/2586/2701, http://repositori.uji.es/xmlui/bitstream/10234/175226/1/Grijalba_Uche.pdf	es		¿Dónde está el error? La epistemología de la verdad en la neurociencia de A. Damasio y la filosofía de R. Descartes	Recerca	2,018	cc-by	8,872	RECERCA, REVISTA DE PENSAMENT I ANÀLISI, NÚM. 22. 2018. ISSN: 1130-6149 – pp. 69-89 doi: http://dx.doi.org/10.6035/Recerca.2018.22.5 ¿Dónde está el error? La epistemología de la verdad en la neurociencia de A. Damasio y la filosofía de R. Descartes Where is the error? The epistemology of the truth in the neuroscience of A. Damasio and the philosophy of R. Descartes MIGUEL GRIJABA UCHE Cátedra Nacional Hercritia. Facultad de Filosofía. Universidad Nacional a Distancia (uned) Artículo recibido: 30 de junio de 2017 Solicitud de revisión: 7 de septiembre de 2017 Artículo aceptado: 10 de enero de 2018 Resumen El presente texto trata de analizar la crítica que Antonio Damasio realiza a René Descartes (el error de Descartes) desde el empleo de una racionalidad neurocientífica. Damasio cae en un neurorreduccionismo para combatir la filosofía cartesiana. La crítica de Damasio al dualismo cartesiano olvida el nivel ontológico y epistemológico para abordar exclusivamente la cuestión antropológica. Este texto propone una defensa de Descartes y un rechazo a un dualismo absoluto a partir de la lectura de Las pasiones del alma. Palabras clave: Damasio, Descartes, neurobiología y pasiones. Abstract The present text tries to analyze the criticism that Antonio Damasio does to René Descartes (the error of Descartes) from the use of a neuroscientific rationality. Damasio falls into a neuroreductionism to combat Cartesian philosophy. Damasio’s critique of Cartesian dualism neglects the ontological and epistemological level to address exclusively the anthropological question. This text proposes a defense of Descartes and a rejection of an absolute dualism based on the reading of Passions of soul. Keywords: Damasio, Descartes, Neurobiologya and passions. 70 RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2018.22.5 · ISSN: 1130-6149 - pp. 66-89 INTRODUCCIÓN Si Descartes hubiera escrito «Siento que pienso, luego existo» (Kushner, 1998: 1) quizás Damasio no hubiera necesitado escribir su libro El error de Descartes. Pero el filósofo de Turena no solo defendió la dicotomía razónsentimiento, sino también sostuvo que los sentidos ofrecían información poco digna de crédito. La razón era sencilla, estos solían acompañarse de cambios corporales tan provisionales y dependientes del contexto como son también el rubor, el sudor o las palpitaciones. En efecto, Descartes vincula el conocimiento de los sentidos a procesos fisiológicos y no mentales, por tanto, de calado subjetivo, poco apropiados para llegar a la verdad. No forman parte, en consecuencia, del proceso de conocimiento propio de la racionalidad, quedando relegados a un orden inferior, el corporal. El neurocientífico Antonio Damasio considera un verdadero desatino creer que el razonamiento y el juicio moral pueden existir separados del cuerpo, que las operaciones mentales pueden realizarse ajenas a la estructura del organismo. Para Damasio, esta falsa creencia, que achaca en origen a Descartes, ha impregnado el pensamiento y la cultura occidentales durante siglos, retrasando el esfuerzo por comprender la mente en términos biológicos. Precisamente, ese es el reto que se propone a sí mismo: explicar la esencial dependencia de las operaciones mentales y los procesos neuronales. En el presente trabajo comenzaré por exponer la cuestión de la interdisciplinariedad entre ciencia y filosofía, necesaria para nuestro autor pero que le ha hecho caer en un reduccionismo científico y neuronal para expresar su anticartesianismo teórico. La crítica que Damasio establece al dualismo cartesiano, en donde nuestro autor comete su propio error al ceñirse a un nivel antropológico olvidando la cuestión ontológica sobre el conocimiento, le lleva a cometer la falacia de la objetividad, junto con la naturalización de la mente que realiza Damasio y que le lleva a aislar subjetividad y verdad. Por el contrario, lo que trato de defender en el texto es que la visión cartesiana no olvida lo material, lo corporal a diferencia de lo afirmado por Damasio. El pensar acontece en la confluencia entre el cuerpo y el alma. Por ello, tanto en Descartes como en Damasio, acontece que primero somos, y después pensamos. Para este motivo analizaré varios textos cartesianos como Las meditaciones metafísicas, Las pasiones del alma o Las reglas para la dirección del espíritu, en donde se demuestra esa interdependencia entre cuerpo y mente-alma en Descartes, en contra de lo defendido desde los primeros textos por Damasio. MIGUEL GRIJABA UCHE ¿Dónde está el error? La epistemología de la verdad en la neurociencia 1. LA INTERDISCIPLINARIEDAD BAJO SOSPECHA Para Damasio, la cultura occidental arrastra un dualismo de origen cartesiano que nos lleva a percibir lo humano a través de una división que pone, de un lado, el cuerpo, y del otro la mente. Desde esta perspectiva se tiende frecuentemente a identificar la mente como lo distintivamente humano. Por el contrario, nos dice Damasio que «alma y espíritu, con toda su dignidad y escala humanas, son estados complejos y únicos de un organismo» (Damasio, 2001a: 231). El científico portugués es consciente de que un gran número de filósofos han tratado de salvaguardar, a lo largo de la historia, aquello que consideran el rasgo esencial de todo ser viviente: la unidad de lo heterogéneo. Desde Hipócrates hasta el Renacimiento, asegura, esta ha sido la corriente de pensamiento predominante, corriente que un solo hombre, René Descartes, fue capaz de cambiar. La discusión acerca de la mente y el cerebro, la naturaleza del alma, hunde sus raíces en la filosofía griega, en la escolástica medieval y en el cartesianismo racionalista de los siglos xvii y xviii, pero se expresa con inu sitado interés en los más recientes estudios científicos neurobiológicos y en la subsiguiente reflexión filosófica acerca de ellos. Así, por ejemplo, Platón, que concibe el cuerpo como la cárcel del alma, piensa que esta última se comunica con lo físico. Pero el estudio y la investigación de las cosas físicas son de importancia secundaria respecto a los asuntos del mundo de las ideas que, siendo más permanente, es más real. La posición de su discípulo Aristóteles es más moderada. Sitúa el pensamiento como producto de la experiencia del cuerpo y, en este sentido, atribuye mayor grado de realidad a los objetos sensibles. En primer lugar, porque la idea que tenemos de todo objeto tiene que tener su origen en la impresión de nuestros sentidos. Y, en segundo lugar, porque es necesario que los mismos fenómenos que producen la sensación, existan independientes de ella. Es decir, acaba otorgando cierta objetividad a los sentidos. En la actualidad, uno de los temas que más atrae la atención de los investigadores en neurociencia, psicología y filosofía de la mente es precisamente la naturaleza de la conciencia –mente o alma. En el umbral del siglo xxi todavía no sabemos desde la neurobiología cómo pensamos, ni qué es el acto propio de pensar, ni tenemos ninguna teoría que lo explique satisfactoriamente. Damasio, por ejemplo, no lo aborda de modo claro. No hay una respuesta a la pregunta sobre si la mente es una propiedad emergente de la actividad eléctrica y metabólica del cerebro, y, en el caso de que eso 71 72 RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2018.22.5 · ISSN: 1130-6149 - pp. 66-89 fuera así, no se conoce tampoco de qué forma la red nerviosa da origen a los fenómenos mentales. Como describe Daniel Dennett, «estamos apenas comenzando a ver los primeros puentes que se están tendiendo sobre el gran abismo de ignorancia entre la psicología y la neuroanatomía» (Dennett, 1995: 3). Debo destacar que el llamado problema mente-cerebro no es, en modo alguno, el problema alma-cuerpo. El debate contemporáneo acerca de las relaciones entre la mente y el cerebro no tiene nada que ver con aquella otra cuestión acerca de la articulación de alma y cuerpo. La confusión se debe a que el efectivo carácter unitario del ser humano hace que todo lo humano tenga alguna relación. El problema alma-cuerpo versa sobre la posibilidad de que en el cerebro de cada ser humano exista un homúnculo, un hombrecillo, al que llamamos alma, que gobierne el funcionamiento cerebral. Ese es un mito de cuño cartesiano que, a fin de cuentas, no explica nada, puesto que la mente no existe ni opera sin el cuerpo. Por el contrario, el problema mente-cerebro está relacionado con la naturaleza de las correlaciones neurofisiológicas que subyacen al fenómeno mental. Y si bien ambas polémicas comparten puntos de anclaje argumental, su objeto es distinto. En opinión de Damasio, Descartes es el enemigo que el intelectual moderno ha de combatir. Y ahí es donde yace, a mi juicio, el primer serio desliz de su planteamiento: tratar de rebatir la filosofía cartesiana apelando casi exclusivamente a mediciones y experimentos neuronales. Damasio es bien explícito en este sentido: en sus obras hace constantes alusiones a filósofos de todas las épocas, pero como motivos de inspiración y no tanto como serias fuentes de conocimiento objetivo. Con todo, Damasio se distingue por su defensa de una más sofisticada teoría organicista, que se funda en lo neuronal, pero para luego sublimarla. Pero una atenta lectura de su obra no deja lugar a dudas sobre su opinión acerca del papel hegemónico que tiene la Neurociencia en la búsqueda de los misterios últimos de la existencia humana. El problema es que Damasio identifica las tesis cientificistas con tesis científicas, es decir, con hipótesis susceptibles de ser sometidas y probadas con métodos experimentales. En otras palabras, los misterios últimos de la humanidad van a ser resueltos gracias a la Neurobiología, según su opinión. Rompiendo una lanza en favor de Damasio, es justo reconocer, en primer lugar, que los límites entre la ciencia experimental y la filosofía siempre han sido difusos. Pero es que, además, la tierra de nadie entre ambas se ha visto significativamente ampliada en una época como la nuestra en la que MIGUEL GRIJABA UCHE ¿Dónde está el error? La epistemología de la verdad en la neurociencia la formalización de las hipótesis científicas ha alcanzado unos niveles de abstracción sin precedentes. Por supuesto, Damasio no es el primero en alistarse en la cruzada anticartesiana del siglo xx, iniciada por Gilbert Ryle y Ludwig Wittgenstein. Pero probablemente sí que lo sea en utilizar las armas de la Neurociencia para tal fin. El problema, como voy a tratar de mostrar a continuación, es que Damasio desconoce el terreno de batalla, y acaba introduciendo mayores malentendidos en el diálogo entre científicos y filósofos que aquellos que intentaba disolver, como se podrá colegir al final de la lectura de este artículo. Esta es la razón por la que, paradójicamente, este tipo de conductas abiertamente interdisciplinares están promoviendo en la ciencia y en la filosofía, como efecto rebote, actitudes reduccionistas que podríamos definir como defensivas. Por último, en lo que se refiere al comentario sobre la posible influencia de Descartes en los males de la modernidad, G. Kirkeboen defiende que el dualismo cartesiano no fue el responsable del abismo entre ciencia natural y el dominio del alma, como achaca Damasio, sino que la causa hay que buscarla en el mecanicismo de la nueva ciencia natural de la época, entre cuyos principales promotores estarían Galileo y Kepler (Kirkeboen, 2000: 318). Al contrario, para Descartes, la naturaleza del alma o espíritu es más fácil de conocer que el cuerpo, y eso le impulsa a adentrarse en el estudio de la mente y a escribir su libro Las pasiones del alma. Por tanto, el retraso que le atribuye Damasio en Medicina no es por culpa de Descartes, sino un error grosero de conocimiento en Damasio. Otra prueba, por último, de que Damasio se equivoca es que, pese a la superación del cartesianismo, el dualismo mente-cerebro no ha sido eliminado ni de la ciencia ni de la filosofía actual. Uno de los neurocientíficos prominentes de este grupo es el premio Nobel John C. Eccles, que defiende un dualismo radical interaccionista entre la mente y el cerebro. Su hipótesis general es que la mente autoconsciente es distinta del cerebro, pero ejerce una función superior de interpretación y control de los procesos neuronales.1 1 Eccles resume su tesis dualista en varios principios. En suma, para Eccles, el problema mentecerebro refleja una división real: los procesos de la mente autoconsciente son distintos de los procesos neuronales. Su solución consiste en sostener una interacción entre mente autoconsciente y maquinaria neuronal a través de la corteza asociativa del hemisferio cerebral dominante. 73 74 RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2018.22.5 · ISSN: 1130-6149 - pp. 66-89 2. LA FALACIA DE LA OBJETIVIDAD El proyecto asumido por Damasio de querer criticar el dualismo cartesiano desde la Neurociencia es, cuanto menos, valiente. Su opinión es clara y ha levantado numerosas ampollas: Es interesante, y a la vez paradójico, que muchos científicos cognitivos que creen que pueden investigar la mente sin recurrir a la neurobiología no se considerarían dualistas. Puede que también haya algo de separación cartesiana de mente y cuerpo detrás del pensamiento de aquellos neurocientíficos que insisten en que la mente puede ser explicada exclusivamente en términos de acontecimientos cerebrales, dejando de lado al resto del organismo y al ambiente físico y social que lo rodea (Damasio, 2001a: 230). De hecho, no pocos colegas de profesión consideraron El error de Descartes un demérito en lo que había sido hasta entonces una brillante carrera científica. En segundo lugar, es admirable su capacidad para captar cuán impregnada está la Neurociencia de sucintos dualismos, así como para entender lo perjudiciales que son algunos de ellos para su buen desarrollo. Fijémonos en el modo concreto en el que Damasio enuncia el error de Descartes: «Pienso luego existo»… Tomada en sentido literal, la afirmación ilustra precisamente lo contrario de lo que creo que es cierto acerca de los orígenes de la mente y acerca de la relación entre mente y cuerpo. Sugiere que pensar, y la consciencia de pensar, son los sustratos reales del ser.Y puesto que sabemos que Descartes imaginó que el pensar es una actividad muy separada del cuerpo, celebra la separación de la mente, la «cosa pensante» (res cogitans), del cuerpo no pensante, el que tiene extensión y partes mecánicas (res extensa) (Damasio, 2001a: 228). La formulación que hace Damasio del planteamiento cartesiano da pie a varios equívocos. El más importante tiene por causa el desconocimiento del autor acerca de las intenciones de partida que llevan a Descartes a proponer su máxima sobre la relación entre el conocimiento y el ser, clave para entender la filosofía cartesiana. Porque, en Descartes, por orden de importancia y de dedicación, lo primero es hallar «un conocimiento cierto y evidente» –tarea que pertenece al nivel ontológico. Lo segundo consistiría en construir una filosofía con la que responder, antes que a muchas otras cuestiones, a la pregunta sobre cómo es posible que el ser humano sea capaz de alcanzar conocimientos ciertos –el nivel epistemológico. En este nivel es donde formula la teoría mente-cuerpo. Solo en tercer lugar, se mete Descartes a resolver las paradojas que planean sobre la solución dualista –el nivel antropológico. MIGUEL GRIJABA UCHE ¿Dónde está el error? La epistemología de la verdad en la neurociencia Pero, ¿es posible responder a la pregunta por la identidad humana sin atender previamente las cuestiones ontológicas y epistemológicas? Esta afirmación se comprende mejor reformulando la cuestión en los siguientes términos: ¿qué relación guarda el cerebro con la capacidad humana para alcanzar conocimientos verdaderos? Descartes reconoce la circularidad del problema, y trata de resolverla con una respuesta claramente negativa: antes de preocuparnos por el nivel antropológico, hemos de atender la cuestión ontológica. Precisamente, es este el punto de partida que Damasio desconoce, olvido que le lleva a malinterpretar la relación cartesiana entre pensamiento y existencia. Descartes precisa que desde el «yo pienso» se sigue, clara y distintamente, el «yo existo» en el acto mismo de pensar. Así, el conocimiento que uno tiene de sus propios estados mentales será incorregible, pues la creencia de estar en un cierto estado mental no puede ser errónea porque, si los tenemos, estos son verdaderos.2 Pero formar una idea clara y distinta de uno mismo no exige pensar en el cuerpo, y para formar una idea clara y distinta del cuerpo no se tiene que pensar en sí mismo.3 De la existencia del conocer, Descartes pasa a las causas del acto cognoscitivo, para lo que ahonda en sus cualidades. Dentro de estas reflexiones cualitativas surgen dos importantes distinciones en Descartes: la que diferencia la sustancia pensante de la sustancia material y la que diferencia el conocimiento objetivo del conocimiento subjetivo. En la primera, Descartes identifica lo esencial al pensar y, en la segunda, los modos de acceso al conocimiento –en primera y en tercera persona, subjetivo frente a objetivo, respectivamente. Nos interesa la segunda distinción para entender otra de las ramificaciones del error cometido por Damasio. El acto cognoscitivo es, para Descartes, previo a la consecución de saberes objetivos, o lo que es lo mismo, no hay enunciados objetivos que no sean los del pensar. Siguiendo este razonamiento, Descartes concluye que el acceso a la primera certeza es un acto de introspección, el despertar a un tipo de realidad 2 3 Dice Descartes en el Discurso del método, cuarta parte, que es imposible estar pensando y estar equivocado de que pienso. Si estoy dudando, no puedo dudar que estoy dudando. Y del hecho de pensar no podemos estar equivocados de que existimos, puesto que no se puede pensar sin que alguien los realice. Y aun cuando, […] tengo yo un cuerpo al que estoy estrechamente unido, sin embargo, puesto que por una parte tengo una idea clara y distinta de mí mismo, según la cual soy solo algo que piensa y no extenso, y, por otra parte, tengo una idea distinta del cuerpo, según la cual, este es una cosa extensa, que no piensa, resulta cierto que yo, es decir, mi alma, por la cual soy, es entera y verdaderamente distinta de mi cuerpo, pudiendo ser y existir sin el cuerpo (Descartes, Meditaciones metafísicas, meditación sexta). 75 76 RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2018.22.5 · ISSN: 1130-6149 - pp. 66-89 desde la posición privilegiada que tiene el cognoscente respecto de su propio conocimiento. La primera y más segura certeza es, en definitiva, un tipo de conocimiento subjetivo. El motivo por el que a Damasio le parece paradójica la conclusión acabada de presentar es porque identifica subjetividad con un saber poco fiable, una falsa apreciación que se debe, como se ha mencionado, a no articular los niveles ontológico y epistemológico del pensamiento cartesiano. Apoya esta crítica, en primer lugar, el hecho de que Damasio cae en la confusión de pensar que lo que sí existen son las neuronas en interacción y que, por tanto, su estudio debe ser el punto de partida de toda investigación que pretenda explicar los fenómenos mentales. Existo –tengo neuronas… y un cuerpo–, luego pienso. El oscuro enigma es el pensamiento –la conciencia–, y el mejor modo de resolverlo son las claras certezas objetivas que proporciona la Neurobiología. Damasio no parece ser consciente de que los conocimientos neurales, aun siendo ciertos, nada dicen de la certeza. Las vivencias psíquicas tienen un papel en la Neurociencia; sin embargo, el rasgo propio del pensar, la intencionalidad del acto cognoscitivo, no acontece. No puede ser de otro modo porque la cualidad intencional –tiene razón Descartes–, solo se puede conocer en la soledad de quien es capaz de abstraer el acto de conocer de lo conocido. Así, una de las más importantes consecuencias del error de Damasio es la reducción del problema cognoscitivo o de la subjetividad –lo que Descartes entiende por mente– al problema de la conciencia, tal como este término es utilizado habitualmente en la Neurociencia contemporánea. La pregunta cartesiana por la relación cuerpo-mente queda transformada en la pregunta por la relación entre el cuerpo y las experiencias fenoménicas del cuerpo –la sensación de lo que ocurre, utilizando la expresión de Damasio. Es casi inevitable que, partiendo de esta postura, muchos neurobiólogos acaben negando el estatuto ontológico de los eventos conscientes o, al menos, que lleguen a considerarlos meros epifenómenos sin fuerza causal. La razón es la misma que atribuimos a Damasio, la creencia de que lo subjetivo es lo que no es susceptible de objetividad. Las lecturas de Descartes son interesantes para comprender que lo mental es el más real de todos los fenómenos y además prerrequisito para la objetividad. Pero hay una consecuencia más grave aún que la negación de la primera de las certezas, y de la que científicos como Damasio no son MIGUEL GRIJABA UCHE ¿Dónde está el error? La epistemología de la verdad en la neurociencia conscientes. La reducción de lo mental a lo consciente implica conculcar, en último término, el valor de verdad de los enunciados objetivos.4 3. DESNATURALIZANDO LA MENTE… Y LAS EMOCIONES Los éxitos editoriales de Damasio están contribuyendo a desplazar el problema de la verdad lejos del campo de atención de la más formalizada Neurobiología. Pero, ¿hace falta rehabilitar el dualismo cartesiano para salvar la ciencia? No necesariamente, numerosas propuestas filosóficas, desde clásicas –como el hilemorfismo de Aristóteles– hasta modernas –como la fenomenología de Husserl– han puesto de manifiesto la dicotomía subjetividad-objetividad y han propuesto heterogéneas teorías de la identidad de cariz realista. Las lecturas de Husserl, mejor incluso que las de Descartes, ayudan a entender por qué lo que intenta Damasio –esto es, aislar la cuestión de la conciencia de la cuestión de la verdad– conduce a errores categoriales que no hacen sino generar esas paradojas y discusiones absurdas e interminables que paralizan el progreso científico, por lo menos en aquellos investigadores que duermen todavía, utilizando la expresión de Rorty, el sueño metafísico. Ser capaz de pensar en las apariencias, en términos de apariencias, es ser capaz de pensar en el pensar mismo y de entender, intuitivamente, por qué Descartes separa el pensamiento del mundo físico: las apariencias no son susceptibles de mediciones espaciales. La cuestión es relevante puesto que una mirada libre de prejuicios nos presenta los fenómenos físicos y psíquicos como realidades patentemente distintas. En efecto, es posible detectar los correlatos físicos del pensamiento, pero llegar a la conclusión de que los unos son los otros requiere un discurso que no se encuentra dado en el punto de partida de Damasio. Damasio confunde el ser fenoménico –por ejemplo, mi tristeza de…– con el contenido proposicional, que sí puede ser físico –…no tener quien me quiera…– o con su causa, que también puede ser física –…por enfermedad depresiva–. Esta confusión le lleva a identificar la causa del contenido proposicional –el escaso número de receptores de serotonina puede ser la causa de mis pensamientos tristes– con la causa del ser de la rela4 Como Richard Rorty ha sabido muy bien argumentar, no es posible mantener una actitud realista fundada en la Neurofisiología. El cerebro no funciona como el espejo de la naturaleza gracias al cual podamos trascender nuestros propios límites biológicos para llegar a saber quiénes somos. La conclusión de Rorty, considerado uno de los padres del materialismo eliminativo, es que la Neurociencia solo puede ofrecer ficciones (Rorty, 1979: 392-393). 77 78 RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2018.22.5 · ISSN: 1130-6149 - pp. 66-89 ción –lo que me permite tener conciencia de... En otras palabras, por el acceso privilegiado que tenemos al fenómeno intencional, los conocimientos de lo mental son más fiables que los conocimientos de lo físico. 4. DESMITIFICANDO A DESCARTES Descartes no defiende la escisión cartesiana tal como se enuncia y le atribuyen autores como Antonio Damasio. Él consideró que siendo entidades separadas tienen el mismo significado: yo soy, yo existo como cosa pensante. Y puesto que sabemos que consideraba el pensar como una actividad separada del cuerpo, Descartes lo que celebra es la separación entre la mente, la cosa pensante –res cogitans– del cuerpo no pensante (Damasio, 2003a: 229-230).5 De hecho, cuando considera la percepción de otros cuerpos y la influencia de estos en el sujeto, habla de una afectación de este como de un todo compuesto de cuerpo y mente. Pero este combinado cuerpo-mente no parece que posea, para Descartes, ninguna condición ontológica, ya que su relación existente no es una relación de identidad ni tampoco se trata de una relación causal (Lowe, 2000: 40). Lo único que sabe Descartes es que la interacción tiene lugar (Priest, 1994: 20-52). El acto de pensamiento se relaciona con la interacción entre el cuerpo y sus partes mecánicas y la mente –alma. Descartes fue consciente de la dificultad de aceptar, al mismo tiempo, la distinción sustancial y la unión entre cuerpo y mente, y no teniendo para ello una explicación filosófica convincente, recurrió a una creencia para resolver de modo misterioso la manera en que materia y mente interaccionan. Vivía en un contexto muy influenciado por el contexto filosófico y religioso que ejercieron influencia en él y en sus contemporáneos, lo que determinaba una fuerte tradición de dualismo social, cultural y religiosamente fundamentado. Sobre dicho misterio, dice N. Grimaldi, el espíritu cartesiano está fuertemente vinculado a la disposición y funcionamiento de los órganos del cuerpo (Grimaldi, 1987: 415), lo cual vendría a significar que Descartes, en realidad, tiene la misma postura que la adoptada posteriormente por Damasio: primero somos y, en la medida en que somos, 5 «También me enseña la naturaleza, por medio de esos sentimientos de dolor, hambre, sed, etc., que no estoy metido en mi cuerpo como un piloto en su navío, sino tan estrechamente unido y confundido y mezclado con él, que formo un solo todo con mi cuerpo. Pues si esto no fuera así, no sentiría yo dolor cuando mi cuerpo está herido, puesto que soy solamente una cosa que piensa» (Descartes 1642, Meditaciones metafísicas, meditación sexta). MIGUEL GRIJABA UCHE ¿Dónde está el error? La epistemología de la verdad en la neurociencia pensamos. No en vano, algunos autores han considerado al «pienso, luego existo» como un pleonasmo en donde la palabra luego sería inútil. Para otros podría equipararse con una ecuación química, en donde el luego podría ser reemplazado por el signo =. Víctor Hugo considera que Descartes podría haber evitado incluso decir yo soy: ego ergo ego. Se sabe ser antes que se sabe pensar (Hugo, 1959: 234). G. Kirkeboen tampoco considera que Descartes creyera que el pensamiento fuera una actividad separada del cuerpo. Según él, las opiniones de Damasio han de ser comprendidas como una versión avanzada de las especulaciones de Descartes, ya que este presenta una psicología en la cual el alma inmaterial usa y se relaciona con el cuerpo (Kirkeboen, 2001a: 174). Por ejemplo, Kirkeboen señala que ya en las Reglas para la dirección del espíritu, Descartes plantea que el razonamiento matemático abstracto del intelecto es un proceso cerebral y corporal (Kirkeboen, 2001a: 174). Para Descartes, algunos procesos psicológicos como memoria, imaginación y percepción sensorial requieren una mente tanto como asiento de la conciencia, como vehículo para la formación de estados mentales, juicio y otras operaciones (Hatfield, 2007: 17). Véase que este aspecto fue mal comprendido por Damasio en la siguiente cita: El control de la inclinación animal mediante el pensamiento, la razón y la voluntad es lo que nos hizo humanos, según Las pasiones del alma, de Descartes. Estoy de acuerdo con su formulación, con la excepción de que allí donde Descartes especificó un control conseguido mediante un agente no físico yo veo una operación biológica estructurada en el interior del organismo humano y ni un ápice menos compleja, admirable o sublime (Damasio, 2003a: 122). Descartes, tanto en Las pasiones del alma, como en otros escritos, afirma la existencia de estas operaciones en el organismo y que sirven para estructurar y ordenar nuestras inclinaciones, hábitos o pensamientos (Hatfield, 2007: 33). Autores de la talla de Merleau-Ponty resaltan las frecuentes aproximaciones simplistas al planteamiento cartesiano. Según él, la cuestión de la unión entre alma y cuerpo en Descartes no es una mera dificultad especulativa, sino que da cuenta de un hecho paradójico: la existencia del cuerpo humano. Lo que se extrae de los textos de Merleau-Ponty es que Descartes se plantea el problema de un cuerpo vivo y mezclado con un alma (Merleau-Ponty, 2000: 17).6 6 Merleau-Ponty refuerza la defensa que hace de Descartes con algunas citas. En la carta de agosto de 1641 a Hyperaspistes, Descartes considera que, si lo corpóreo es lo relativo al cuerpo, también será corpórea el alma. En la carta a Elisabeth, del 28 de junio de 1643, explica que se debe concebir 79 80 RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2018.22.5 · ISSN: 1130-6149 - pp. 66-89 5. LAS PASIONES DEL ALMA Para Descartes, el término alma7 apenas se distingue de la facultad de pensar o del pensamiento mismo, localizado en la epífisis, bajo el pretexto de que esta glándula está centrada en el cerebro y, por lo tanto, equidistante a todas las radiaciones que vienen de cualquier lugar del cuerpo: Pienso que cuando Dios una un alma racional a esta máquina, como a continuación expondré, otorgará a esta alma como sede principal el cerebro y hará que su naturaleza sea tal que tenga sensaciones diversas, según las distintas formas en que estén abiertas las entradas de los poros situadas en la superficie del cerebro (Descartes, El tratado del hombre, 50). A esa alma, Descartes le asocia las pasiones, entendidas estas como verdaderos estados del alma o sentimientos. Pero el origen de las pasiones, en contra de la idea que le atribuye Damasio, se encuentra no solo en el cerebro, sino en todas las partes del cuerpo, en cuanto que sirven para la «producción de sangre y, por ende, de los espíritus»8. El alma se sumerge en estos estados a través de representaciones que le son dadas normalmente por los movimientos de la glándula: a cada movimiento le corresponde una representación, que induce al alma a caer en un determinado estado. La razón por la que el alma no tiene poder directo sobre las pasiones reside, según Descartes, en que están asociadas a elementos corporales.9 De manera similar a como Damasio defiende, también para Descartes la voluntad no puede apaciguar o avivar una pasión de modo inmediato, sino a través de las imágenes de los objetos que van asociados a las pasiones opuestas a las imágenes de las que nos queremos desembarazar. De este modo, se instaura en la filosofía cartesiana una batalla por el control de la glándula, en donde esta puede ser impulsada simultáneamente por el alma 7 8 9 la unión alma y cuerpo a imagen de la unión peso y extensión en la física escolástica. Por este motivo no queda otro remedio que conceder al alma algún tipo de materialidad y, por eso, el alma se concibe como unida al cuerpo (Merleau-Ponty, 2000: 18). La diferencia entre espíritu y alma no es un problema de traducción. Las palabras inglesas mind y soul nos envían a una distinción similar a la comentada, la cual alcanza los límites del campo de investigación de las ciencias neuropsicológicas. Para algunos filósofos, decir alma significa algo más que decir mente. Viene a representar una mente inmortal. Para Aristóteles, alma quiere decir principio de vida, sin presunción de nada espiritual (Kenny, 2000: 48-49). Tras la lectura de los artículos 36, 38 y 39 de Las pasiones del alma tiendo a pensar que Descartes sostenía más bien, y tal como defiende Hatfield, que un único centro cerebral produce, en diferentes individuos, distintos estados mentales (Hatfield, 2007: 15). Bien es cierto que, para Descartes, algunas formas de razonamiento tienen lugar sin la participación del cuerpo. Pero nuestros juicios morales, que dependen de nuestra voluntad, pueden ser ayudados por las pasiones, las cuales están inherentemente causadas por el cuerpo (Hatfield, 2007: 1). MIGUEL GRIJABA UCHE ¿Dónde está el error? La epistemología de la verdad en la neurociencia y por el cuerpo (Descartes, Las pasiones del alma, art. 47). Podemos apreciar en estas afirmaciones que Descartes intuye las correlaciones entre sustancias humorales y fenómenos vasomotores y nerviosos, pudiendo ser tratado como precursor de las ideas que luego desarrollará Claude Bernard (Campan, 1996: 138). Otra cuestión interesante es que, a pesar de lo que suele creerse, la idea de la glándula pineal como asiento del alma no es exclusiva de Descartes. De hecho, fue defendida previamente por Jean Cousin, en 1641, quien afirmaba que la glándula pineal era el asiento del sentido común (Lokhorst, 2001: 7). Lo que es original en Descartes, y escandalosamente ignorado por Damasio, es su tesis sobre que la mente se ve afectada por la actividad de dicha glándula (Campan, 1996: 138). Por tanto, queda claro que Descartes no defendió un dualismo absoluto, sino una real interdependencia entre ambas sustancias, cuerpo y alma o conciencia, sin la cual no existiría el ser humano. Damasio critica a Descartes el haberse plegado a la mentalidad de la época, acusación ominosa. Descartes tiene el coraje de presentar el almamente como manifestación del cerebro, y de exponer una relación dual –el cuerpo es el cuerpo, la mente es la mente–, pero aceptando una interdependencia entre estas dos entidades. Pero, además, lejos de generar un retraso en el mundo de la neuropsicología, como también le achaca Damasio, Descartes desarrolla en Las pasiones del alma, una innovadora asociación entre hipótesis de índole fisiopatológica con otras de índole psicopatológica. Este hecho da testimonio de su deseo de establecer uniones entre los procesos mentales y corporales. En esta misma línea de hipótesis, Hatfield afirma que el funcionamiento del cuerpo humano, en Descartes, siempre conlleva una mente interactuando con el cuerpo, que solo cesa cuando muere este. Aún más, los mecanismos corporales no se limitan a causar estados mentales en una sustancia mental, sino que también funcionan como mecanismos psicológicos que pueden causar comportamientos que no dependen de la mente.10 Otra obra que desmiente a Damasio son las Meditaciones metafísicas, en donde Descartes habla de la mezcla entre la mente y el cuerpo. De hecho, es el pionero en el tipo específico de neurobiología de la racionalidad 10 Por lo tanto, desde una visión cartesiana, psicológico –como adjetivo que denota mecanismos que guían comportamientos adaptativos– no es sinónimo de mental –en cuanto que actividad de una mente inmaterial. Por supuesto que hay estados psicológicos que precisan de una mente –e incluso de una interacción mente-cuerpo: percepción sensorial consciente, conocimiento intelectual, emociones intelectuales o actos de la voluntad, entre otros (Hatfield, 2007: 2). 81 82 RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2018.22.5 · ISSN: 1130-6149 - pp. 66-89 que el mismo Damasio propone en su libro El error de Descartes (Kirkeboen, 2001b: 196). Y cuando mi cuerpo necesita comer, beber, tendría yo un simple conocimiento de esta necesidad, sin que de ella me avisaran confusos sentimientos de hambre o sed; pues, en efecto, todos esos sentimientos de hambre, sed, dolor, etc., no son sino ciertos confusos modos de pensar, que proceden y dependen de la íntima unión y especie de mezcla del espíritu con el cuerpo (Descartes, Meditaciones metafísicas, meditación sexta). De nuevo, ningún escrito posterior de Descartes viene a contradecir esta posición clara y aparentemente opuesta a lo comentado por Damasio sobre Descartes. Para ilustrar este propósito basta leer algunos de los títulos de artículos del libro de Descartes, Las pasiones del alma: –Art. 122: De los desvanecimientos. –Art. 128: Del origen de las lágrimas. –Art. 131: Cómo se llora de tristeza. –Art. 132: De los gemidos que acompañan a las lágrimas. –Art. 133: Por qué los niños y los viejos lloran con facilidad. –Art. 134: Por qué algunos niños palidecen en vez de llorar. –Art. 135: De los suspiros. En cada uno de estos artículos Descartes propone una interpretación, asociada a una hipótesis fisiopatológica, del registro cognitivo de cada fenómeno estudiado. Ello testimonia su deseo de establecer lazos entre los procesos del pensamiento y los fenómenos somáticos. En Las pasiones del alma, Descartes da cuenta también de diferentes cuestiones importantes en la Neurociencia actual, incluyendo mecanismos neurofisiológicos descritos en De homine, como una interesante teoría precursora de la teoría del reflejo, aspectos de psicología introspectiva basados en la experiencia personal o conceptos como emociones –pasiones– intelectuales, así como estudios sobre estados afectivos del hombre, tanto normales como patológicos.11 Por último, es precisamente en las Meditaciones metafísicas donde encontramos el principio de habituación cartesiano, en el que Descartes expone una teoría de la racionalidad en la que aparecen integradas las emociones de un modo muy similar a como lo hace Damasio en su hipótesis del marcador somático (LeDoux, 1986: 303; Irons, 1985: 291). También en Reglas para la dirección del espíritu, Descartes considera al cuerpo y a las emociones como elementos fundamentales en el proceso de 11 El artículo 134 es de sorprendente modernidad, ya que describe de modo muy preciso lo que se denomina, en términos de psicosomática infantil, como espasmo del sollozo. MIGUEL GRIJABA UCHE ¿Dónde está el error? La epistemología de la verdad en la neurociencia razonamiento y racionalidad. Más concretamente, sostiene que nuestros pensamientos (acciones del alma) también causan (o se correlacionan con) cambios de los movimientos de los espíritus animales en el cerebro, y estos cambios pueden activar diferentes pasiones. Hay que reconocerle a Damasio que su idea enfatiza más el hecho de que los marcadores somáticos puedan operar de forma encubierta –sin aparecer a la conciencia– (Damasio, 2003a: 166). Por el contrario, para Descartes, la reacción emocional no posee motivos conscientes como determinantes del comportamiento y la toma de decisiones. En el mismo orden de cosas, en la regla xii de sus Reglas para la dirección del espíritu, Descartes explica cómo es posible relacionar el comportamiento reflejo, la percepción, la memoria y la cognición con procesos en un cerebro corporal. Descartes asume que el intelecto no tiene acceso directo al exterior, solo trabaja con naturalezas simples que habitan el cerebro de forma causal u ocasional. Por tanto, también podemos decir que para Descartes el cuerpo es una referencia para la mente (Hatfield, 2007: 31). En fin, no parece tener razón Damasio al querer atribuir a Descartes y a sus sucesores el supuesto divorcio moderno entre el cuerpo y el espíritu y, en especial, en la medicina occidental.12 Incluso podemos llegar a afirmar que la aportación de William James sobre la naturaleza de la emoción y del sentimiento, como un proceso que envuelve al cuerpo, era también la hipótesis de Descartes. Así, este último es claro al considerar las pasiones como una percepción directa de un terreno específico: el del cuerpo. En efecto, como apunta Merleau-Ponty, el cuerpo si capta y siente lo que ocurre fuera de él es por la pasión que lo constituye, donde la pasión es entendida aquí como un tipo de percepción de los sentidos o sensación interna13 (Kirkeboen, 2001a: 183). Es decir, son estados mentales que tienen su causa en procesos corporales. Y las denomina pasiones porque la mente es pasiva respecto de un cuerpo activo (Descartes, Las pasiones del alma, arts. 1-2). En este sentido, al leer Las pasiones del alma, encontramos: Por lo demás, al igual que el curso que siguen los espíritus hacia los nervios del corazón basta para mover la glándula, […], así también, por el simple hecho de que algunos espíritus van al mismo tiempo hacia los nervios que sirven para mover las piernas para huir, 12 Muy significativos son también los títulos de los artículos del capítulo V de la obra Sobre la búsqueda de la verdad de uno de sus más importantes discípulos, N. Malebranche. Véanse, por ejemplo, los siguientes: Sobre la unión de las ideas del espíritu con el cerebro; Sobre la unión recíproca que existe en las imágenes (huellas) del cerebro; Sobre la memoria. 13 Contrástese con las ideas aparecidas en Descartes, Las pasiones del alma (arts. 27-28). 83 84 RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2018.22.5 · ISSN: 1130-6149 - pp. 66-89 provocan otro movimiento en la misma glándula mediante el cual el alma siente y percibe esa huida, la cual puede, de esta forma, ser excitada en el cuerpo únicamente por la disposición de los órganos y sin que el alma participe (Descartes, Las pasiones del alma, art. 38). Y basta compararla con la exposición de W. James: Mi teoría [...] es que los cambios corporales que siguen directamente a la percepción de un hecho excitador y el sentimiento de esos mismos cambios que ocurren es una emoción (W. James. 1884/1969: 247). La diferencia con James estriba en la explicación de cómo dos personas que perciben un objeto de una manera aproximada, pueden tener reacciones emocionales muy diferentes. Incluso una misma persona suele modificar su reacción emocional en diferentes tiempos. Para Descartes, la causa está en que los mismos movimientos de la glándula pueden provocar también diferentes movimientos del cuerpo asociados a las diversas pasiones excitadas en el alma (cfr. Descartes, Las pasiones del alma, art. 39). En efecto, Descartes es el primero en ensayar lo que Damasio pide: un esfuerzo sistemático en comprender la compleja maquinaria que sirve de base a las emociones (Kirkeboen, 2001a: 182). De manera que las pasiones se convierten en esenciales en la preservación del cuerpo y deben ser controladas por el alma (Smith, 1999: 225). Su función es disponer el alma hacia lo que nos sea beneficioso, útil o bueno para el cuerpo (Descartes, Las pasiones del alma, art. 52). Esta es una de las razones por las que el filósofo acepta que la materia produce efectos sobre el espíritu, que lo racional puede ser preso de lo irracional y el espíritu de las pasiones. También la teoría cartesiana es, en último término, un método para alcanzar una vida mejor. Así lo expresa explícitamente en el artículo 148 de Las pasiones del alma. Pero, de nuevo, a pesar de las múltiples evidencias bibliográficas, Damasio califica de desafortunado el influjo de la filosofía cartesiana en la medicina que tendría continuidad con la metáfora de la mente como programa informático, la explicación de la mente en términos exclusivamente cerebrales o las consecuencias en la medicina tradicional acerca del estudio y tratamiento de la patología psicosomática, entre otros asuntos (Damasio, 2003a: 230-231). En referencia a la crítica de Damasio sobre el abandono del estudio de las enfermedades psicosomáticas en la teoría cartesiana, hay que decir que Descartes había detectado que la mente depende del temperamento y de las disposiciones de los órganos corporales mucho antes MIGUEL GRIJABA UCHE ¿Dónde está el error? La epistemología de la verdad en la neurociencia que nadie. Por ejemplo, así ocurre cuando enfatiza en los efectos de las emociones sobre el cuerpo (Kirkeboen, 2001a: 184). El principio en el que se apoya todo lo que he escrito, a saber, que la relación entre nuestra alma y nuestro cuerpo es tan profunda que, una vez hemos unido alguna acción corporal con algún pensamiento, ninguno de los dos se presenta sin que el otro se presente también [...]. Es fácil pensar que las extrañas aversiones de algunas personas, que les impiden tolerar el olor de las rosas, o la presencia de un gato, o cosas parecidas, sólo responden a que al comienzo de su vida fueron gravemente afectados por esos objetos (Descartes, Las pasiones del alma, art. 136). El enfoque psicosomático de Descartes se basa en la aceptación de ciertas conexiones entre emociones y movimientos corporales. Lindeboom observa al respecto: La concepción de la conexión continua de pensamientos espaciales y de fenómenos espaciales del cuerpo [...] representa, junto a la influencia de memorias inconscientes, la base cartesiana de la psicosomática. Podría apenas ser negado que estas nociones esencialmente constituyen, con una considerable extensión, la base del modelo de la moderna psicosomática (Lindeboom, 1979: 91). En efecto, en la fisiología cartesiana, una terapéutica centrada en el cuerpo será solo una parte de la solución hacia la consecución de la salud, ya que un alma sana es esencial en el mantenimiento y alcance de la supervivencia. Por esto, cuando una pasión nos desordena y nos vuelve tristes, es necesario estar advertidos de todas las imágenes que puede crear en nuestra alma y que nos van a inquietar, e intentar contraponer otras representaciones que terminen expulsando de nuestro interior esas imágenes que no dejan de ser engañosas. Nuestras pasiones tampoco pueden ser excitadas ni sofocadas por la acción directa de nuestra voluntad, pero pueden serlo indirectamente merced a la representación de las cosas que suelen ir unidas con las pasiones que queremos tener, y que son contrarias a las que queremos rechazar. Así, para excitar en uno mismo la audacia y vencer al miedo, no basta con tener esa voluntad, sino que hay que aplicarse en considerar las razones, los objetos o los ejemplos que convencen de que el peligro no es grande; que siempre hay más seguridad en la defensa que en la huida (Descartes, Las pasiones del alma, art. 45). Descartes señala que uno de los modos para adquirir una vida mejor es a través del aprendizaje cuidadoso y el control de los mecanismos emocionales. Por tanto, la propuesta cartesiana es dominar las pasiones de tal forma, y manejarlas con tanta habilidad, que los males que causan sean muy soportables, y que de todas ellas se pueda obtener la alegría, como forma 85 86 RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2018.22.5 · ISSN: 1130-6149 - pp. 66-89 más deseable de vida. Nótese que, en lo que se refiere a la gestión de las emociones, la propuesta cartesiana es muy parecida a la expresada por Spinoza y, por ende, a la de Damasio. En síntesis, es difícil entender la mala interpretación que hace Damasio de Descartes. Sospecho que el problema principal se debe a que no ha consultado las fuentes originales. Un indicio de este problema se halla en el último capítulo del libro, donde en parágrafos sucesivos Damasio emplea indistintamente dos términos a los que Descartes no había dado igual significado. En el primer parágrafo, Damasio cita un texto del Discurso del método: Por ello supe que yo era una sustancia, cuya misma esencia o naturaleza es pensar, y que para su existencia no hay necesidad de ningún lugar, ni dependencia de ninguna cosa material; de manera que este «yo», es decir, el alma por la que soy lo que soy, es completamente distinta del cuerpo, e incluso es más fácil de conocer que éste; e incluso si no existiera el cuerpo, el alma no cesaría de ser lo que es (R. Descartes, El discurso del método, cuarta parte, p. 58) A continuación, este pasaje es comentado por Damasio de la siguiente forma: Éste es el error de Descartes: la separación abismal entre el cuerpo y la mente, entre el material del que está hecho el cuerpo, medible, dimensionable, operado mecánicamente, infinitamente divisible, por un lado, y la esencia de la mente, que no se puede medir, no tiene dimensiones, es asimétrica, no divisible (Damasio, 2003a: 230). CONCLUSIONES Damasio aísla la noción de subjetividad (conciencia) de la idea de verdad y presenta una antropología sin ontología. Esta situación le conduce a un monismo fisicalista sui generis. Tal como apunta S. Holm, Damasio pretende explicar el mecanismo por el cual la mente realiza el razonamiento. Pero si dicho mecanismo deja al cuerpo en un lugar secundario, y al cerebro como una posible entidad separada, entonces Damasio no alcanza a refutar lo que él denomina el error de Descartes, es decir, la separación entre cuerpo y alma. La mayor parte de los pensamientos, sensaciones, imágenes, memoria, sentimientos e incluso las pasiones tienen una causa corporal en Descartes y requieren que la mente interactúe con el cerebro, algo que olvida Damasio. Damasio desconoce el campo de batalla que es la metafísica cartesiana y cae en un reduccionismo neuronal porque desco- MIGUEL GRIJABA UCHE ¿Dónde está el error? La epistemología de la verdad en la neurociencia noce realmente la Filosofía además de una naturalización de la mente. Su teoría se encuadra dentro de un monismo materialista de corte cerebrista o neural como explicación del acontecer emocional o de conciencia, tal como se deriva de la lectura de sus libros. El error de Damasio consiste en creer en la Neurobiología como fundamento único de su teoría de la mente. En contraste, sí que hay razones para sospechar que la Neurología no pueda acabar por rellenar el abismo entre lo material y lo espiritual. Damasio no es el único investigador del siglo xx que afirma que Descartes es el enemigo que el intelectual moderno ha de combatir. Sin embargo, la lectura que hace Damasio de Descartes no es correcta: trata de rebatir la filosofía cartesiana apelando casi exclusivamente a la Neurología y acaba introduciendo mayores malentendidos en el diálogo entre científicos y filósofos que aquellos que intentaba disolver. ¿Defendió Descartes un dualismo absoluto? Tal como trato de reflejar, tras una lectura intensa de sus textos, mi respuesta es que no. Para él, existe una real interdependencia entre ambas sustancias, cuerpo y alma o conciencia, sin la cual no existiría el ser humano. El pensamiento nace en la confluencia entre lo material (el cuerpo) y la mente (alma). Siguiendo el mismo hilo conductor, en Las pasiones del alma, Descartes da cuenta también de diferentes cuestiones importantes en la Neurociencia actual, incluyendo mecanismos neurofisiológicos que explican esta interacción mente y cuerpo. Por último, es precisamente en las Meditaciones metafísicas donde encontramos el principio de habituación, en el que Descartes expone una teoría de la racionalidad en la que aparecen integradas las emociones de un modo muy similar a como lo hará posteriormente Damasio con su hipótesis del marcador somático. Damasio trata de evitar el espejismo homuncular, afirmando que la conciencia –la mente– surge en la interacción entre las representaciones mentales de los objetos y de mi cuerpo en el acto de observar esos objetos. Esta justificación es, a mi parecer, insuficiente. Damasio reconoce que no sabemos con seguridad la manera como el cerebro produce la mente, aceptando un emergentismo débil y un cierto dualismo de punto de vista. Este es otro de los puntos débiles del autor. La antropología de Damasio se alinea con la de Spinoza y es una interpretación no antropocéntrica de hombre. Sin embargo, también Descartes, como Damasio, utiliza el cuerpo como referencia a la mente, y la relación de las emociones con el cuerpo y la mente. Así, no solo es que Descartes sea menos dualista de lo que Damasio imagina, sino que Damasio es más cartesiano de lo que le gustaría. 87 88 RECERCA · DOI: http://dx.doi.org/10.6035/Recerca.2018.22.5 · ISSN: 1130-6149 - pp. 66-89 BIBLIOGRAFÍA Campan, Louis (1996). A propos de «l’année Descartes». Descartes le physiologist. Urgences, XV, 137-139. Damasio, Antonio (1990). Individuals with sociopathic behaviour caused by frontal damage fail to respond autonomically to social stimuli. Behav Brain Res, 41, 81-94. Damasio, Antonio (1994). Descartes’ error and the future of human life. Sci Am, 271 (4), 144. Damasio, Antonio (1998). Investigating the biology of consciousness. Philos Trans R Soc Lond B Biol Sci, 353 (1377), 1879-1882. Damasio, Antonio (1999). How the brain creates the mind. Sci Am, 281 (6), 112-117. Damasio, Antonio (2000a). Creación cerebral de la mente. Investig Cienc, 46, 66-71. Damasio, Antonio (2000b). Eighth C.U. Ariens Kappers lecture. The fabric of mind: a neurobiological perspective. Prog Brain Res, 126, 457-467. Damasio, Antonio (2001a). La sensación de lo que ocurre: cuerpo y emoción en la construcción de la conciencia. Madrid: Debate S.A. Damasio, Antonio (2001b). Descartes’ error revisited. J Hist Neurosci, 10(2), 192-194. Damasio, Antonio (2001c). In reply to Geir Kirkeboen. J Hist Neurosci, 10(2), 197. Damasio, Antonio (2003a). El error de Descartes: La emoción, la razón y el cerebro humano. Barcelona: Editorial Crítica, SL. Damasio, Antonio (2003b). Transparent feelings: A reply to Jaak Panksepp and Douglas Watt. Neuropsychoanalysis, 5(2), 215-218. Damasio, Antonio (2003c). Spinoza’s monism and the idea of the body: A repy to Heidi Ravven. Neuropsychoanalysis, 5(2), 229-230. Damasio, Antonio (2005). En busca de Spinoza. Neurobiología de la emoción y los sentimientos. Barcelona: Harcourt Crítica. Damasio, Antonio & Damasio, Hanna (2006). Minding the body. Daedalus, 135 (3), 15-22. Damasio, Antonio (2007). Neuroscience and Ethics: intersections. Am J Bioethics, 7(1), 3-7. Damasio, Antonio (2010). Y el cerebro creó al hombre. Barcelona: Destino. Damasio, Hanna, Grabowsky,Thomas, Randall, Frank, Galaburda, Albert M. & Damasio, Antonio (1994). The return of Phineas Gage: clues about the brain from the skull of a famous patient. Science, 264, 1102-1105. MIGUEL GRIJABA UCHE ¿Dónde está el error? La epistemología de la verdad en la neurociencia Descartes, Renné (1637/1981). Discurso del método. Castellón: Los Libros de Plon. Descartes, Renné (1628/1984). Reglas para la dirección del espíritu. Madrid: Alianza Editorial, SA. Descartes, Renné (1662/1990). El tratado del hombre. Madrid: Alianza Editorial, SA. Descartes, Renné (1641/2005a). Meditaciones metafísicas. Madrid: Ed.Tecnos, SA. Descartes, Renné (1622/2005b). Las pasiones del alma. Madrid: Editorial Edaf, SA. Grimaldi, Nicholas (1987). Les ambitions médicales de Descartes et sa découverte en 1637 de la principale maladie de l’homme. Hist Sc Méd, XVI, 415-420. Harris, Edmund (2003). Descartes’ error and Spinoza’s truth. NASS monograph, 11, 6-23. Hatfield, George (2007). The Passions of the soul and Descartes’s machine psychology. St Hist Philos Sc Part A, 38 (1), 1-35. James, William (1884/1969). What is an emotion? En William James. Collected essays and reviews (244-280). New York: Russell and Russell. Kenny, Anthony (2000). La metafísica de la mente. Barcelona: Ed. Paidós. Kirkeboen, George (2001a). Descartes’ embodied psychology: Descartes’ or Damasio’ error? J Hist Neurosci, 10(2),73-191. Kirkeboen, George (2001b). Sources of Damasio’s error. A reply to Damasio. J Hist Neurosci, 10(2), 195-196. LeDoux, Joseph (1986). The neurobiology of emotion. En LeDoux, Joseph (Ed.). Mind and Brain. Dialogues in Cognitive Neuroscience (301324). Cambridge: Cambridge University Press. Lindeboom, Gerrit Arie (1979). Descartes and medicine. Amsterdam: Rodopi. Lowe, Edward Jonathan (2000). Filosofía de la mente. Barcelona: Idea Books, SA. Merleau-Ponty, Maurice (2000). Fenomenología de la percepción. Barcelona: Ed. Península. Priest, Stephan (1994). Teorías y filosofías de la mente. Madrid: Cátedra. Rorty, Richard (1979). Philosophy and the mirror of nature. Princeton: Princeton University Press. Smith, C.U.M. (1999). 350th anniversary of Les passions de l’ame. J HistNeurosci, 8(3), 221-226. 89
https://openalex.org/W2884538833	http://www.phcogj.com/sites/default/files/PharmacognJ-10-5-963.pdf	English	null	Pharmacognostic Evaluation and HPTLC Finger Printing of Rhizome of Chlorophytum borivilianum Sant. and F. from Nepal	Pharmacognosy journal	2,018	cc-by	4,211	Kopila Adhikari1* KN Anuradha1 N. Prabhu Suchitra2 1Department of Dravyaguna, SDM College of Ayurveda and Hospital, Hassan, Karnataka, INDIA. 2Pharmacognosy and Phytochemistry, SDM Research Center for Ayurveda and Allied Science, Kuthpady, Udupi, Karnataka, INDIA. Cite this article: Kopila A, Anuradha KN, Prabhu Suchitra N. Pharmacognostic Evaluation and HPTLC Finger Printing of Rhizome of Chlorophytum borivilianum Sant. and F. from Nepal. Pharmacogn J. 2018;10(5):963-8. Pharmacogn J. 2018; 10(5):963-968 A Multifaceted Journal in the field of Natural Products and Pharmacognosy www.phcogj.com \| www.journalonweb.com/pj \| www.phcog.net Pharmacogn J. 2018; 10(5):963-968 A Multifaceted Journal in the field of Natural Products and Pharmacognosy www.phcogj.com \| www.journalonweb.com/pj \| www.phcog.net Pharmacogn J. 2018; 10(5):963-968i Pharmacogn J. 2018; 10(5):963-968i Original Article Original Article INTRODUCTION Chlorophytum includes nearly 300 species which are distributed throughout tropical and subtropical parts of the world. Seventeen species of Chlorophytum is recorded in India.1 Among them Chlorophytum boriv ilianum Sant. and F. is having highest saponin content which is responsible for its therapeutic utilities.2 It was first reported in India in 1954 and reached rare status in nature due to over exploitation.3 C. arundinaceum Baker, C. laxum R.Br., C. tuberosum Baker, C. orchi dastrum Hook.f., p.p. non Lindl. are used as adulter ant and substitutes.4 C. borivilianum is commercially cultivated and commonly used by pharmaceuticals.5 Chlorophytum borivilianum Sant. and F., also known as Shveta Musali, from the family Liliaceae, is a peren nial herb, 10-35 cm in height; rhizome elongated, cylindrical, fleshy. Leaves are basal, linear-lanceolate and membranous with short petioles. White flowers with six petals, small, black seeds enclosed in flower ing boles.6–8 It is used in Ayurveda, Traditional Chi nese Medicine, Unani and in folklore practice as an aphrodisiac herb. It‘s rhizome is Shukrala, Rasayana, Vrisya, Balya, Brimhana, Madhura, Tikta, Snigdha, Sheeta, and Laghu.4,9,10 It has shown spermatogenic, aphrodisiac, immune-modulatory, anti-diabetic, anti- oxidant, anti-stress, anti-microbial, anti-aging, anti- tumor and anti-inflammatory activities.11–15 Saponins (borivilianosides E-H), flavonoids, proteins, carbohy drate, phenolics, triterpenoids, tannis, sucrose, glu cose, fructose, galactose, mannose and xylose have been reported from C. borivilianum.2-3,15–16 Kopila Adhikari1* KN Anuradha1 N. Prabhu Suchitra2 f Key words: Chlorophytum borivilianum, Pharmacognostic, Phytochemical, Shveta Musali, Qual ity Control. Key words: Chlorophytum borivilianum, Pharmacognostic, Phytochemical, Shveta Musali, Qual ity Control. Adhikari Kopila Department of Dravyaguna, SDM Col lege of Ayurveda and Hospital, Hassan, Karnataka, INDIA. Plant material Fresh rhizomes were collected from Chitwan Dis trict, Nepal in the month of November - December. The plant material was authenticated at Department of Dravyaguna, SDM College of Ayurveda Hassan, Karnataka and the voucher specimen was depos ited in the respective herbarium for future refer ence (SDMCAH-DG/2017/14). The rhizomes were cleaned and shade dried. The dried rhizomes were coarsely powdered and used for macroscopic, micro scopic characterization, phyto-chemical analysis and HPTLC. Article Available online http://www.phcogj.com/v10/i5 Pharmacognostic Evaluation and HPTLC Finger Printing of Rhizome of Chlorophytum borivilianum Sant. and F. from Nepal Kopila Adhikari1,, KN Anuradha1, N. Prabhu Suchitra2 ABSTRACT Introduction: Chlorophytum borivilianum Sant. and F.; commonly known as Shveta Musali from the family Liliaceae is a perennial herb. It is used in Ayurveda, Traditional Chinese Medicine, Unani and in folklore practice as an aphrodisiac herb. Present study depicts pharmacognostic features of Chlorophytum borivilianum Sant. and F. collected from Nepal. Methods: Macro- microscopic analyses, physico-chemical studies and HPTLC finger printing of rhizomes of Chlo rophytum borivilianum Sant. and F. were carried out according to pharmacopoeial procedures. Results: Microscopic analysis has shown presence of epidermis, cork, cortex, collenchymatous cells, starch grains, cluster crystals of calcium oxalate, idioblast, phloem, vascular bundles, pit ted xylem parenchyma, sclereids, stone cells, fragment of epiblema, and acicular needles. Pre liminary phytochemical analysis revealed presence of alkaloid, carbohydrate, carboxylic acid, resins and saponins. TLC photo-documentation revealed presence of many phyto-constituents with different Rf values and HPTLC densitometric scan of the plates showed numerous bands under short UV, long UV and 620 nm (after derivatisation). Conclusion: Chlorophytum borivil ianum Sant. and F. was evaluated for its pharmacognostic features and HPTLC. These specific identities will be useful in identification and authentication of the raw drug. Kopila Adhikari1 KN Anuradha1 N. Prabhu Suchitra2 1Department of Dravyaguna, SDM College of Ayurveda and Hospital, Hassan, Karnataka, INDIA. 2Pharmacognosy and Phytochemistry, SDM Research Center for Ayurveda and Allied Science, Kuthpady, Udupi, Karnataka, INDIA. Microscopic evaluation Microscopic evaluation of transverse section of rhizome of Chlorophy tum borivilianum Sant. and F. revealed the presence of epidermis, cork, starch grains, idioblast cells, outer cortex, xylem, phloem, radial vascular bundle, collenchyma cells, single layered endodermis, stellar region, pith, and numerous cluster crystals. The details of the microscopic evaluation of transverse section of rhizome are presented in Figure 1. Microscopic Organoleptic and macroscopic observations Results obtained from organoleptic and macroscopic observations of fresh rhizome and powder are illustrated in the Table 1. evaluation of powder of rhizome of Chlorophytum borivilianum Sant. and F. revealed the presence of starch grains, pitted xylem parenchyma, vessels, cork, sclereids, stone cells, fragment of epiblema, acicular needles. The details of the powder microscopy of rhizome are presented in Figure 2. Preliminary phyto-chemical screening Ethanolic extract of C. borivilianum Sant. and F. was subjected to quali tative evaluation for the presence or absence different groups of phyto- constituents such as alkaloids, flavonoids, saponins, carbohydrates, carboxylic acid, coumarins, phenol, quinine, resins, steroid, tannin, ter penoid, and amino acids.20 Detail of phyto-chemical evaluation is illus trated in Table 3. Organoleptic and Macroscopic Evaluation Fresh and dried rhizomes along with the powder were evaluated for their organoleptic and macroscopic features i.e. size, shape, color, odor, taste, texture and specific botanical characters were evaluated as per the stan dard procedure.17 The external features of the test samples were docu mented using Canon IXUS digital camera. Powder microscopy Pinch of powder of rhizome previously sieved was put on the slide and mounted in glycerin. Powder characters are observed under the Zeiss AXIO trinocular microscope attached with Zeiss Axio Cam camera under bright field light. Microscopic evaluation Transverse section of rhizome: Sample was preserved in fixative solu tion. The fixative used was FAA (Formalin-5ml + Acetic acid-5ml + 70% Ethyl alcohol-90ml). The materials were left in FAA for more than 48 h.18–19 The preserved specimens were cut into thin transverse section using a sharp blade and the sections were stained with safranin. The slides were also stained with iodine in potassium iodide for detection of starch. Transverse sections were photographed using Zeiss AXIO trin ocular microscope attached with Zeiss Axio Cam camera under bright field light. Magnifications of the figures are indicated by the scale-bars. Figure 1: Macro-microscopic features of Rhizome of Chlorophytum borivilianum Sant. and F. Fig. la - Rhi/ome; Fig. lb - Powder; Fig. Ic - TS through outer region; Fig. Id - Enlarged TS through outer region; Fig. le - TS through inner region; Fig. If - Enlarged xylem and phloem; E - Epidermis; Ck - Cork; Id - Idioblast; OC - Outer cortex; ACr - Acicular crystal; SG - Starch grain: Col - Collenchyma: End - Endodermis; IC - lnner cortex; Pi - Pith; Xy - Xylem; Ph - Phloem. RESULTS Organoleptic and macroscopic observations Kopila et al.: Pharmacognostic Evaluation of Rhizome of Chlorophytum borivilianum Sant. and F. Kopila et al.: Pharmacognostic Evaluation of Rhizome of Chlorophytum borivilianum Sant. and F. Kopila et al.: Pharmacognostic Evaluation of Rhizome of Chlorophytum borivilianum Sant. and F. Table 1: Organoleptic and Macroscopic Evaluation of Chlorophytum bori vilianum Sant. and F. Observations Rhizome Powder Size 5-12cm long, 1.5-2cm diameter NA Shape Elongated NA Color Whitish Brownish Odor Not characteristics Not characteristics Taste Madhura, Tikta, Mucilagenous Madhura, Tikta, Mucilagenous Texture Smooth and having horizontal wrinkles on drying Smooth Table 1: Organoleptic and Macroscopic Evaluation of Chlorophytum bori vilianum Sant. and F. Table 1: Organoleptic and Macroscopic Evaluation of Chlorophytum bori vilianum Sant. and F. Physico-chemical analysis Physico-chemical parameters viz. loss on drying at 105ºC, total ash, acid insoluble ash, water soluble ash, ethanol and water soluble extractive val ues were evaluated using standard methods.20 HPTLC finger printing One gm of powdered sample of Chlorophytum borivilianum Sant. and F. was suspended in 10.0ml of alcohol (99.9%) with intermittent shaking for the first few hours and kept for 24 h at room temperature followed by filtration, made up to 10.0ml with ethanol; 8µl of the above extract was applied on a pre-coated silica gel F254 on aluminum plates to a band width of 7 mm using Linomat 5 TLC applicator. The plate was developed in Toluene: Ethyl acetate (7.0: 3.0). The developed plates were visual ized in short UV, long UV and then derivatised with vanillin sulphuric acid and scanned under UV 254nm, 366nm and 620nm. Retention fac tor (Rf), color of the spots and densitometric scan were recorded using CAMAG Scanner 4.21–22 Figure 1: Macro-microscopic features of Rhizome of Chlorophytum borivilianum Sant. and F. Fig. la - Rhi/ome; Fig. lb - Powder; Fig. Ic - TS through outer region; Fig. Id - Enlarged TS through outer region; Fig. le - TS through inner region; Fig. If - Enlarged xylem and phloem; E - Epidermis; Ck - Cork; Id - Idioblast; OC - Outer cortex; ACr - Acicular crystal; SG - Starch grain: Col - Collenchyma: End - Endodermis; IC - lnner cortex; Pi - Pith; Xy - Xylem; Ph - Phloem. Copyright Copyright © 2018 Phcog.Net. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. 963 Pharmacognosy Journal, Vol 10, Issue 5, Sep-Oct, 2018 Pharmacognosy Journal, Vol 10, Issue 5, Sep-Oct, 2018 Kopila et al.: Pharmacognostic Evaluation of Rhizome of Chlorophytum borivilianum Sant. and F. Physico-chemical analysis Physico-chemical characters were evaluated and the results obtained are illustrated in Table 2. Pharmacognosy Journal, Vol 10, Issue 5, Sep-Oct, 2018 Pharmacognosy Journal, Vol 10, Issue 5, Sep-Oct, 2018 964 Kopila et al.: Pharmacognostic Evaluation of Rhizome of Chlorophytum borivilianum Sant. and F. Kopila et al.: Pharmacognostic Evaluation of Rhizome of Chlorophytum borivilianum Sant. and F. Figure 2: Powder Microscopy of Rhizome of Chlorophytum borivilia num Sant. and F. Fig: 2a - Parenchyma with starch grains; Fig: 2b - Parenchyma with bunch of starch grains; Fig: 2c - Isolated starch: Fig: 2d - Fragments of Epiblema: Fig: 2e - Vessels; Fig: 2f - Bundle of fibres; Fig: 2g - Cork cells; Fig: 2h - Group of Stone cells: Fig: 2i - Pitted xylem parenchyma; Fig: 2j - Group of Sclereids; Fig: 2k - Isolated sclereids; Fig: 21 - Aciculur needles, of the plates showed numerous bands under short UV, long UV and 620 nm (after derivatisation). of the plates showed numerous bands under short UV, long UV and 620 nm (after derivatisation). Phytochemical Analysis Phytochemical analysis of Chlorophytum borivilianum Sant. and F. had shown presence of alkaloid, carbohydrate, carboxylic acid, resins and saponins. Previous studies had shown presence of steroid, glycosides, saponins, and triterpenoid starch in Chlorophytum borivilianum Sant. and F.23–24 Macroscopic characters Figure 2: Powder Microscopy of Rhizome of Chlorophytum borivilia num Sant. and F. Rhizome of C. borivilianum was elongated, with whitish external sur face. It was smooth and having horizontal wrinkles on drying. It was not having any characteristics odor. It was having Madhura (Sweet), Tikta (Bitter) and mucilaginous taste. Powder of C. borivilianum was brownish in color. Fig: 2a - Parenchyma with starch grains; Fig: 2b - Parenchyma with bunch of starch grains; Fig: 2c - Isolated starch: Fig: 2d - Fragments of Epiblema: Fig: 2e - Vessels; Fig: 2f - Bundle of fibres; Fig: 2g - Cork cells; Fig: 2h - Group of Stone cells: Fig: 2i - Pitted xylem parenchyma; Fig: 2j - Group of Sclereids; Fig: 2k - Isolated sclereids; Fig: 21 - Aciculur needles, PHOTO DOCUMENTATION (SOLVENT SYSTEM PET ETHER: ETHER ACETATE) Two spots were detected under short UV - 254 nm in Chlorophytum borivilianum Sant. and F. (Rf 0.70, 0.77). All of them were having light green color. Four spots were detected under long UV - 366 nm (Rf 0.04, 0.38 – fluorescent light blue color, Rf 0.72, 0.84 – fluorescent dark blue color). Three spots were detected after derivatisation - 620 nm (Rf 0.24, 0.54, 0.77 – light purple color). Detail of HPTLC photo documentation are illustrated in Figure 3. Microscopic characters Transverse section of rhizome of C. borivilianum was having epidermis, cork, starch grains, idioblast cells, outer cortex, xylem, phloem, radial vascular bundle, collenchyma cells, single layered endodermis, stellar region, pith, and numerous cluster crystals. Powder microscopy of the given sample had pitted xylem parenchyma, starch grains, cork cells, sclereids, bundle of fibers, vessels, acicular needles, fragments of epi blema and stone cells. Table 2: Physico-chemical Evaluation of Rhizome of Chlorophytum boriv ilianum Sant. and F. Observations Observed values Foreign matter 7.93±0.06% Loss on drying 8.70±0.02% Total ash 8.91±0.05% Acid insoluble ash 4.51±0.01% Water soluble ash value 06.45±0.02% Alcohol soluble extractive 3.97±0.04% Water soluble extractive 24.78±0.11% Table 2: Physico-chemical Evaluation of Rhizome of Chlorophytum boriv ilianum Sant. and F. Relevance of finding from current study related to microscopic evalua tion can be also substantiated from the earlier works.23 Phytochemical Evaluation Phytochemical evaluation of Chlorophytum borivilianum Sant. and F. revealed the presence of alkaloid, carbohydrate, carboxylic acid, resins and saponins in the rhizome. Detail of phyto-chemical evaluation is illustrated in Table 3. HPTLC DENSITOMETRIC SCAN Total 17 numbers of active components were detected in Chlorophytum borivilianum Sant. and F. having Rf value (0.01, 0.04, 0.25, 0.27, 0.33, 0.39, 0.60, 0.79, 0.85, 0.05, 0.42, 0.79, 0.03, 0.21, 0.28, 0.56, 0.68, 0.86). The detail of HPTLC photo documentation are illustrated in Figure 4. Nine peaks were detected under short UV - 254 nm; among them maximum percentage of area were occupied by Rf 0.01 (35.26%), 0.04 (12.94%), 0.33 (11.11%). Four peaks were detected under long UV - 366nm; among them maximum percentage of area were occupied by Rf 0.01 (67.65%), 0.05 (21.20%), 0.79 (7.54%). Six peaks were detected after derivatization - 620nm; among them maximum percentage of area were occupied by Rf 0.03 (65.06%), 0.68 (13.38%), 0.86 (9.51%). HPTLC Finger printing Rf values and color of the spots in chromatogram developed in toluene: ethyl acetate (7.0:3.0) for ethanolic extract of rhizomes of Chlorophytum borivilianum Sant. and F. were recorded. TLC photo-documentation revealed presence of many phytoconstituents with different Rf values and HPTLC densitometric scan of the plates showed numerous bands under short UV, long UV and 620 nm (after derivatisation). Rf values and color of the spots in chromatogram developed in toluene: ethyl acetate (7.0:3.0) for ethanolic extract of rhizomes of Chlorophytum borivilianum Sant. and F. were recorded. The details of Rf value is given in Table 4. TLC photo-documentation revealed presence of many phy toconstituents with different Rf values and HPTLC densitometric scan 965 Pharmacognosy Journal, Vol 10, Issue 5, Sep-Oct, 2018 Kopila et al.: Pharmacognostic Evaluation of Rhizome of Chlorophytum borivilianum Sant. and F. Kopila et al.: Pharmacognostic Evaluation of Rhizome of Chlorophytum borivilianum Sant. and F. Kopila et al.: Pharmacognostic Evaluation of Rhizome of Chlorophytum borivilianum Sant. and F. Photo documentation (solvent system pet ether: ether acetate) Table 3: Phytochemical evaluation of Chlorophytum borivilianum Sant. and F. Test Color if positive Result Remarks 1. Alkaloids Dragendrof‘s test Orange precipitate Orange precipitate Present Wagners test Red precipitate Red precipitate Present Mayers test Dull white precipitate Dull white precipitate Present 2. Steroids Liebermann- buchard test Bluish green No Bluish green Absent Salkowski test Bluish red to cherry red Red color in the chloroform Absent 3. Carbohydrate Molish test Violet ring Violet ring Present Fehlings test Brick red precipitate Bluish color Absent Benedicts test Red precipitate Bluish color Absent 4. Tannin With FeCl3 Dark blue or green or brown Golden Yellow Color Absent 5. Flavanoids Shinoda‘s test Red to pink Golden Yellow Color Absent 6. Saponins With NaHCO3 Stable froth Stable froth Present 7. Triterpenoids Tin and thionyl chloride test Red Black color Absent 8. Coumarins With 2 N NaOH Yellow Colorless solution Absent 9. Phenols With alcoholic ferric chloride Blue to blue black, brown Golden Color solution Absent 10. Carboxylic acid With water and NaHCO3 Brisk effervescence Brisk Effervescence Present 11. Resin With aqueous acetone Turbidity Turbidity Present 12. Quinone 5% NaOH Pink/purple/red Color less solution Absent 13. Amino acids Ninhydrine reagent Purple color Color less solution Absent Figure 3: HPTLC Photo Documentation of Rhizome of Chlorophytum borivilianum Sant. and F. Table 4: Rf values of Ethanolic Extract of Rhizome of Chlorophytum borivilianum Sant. and F. ABBREVIATIONS HPTLC Densitometric scan HPTLC Finger printing Short UV Long UV After derivatisation - 0.04 (FL. blue) - - - 0.24 (L. purple) - - - - - - - 0.38 (FL. blue) - - - - - - - - - 0.59 (L. purple) - - - 0.70 (L. green) - - - 0.72 (FD. blue) - 0.77 (L. green) - 0.77 (L. purple) - 0.84 (FD. blue) - D-Dark; L-Light; F- Fluorescent Table 4: Rf values of Ethanolic Extract of Rhizome of Chlorophytum borivilianum Sant. and F. Short UV Long UV After derivatisation - 0.04 (FL. blue) - - - 0.24 (L. purple) - - - - - - - 0.38 (FL. blue) - - - - - - - - - 0.59 (L. purple) - - - 0.70 (L. green) - - - 0.72 (FD. blue) - 0.77 (L. green) - 0.77 (L. purple) - 0.84 (FD. blue) - D-Dark; L-Light; F- Fluorescent Table 4: Rf values of Ethanolic Extract of Rhizome of Chlorophytum borivilianum Sant. and F. Short UV Long UV After derivatisation - 0.04 (FL. blue) - - - 0.24 (L. purple) - - - - - - - 0.38 (FL. blue) - - - - - - - - - 0.59 (L. purple) - - - 0.70 (L. green) - - - 0.72 (FD. blue) - 0.77 (L. green) - 0.77 (L. purple) - 0.84 (FD. blue) - D-Dark; L-Light; F- Fluorescent Table 4: Rf values of Ethanolic Extract of Rhizome of Chlorophytum borivilianum Sant. and F. Table 4: Rf values of Ethanolic Extract of Rhizome of Chlorophytum borivilianum Sant. and F. Table 3: Phytochemical evaluation of Chlorophytum borivilianum Sant. and F. Figure 3: HPTLC Photo Documentation of Rhizome of Chlorophytum borivilianum Sant. and F. 4. Tannin Dark blue or green or brown Golden Yellow Color Absent With FeCl3 5. Flavanoids Shinoda‘s test 6. Saponins 7. Triterpenoids Figure 3: HPTLC Photo Documentation of Rhizome of Chlorophytum borivilianum Sant. and F. 8. Coumarins among them maximum percentage of area were occupied by Rf 0.01 (35.26% - 254nm); 0.01 (67.65% - 366nm), 0.03 (65.06% - 620nm). Rel evance of finding from current study related to HPTLC fingerprinting can be also substantiated from the earlier works.23 9. Phenols CONFLICT OF INTEREST Total eight spots detected in Chlorophytum borivilianum Sant. and F in different Rf value. Number of spots indicates the total number of active chemical components present in the given sample. The authors declare no conflict of interest. ACKNOWLEDGEMENT 13. Amino acids Authors are highly grateful to Department of Dravyaguna, SDM College of Ayurveda, Hassan; SDM Center for Research in Ayurveda and Allied science, Udupi for providing the lab facilities. CONCLUSION 10. Carboxylic acid Macro-microscopic, physico-chemical, preliminary phytochemical and HPTLC finger printing of Chlorophytum borivilianum Sant. and F. has been carried out as per pharmacopoeial methodology. The detail presented in the study shows the qualitative presence of various secondary metabolites and in the rhizome of Chlorophytum borivilianum Sant. and F. Thus, the study was helpful in the qualitative analysis of genuinity of the given drug. Macro-microscopic, physico-chemical, preliminary phytochemical and HPTLC finger printing of Chlorophytum borivilianum Sant. and F. has been carried out as per pharmacopoeial methodology. The detail presented in the study shows the qualitative presence of various secondary metabolites and in the rhizome of Chlorophytum borivilianum Sant. and F. Thus, the study was helpful in the qualitative analysis of genuinity of the given drug. 11. Resin Pharmacognosy Journal, Vol 10, Issue 5, Sep-Oct, 2018 dradeva. Krisnadasa Academy, Varanasi, 1982; 208-209. dradeva. Krisnadasa Academy, Varanasi, 1982; 208-209. REFERENCES 10. Gupta AK, Madhu S. Reviews on Indian medicinal plants, vol.6. Medicinal plants unit, Indian council of medical research; New Delhi, India, 2008;87-90. 1. Patil VN, Deokule SS. Pharmacognostic evaluation of Chlorophytum orchidas trum Lindl. Annals of Biological Research. 2010;1(4):126-37. 11. Kenjale R, Shah R, Sathaye S. Effects of Chlorophytum borivilianum on sexual be havior and sperm count in male rats. Phytotherapy Research. 2008;22(6):796-801. 2. Sharma R, et al. Saponin: A wonder drug from Chlorophytum species. Global Journal of Research on Medicinal Plants and Indigenous Medicine. 2012;1(10):503-15. 12. Mohd M, Shah AK, Mohd A, Abhishek M, Aftab A. Antidiabetic activity of the aqueous extract of Chlorophytum borivilianum in Streptozotocin induced- hyperglycemic rats- A preliminary study. Journal of Pharmacy Research. 2009;2(1):51-3. 3. Thakur GS, Bag M, Sanodiya BS. Debnath M, Bhadouriya P, Prasad GBKS, et al. Chlorophytum borivilianum A white gold for biopharmaceuticals and Neutraceu ticals. Current Pharmaceutical Biotechnology. 2009;10(7):650-66. 13. Kenjale RD, Shah RK, Sathaye SS. Anti-stress and anti-oxidant effects of roots of Chlorophytum borivilianum Sant. and F. Indian Journal of Experimental Biol ogy. 2007;45:974-9. 4. Sri Bhava Misra. Bhavprakash Nighantu, Guduchyadi Varga, edited by Misra Brahmasankara, Vaisya Rupalal, part 1, Chaukhamba Sanskrit Samsthana, Va ranasi, 1999;389-91. 14. Sundaram S, Dwivedi P, Purwar S. Antibacterial activities of crude extracts of Chlorophytm borivilianum to bacterial pathogens. Research Journal of Medici nal Plants. 2011;5(3):343-7. 5. Singh A, Khanuja SPS, Singh S. Agronomic practices for the production of Safed musli (Chlorophytum borivilianum Sant. and F.) in India. Natural Product Radi ance. 2000;2(6):308-13. 15. Sreevidya N, Govindarajan R, Vijayakumar M, thakur M, Dixit VK, Mehrotra S, Madhusudan KP. Action of fructo-oligo polysaccharide fraction of Chlorophytum borivilianum against streptozotocin induced oxidative stress. Planta Medica. 2006;72(15):1421-4. 6. Mandal DG, Nandi AK. Morphological and anatomical circumscription for the identification of two source of aphrodisiac medicine Chlorophytum borivilianum Sant. and F. and Chlorophytum tuberosum (Roxb.) Baker. International Journal of Medicinal and Aromatic Plants. 2012;2(3):406-10. 16. Acharya D, Mitaine-Offer AC, Kaushik N, Miyamoto T, Paululat T, MirjoletT, et al. Steroidal Saponin from Chlorophytum borivilianum. Journal of Natural Products. 2009;73:7-11. 7. Sharma R. Agro-Techniques of Medicinal Plants. Daya Publishing House, Delhi. 2004;168. 8. Joshi, Joshi, Prajapati. Seed album of Some Medicinal Plants of India. Asian Medicinal Plants and Health Care Trust, Jodhpur (Rajasthan) India. 2005;16. 17. Kokate CK. Practical pharmacognosy. Vallabh Prakasan, New Delhi. 1986;111. 18. Johansen DA. Plant microtechnique. Mc Graw-Hill Book Company, Inc: London. 1940:530. 9. Pandit Narahari. HPTLC Densitometric scan TLC: Thin layer chromatography; HPTLC: High Performance Thin layer chromatography; Rf: Retention factor; UV: Ultra violet; nm: Nano meter; ml: Mili-liter; µl: Micro-liter; viz: Namely. Total 17 numbers of active components were detected in Chlorophytum borivilianum Sant. and F. having Rf value (0.01, 0.04, 0.25, 0.27, 0.33, 0.39, 0.60, 0.79, 0.85, 0.05, 0.42, 0.79, 0.03, 0.21, 0.28, 0.56, 0.68, 0.86) 966 Pharmacognosy Journal, Vol 10, Issue 5, Sep-Oct, 2018 Kopila et al.: Pharmacognostic Evaluation of Rhizome of Chlorophytum borivilianum Sant. and F. Kopila et al.: Pharmacognostic Evaluation of Rhizome of Chlorophytum borivilianum Sant. and F. Figure 4: Densitometric scan of Rhizome of Chlorophytum borivilianum Sant. and F. Figure 4: Densitometric scan of Rhizome of Chlorophytum borivilianum Sant. and F. Pharmacognosy Journal, Vol 10, Issue 5, Sep-Oct, 2018 REFERENCES Raj Nighantu, Mulakadi Varga, 115-7, translated by Tripathi In 9. Pandit Narahari. Raj Nighantu, Mulakadi Varga, 115-7, translated by Tripathi In 967 Pharmacognosy Journal, Vol 10, Issue 5, Sep-Oct, 2018 Kopila et al.: Pharmacognostic Evaluation of Rhizome of Chlorophytum borivilianum Sant. and F. Publishers and Distributors; 1996. Publishers and Distributors; 1996. 23. Nandi et al. Comparative pharmacognostic and phytochemical studies of two aphrodisiac plants Chlorophytum borivilianum Santapau and Fernandes and Chlorophytum tuberosum (Roxb.) Baker. International Journal of Pharmacy and Pharmaceutical Science. 2013;5(4):517-23. 23. Nandi et al. Comparative pharmacognostic and phytochemical studies of two aphrodisiac plants Chlorophytum borivilianum Santapau and Fernandes and Chlorophytum tuberosum (Roxb.) Baker. International Journal of Pharmacy and Pharmaceutical Science. 2013;5(4):517-23. 20. Khandelwal KR. Practical Pharmacognosy Techniques and Experiments. Nirala Prakashana. 20th ed.2010;23.1-23.7. 21. Stahl I. Thin layer chromatography. A laboratory Hand Book (Student edition), Berlin, Springer-Verlag. 1969;52-86. 24. Panda SK, Das D, Tripathy NK. Pharmacognostical Studies on Root Tubers of Chlorophytum borivilianum Santapau and Fernandes. International Journal of Pharmaceutical and Phytochemical Research. 2010;2(4):13-7. 22. Sethi PD. High performance thin layer chromatography. 1st ed. New Delhi, CBS PICTORIAL ABSTRACT Cite this article: Kopila A, Anuradha KN, Prabhu Suchitra N. Pharmacognostic Evaluation and HPTLC Finger Printing of Rhizome of Chlorophytum borivilianum Sant. and F. from Nepal. Pharmacogn J. 2018;10(5):963-8. 19. Berlyn GP, Mikcsche JP, Sass JE. Botanical microtechnique and cytochemistry. Iowa State University Press. 1976. 20. Khandelwal KR. Practical Pharmacognosy Techniques and Experiments. Nirala Prakashana. 20th ed.2010;23.1-23.7. 21. Stahl I. Thin layer chromatography. A laboratory Hand Book (Student edition), Berlin, Springer-Verlag. 1969;52-86. 22. Sethi PD. High performance thin layer chromatography. 1st ed. New Delhi, CBS 20. Khandelwal KR. Practical Pharmacognosy Techniques and Experiments. Nirala Prakashana. 20th ed.2010;23.1-23.7. 19. Berlyn GP, Mikcsche JP, Sass JE. Botanical microtechnique and cytochemistry. Iowa State University Press. 1976. 21. Stahl I. Thin layer chromatography. A laboratory Hand Book (Student edition), Berlin, Springer-Verlag. 1969;52-86. 22. Sethi PD. High performance thin layer chromatography. 1st ed. New Delhi, CBS 24. Panda SK, Das D, Tripathy NK. Pharmacognostical Studies on Root Tubers of Chlorophytum borivilianum Santapau and Fernandes. International Journal of Pharmaceutical and Phytochemical Research. 2010;2(4):13-7. PICTORIAL ABSTRACT Cite this article: Kopila A, Anuradha KN, Prabhu Suchitra N. Pharmacognostic Evaluation and HPTLC Finger Printing of Rhizome of Chlorophytum borivilianum Sant. and F. from Nepal. Pharmacogn J. 2018;10(5):963-8. 968
https://openalex.org/W2896862709	https://europepmc.org/articles/pmc6232933?pdf=render	English	null	A Review of the Potential Mechanisms of Action of Baclofen in Alcohol Use Disorder	Frontiers in psychiatry	2,018	cc-by	11,843	REVIEW published: 17 October 2018 doi: 10.3389/fpsyt.2018.00506 A Review of the Potential Mechanisms of Action of Baclofen in Alcohol Use Disorder Renaud de Beaurepaire* Groupe Hospitalier Paul-Guiraud, Villejuif, France Baclofen, a GABA-B receptor agonist, is a promising treatment for alcohol use disorder (AUD). Its mechanism of action in this condition is unknown. GABA-B receptors interact with many biological systems potentially involved in AUD, including transduction pathways and neurotransmitter systems. Preclinical studies have shown that GABA-B receptors are involved in memory storage and retrieval, reward, motivation, mood and anxiety; neuroimaging studies in humans show that baclofen produces region-speciﬁc alterations in cerebral activity; GABA-B receptor activation may have neuroprotective effects; baclofen also has anti-inﬂammatory properties that may be of interest in the context of addiction. However, none of these biological effects fully explain the mechanism of action of baclofen in AUD. Data from clinical studies have provided a certain number of elements which may be useful for the comprehension of its mechanism of action: baclofen typically induces a state of indifference toward alcohol; the effective dose of baclofen in AUD is extremely variable from one patient to another; higher treatment doses correlate with the severity of the addiction; many of the side effects of baclofen resemble those of alcohol, raising the possibility that baclofen acts as a substitution drug; usually, however, there is no tolerance to the effects of baclofen during long-term AUD treatment. In the present article, the biological effects of baclofen are reviewed in the light of its clinical effects in AUD, assuming that, in many instances, clinical effects can be reliable indicators of underlying biological processes. In conclusion, it is proposed that baclofen may suppress the Pavlovian association between cues and rewards through an action in a critical part of the dopaminergic network (the amygdala), thereby normalizing the functional connectivity in the reward network. It is also proposed that this action of baclofen is made possible by the fact that baclofen and alcohol act on similar brain systems in certain regions of the brain. Edited by: Liana Fattore, Consiglio Nazionale Delle Ricerche (CNR), Italy Reviewed by: Alessandra Tiziana Peana, University of Sassari, Italy Philippe Rondard, Centre National de la Recherche Scientiﬁque (CNRS), France *Correspondence: Renaud de Beaurepaire debeaurepaire@wanadoo.fr Specialty section: This article was submitted to Addictive Disorders, a section of the journal Frontiers in Psychiatry Received: 31 July 2018 Accepted: 25 September 2018 Published: 17 October 2018 Keywords: GABA receptor b, reward network, amygdala, Pavlovian associations, substitution (morphine, methadone, buprenorphin) Citation: de Beaurepaire R (2018) A Review of the Potential Mechanisms of Action of Baclofen in Alcohol Use Disorder. Front. Psychiatry 9:506. doi: 10.3389/fpsyt.2018.00506 de Beaurepaire R (2018) A Review of the Potential Mechanisms of Action of Baclofen in Alcohol Use Disorder. Front. Psychiatry 9:506. doi: 10.3389/fpsyt.2018.00506 Baclofen is a gamma-aminobutyric acid (GABA) analog that activates the GABA-B receptor subtype, and is used worldwide in neurology for the treatment of spasticity due to its myorelaxant properties (1). Many preclinical [see (2), for review] and clinical studies have demonstrated the eﬃcacy of baclofen in the treatment of several addictive disorders, including October 2018 \| Volume 9 \| Article 506 Frontiers in Psychiatry \| www.frontiersin.org Baclofen Mechanism of Action de Beaurepaire alcohol use disorder (AUD) (3–10), even though negative results have also been published (11, 12). While it is clearly established that the myorelaxant properties of baclofen are related to a dampening of the spinal motor reﬂex (13), its potential mechanism of action in AUD remains elusive. Central GABA-B receptors are involved in the regulation of a large number of systems and functions, including several neurotransmitter systems (dopamine, serotonin, norepinephrine, glutamate), transduction pathways, memory, and other cognitive functions, as well as inﬂammation. All these systems and functions are possibly involved in the anti-addictive eﬀects of baclofen. In the present paper, the biological eﬀects of baclofen are reviewed in the light of its clinical eﬀects, assuming that, in many instances, clinical eﬀects can be reliable indicators of underlying biological processes. AUD is a chronic relapsing disorder characterized by an increased motivation to seek alcohol and drink compulsively, with an increasing loss of control over drinking, progressing from impulsivity to compulsivity (15). In clinical research, the concept of compulsion is generally not used: the word compulsion is absent from the DSM-5 AUD section, where the term craving, deﬁned as “a strong desire or urge to use alcohol” (16), seems to encompass the compulsion to drink. According to the DSM-5, AUD is commonly associated with anxiety, depression, psychotic disorders, cognitive disorders and sleep disorders. Impulsivity is cited as a vulnerability factor for AUD. Regarding biological research, addiction models use the concept of craving (associated with those of preoccupation/anticipation), and add the concepts of positive and negative reinforcement to those of impulsivity and compulsivity. Citation: This diﬀers from those who have been cured by using other methods, for whom abstinence most generally requires a lot of eﬀort, and for whom craving for alcohol often returns when they resume some alcohol drinking or see alcohol cues. However, all AUD patients treated with baclofen do not reach such a state of complete indiﬀerence. My experience (more than a thousand patients treated with a high-dose of baclofen over the last 10 years) is that about one third of the patients reach this state of complete indiﬀerence; while another third experiences a clear decrease in craving, but not its complete suppression (these patients drink very signiﬁcantly less, but still have moments of desire for alcohol); in the last third of patients baclofen treatment is ineﬀective despite often reaching very high doses. In these latter patients, the dose increase may have been limited by adverse eﬀects, but it happens that some patients reach very high doses (superior to 400 mg/day) without achieving a state of indiﬀerence. In any event, a state of indiﬀerence can be reached in a substantial number of patients, and one of the aims of the present article is to try to address the concept of indiﬀerence in biological terms. As mentioned above, baclofen is a selective GABA-B receptor agonist. GABA-B receptors are heterodimeric metabotropic receptors consisting of one GABA-B receptor-1 subunit (GABBR1) and one GABA-B receptor-2 subunit (GABBR2). GABA-B receptors are coupled via G-proteins to potassium and calcium channels, and to adenylate-cyclase (18). Studies have shown that GABA-B receptors are highly expressed all throughout the brain, some regions having a very high density of receptors, other regions a low or a very low density, and some regions insigniﬁcant densities. The variable densities of GABA-B receptors may have important implications regarding the use of baclofen in the treatment of AUD, given that, when a patient takes baclofen activation of regions with high densities should have clearer and more immediate physiological and behavioral consequences than the activation of regions with low densities. Binding and expression of GABA-B receptors have been studied in rodents. The Chu et al. binding study (using baclofen) showed highest densities of GABA-B receptors in the medial habenula, thalamus, cerebellum, cortex and colliculus; while ventral tegmental area and mesolimbic dopaminergic projections were among the structures with the lowest binding (19). Bowery et al. Citation: In positive reinforcement, cues and contexts associated with drinking acquire incentive salience after repeated association with drinking, and increasing strength of salience progressively leads to compulsive alcohol seeking and drinking. In negative reinforcement, craving is induced by the motivational value of the negative states of alcohol withdrawal. This leads to a state of preoccupation/anticipation where craving is intensiﬁed by the anticipation of access to alcohol resulting in compulsive alcohol seeking and drinking. A challenge in alcohol research is to explain how speciﬁc cues or contexts are paired with states of craving, while explaining which mechanisms of brain encoding are involved in these associations. It is hypothesized that the understanding of how speciﬁc neuronal ensembles encode and mediate the recall of learned associations among the cues, contexts, and behaviors during alcohol seeking and drinking will be helpful for the comprehension of how baclofen works in the treatment of AUD (17). The eﬀects of baclofen in the treatment of alcohol dependence have been thoroughly described by a physician suﬀering from AUD, Olivier Ameisen, who reported the cure of his alcohol dependence with a high dose of baclofen, ﬁrst in an article published in 2005, then later in a book published in 2008 (3, 14). He reported that, in his case, the dose of 270 mg/day produced a state of “complete indiﬀerence” toward alcohol. Indiﬀerence is not an operational concept in addictology. It is nevertheless a concept that should be taken into consideration. It is characterized by an eﬀortless suppression of craving, but goes beyond it. In people indiﬀerent to alcohol, the experience of drinking or seeing alcohol cues has changed completely, as if alcohol had no meaning to them anymore. Those who are indiﬀerent to alcohol can drink a glass of an alcoholic beverage, they do not ﬁnish the glass, they do not want to continue drinking, they feel nothing, while they remain unchanged for other aspects of their life, which they enjoy normally. The state of indiﬀerence is not transitory when the eﬀective dose of baclofen is maintained: on the contrary, it is very long lasting, and people completely indiﬀerent to alcohol can generally stop baclofen after one or a few years of treatment, and they most often do not relapse (as if all memories associated with alcohol had vanished). Citation: reported highest densities of GABA-B binding in the cerebellum, interpeduncular nucleus, frontal cortex, and thalamus (20), and highest GABBR1 mRNA transcripts in the hippocampus, thalamus, and cerebellum (21). Billington et al. (22), using immunohistochemistry targeting GABBR1 and GABBR2, found October 2018 \| Volume 9 \| Article 506 Frontiers in Psychiatry \| www.frontiersin.org 2 Baclofen Mechanism of Action de Beaurepaire receptor colocalization in the cerebellum, hippocampus, cortex, thalamus, and basal ganglia; the raphe nucleus was strongly stained for GABBR1 and weakly for GABBR2; there was no signiﬁcant staining in the VTA and mesolimbic dopamine projections. Lu et al. reported strong expression of GABBR1 in the medial habenula, hippocampus, hypothalamus (supraoptic and suprachiasmatic nuclei), and cerebellum; intermediate expression in thalamus and brainstem nuclei containing monoaminergic neurons; and low expression in globus pallidus, ventral pallidum, and substantia nigra pars reticulata (23). Li et al. (24) reported strong expression of both GABBR1 and GABBR2 in medial habenula, cingulate and piriform cortex, cerebellum, and hippocampus; moderate expression in thalamic nuclei, amygdala, and other parts of the cortex; low expression in the basal ganglia and hypothalamus; insigniﬁcant expression in the ventral mesencephalon, where the VTA is located (24). Conversely, a study using immunohistochemistry and focusing selectively on midbrain monoaminergic nuclei showed that GABA-B receptors are present in neurons of the VTA and raphe nuclei (no comparison in terms of density of receptors was made with other parts of the brain) (25). Besides, acute treatment with baclofen in rats produces an activation of a number of brain nuclei, mostly in the hypothalamus, the amygdala, and the brainstem, and has no detectable eﬀect in reward-relevant regions such as the nucleus accumbens, striatum, or ventral tegmental area (26). In conclusion, despite some discrepancies between these studies and despite the fact that it is not known to what extent observations made in rodents are relevant to humans, it is likely that, when given to patients, baclofen action is much stronger in brain structures that contain high or very high densities of GABA-B receptors, such as the cerebellum, medial habenula, hippocampus, some nuclei of the hypothalamus and thalamus, and certain parts of the cortex, than in dopaminergic structures. while short-term baclofen treatment inhibits amygdala reactivity to incentive cues (29). Most importantly, it has recently been shown that alcohol addiction is associated with impaired GABA clearance in the amygdala, with an increase in GABA tone associated with higher anxiety-like behavior (30). Citation: Baclofen could possibly improve anxiety through a rapid normalizing eﬀect on amygdala GABA tone. Clinical studies have shown that baclofen has anxiolytic eﬀects in patients with AUD (31) and it has been hypothesized that the anticraving eﬀects of baclofen could be related to an anxiolytic eﬀect (5, 6, 32). A study by Morley et al. showed no anticraving eﬀect in AUD patients, but a secondary analysis showed that baclofen was eﬀective in the group of anxious patients included in the study (33). Given that craving for alcohol is closely related to states of stress (34, 35), the stress-relieving eﬀects of baclofen may contribute to reduce craving in AUD patients. In addition, activation of GABA- B receptors could normalize the abnormal GABA tone in the amygdala and have signiﬁcant therapeutic eﬀects (36). However, adverse and anxiolytic eﬀects generally occur during the ﬁrst days or weeks of treatment, while a state of complete indiﬀerence most often occurs much later, after one or several months of treatment (depending on the dose needed and the protocol of dose increase). This could imply that complete indiﬀerence to alcohol does not result from an immediate or short-term eﬀect of baclofen on GABA-B receptors, but is rather the result of long-term plastic remodeling of certain brain systems. Preclinical studies have highlighted such plastic eﬀects of baclofen or other GABA-B agonists on brain systems after chronic treatment. Keegan et al. have shown that rats treated chronically with baclofen have signiﬁcant decreases in G-protein-dependent signal transduction (measured by GTP-gamma-S binding) in the frontal cortex, septum, amygdala, and parabrachial nucleus (37). Such decreases demonstrate a general desensitization of G-protein-dependent systems. But chronically treated rats also show signaling alterations via kinase cascades, including increases in activation of focal adhesion kinase (FAK) and of activated glycogen synthase kinase 3 (GSK3ß), and elevations in phosphorylated dopamine- and cAMP-regulated phosphoprotein-32 (DARPP-32), in several brain structures, which could indicate an absence of desensitization in these structures (i.e., no tolerance). According to Keegan et al. neuroadaptation mediated by G-proteins correlates with tolerance, while signaling alterations via kinase cascades shows cross-talk between GABA-B receptors and alternative mechanisms that are resistant to desensitization. Regarding brain areas, chronic baclofen treatment produced a sustained increase in auto-phosphorylation of FAK in the caudate, an increase in phosphorylations of GSK3ß in the caudate and putamen, and an increase of DARPP-32 in the nucleus accumbens. Frontiers in Psychiatry \| www.frontiersin.org Citation: These actions demonstrate that chronic baclofen can induce signiﬁcant plastic eﬀects in dopaminergic structures, and that the eﬀects of baclofen in these dopaminergic structures are associated with an absence of tolerance. The Keegan et al. study shows that chronic baclofen treatment also produces a sustained increase in kinase cascades activity in other regions, namely the cortex, thalamus, and hippocampus for FAK; the cortex, thalamus and septum for GSK3ß; and the Adverse eﬀects generally occur before anticraving eﬀects during baclofen treatment of AUD, especially when baclofen is used in order to make patients reach a state of complete indiﬀerence, for which high or very high doses are most often necessary. Many adverse eﬀects may in large part be explained by an early occurring activation of brain areas containing high densities of GABA-B receptors. For example, the most common and early occurring baclofen adverse eﬀects include fatigue, diurnal somnolence and nocturnal insomnia. Theses symptoms may be explained by an action of baclofen on GABA- B receptors in the brainstem and hypothalamus, which are among the structures most strongly activated by baclofen, and which control basic states of vigilance (in particular through the suprachiasmatic nucleus). Frequently occurring adverse sensory eﬀects (tinnitus, paresthaesias, blurred vision, etc.) may be explained by the high density of receptors in the thalamus, and memory problems by the high density of receptors in the hippocampus. Baclofen can also frequently promote anxiolysis; this could possibly be explained by an early occurring eﬀect of baclofen on serotonin neurons and on the amygdala. Hyperactivity of serotonin raphe neurons or hyperactivity of the amygdala are mechanisms known to produce anxiety; baclofen acutely inhibits serotonin neurons and serotonin release (27, 28) Frontiers in Psychiatry \| www.frontiersin.org October 2018 \| Volume 9 \| Article 506 3 Baclofen Mechanism of Action de Beaurepaire withdrawal signs (53), cue-induced reinstatement of previously extinguished alcohol-seeking behavior (59), and the reinforcing and motivational properties of alcohol (60–65) in diﬀerent validated rodent models of AUD [for review, see (2)]. That baclofen reduces alcohol consumption in animal models has been further strengthened by the demonstration that R(+)-baclofen, and not S(-)-baclofen selectively reduces self-administration of alcohol in rats (66). Regarding the mechanism of action of baclofen in these models, it is generally hypothesized that baclofen reduces alcohol consumption through an anti- dopaminergic eﬀect. The hypothesis is based on two major points. The ﬁrst is the fact that baclofen has clear anti- rewarding eﬀects. THE ANTI-DOPAMINERGIC HYPOTHESIS All addictive substances alter dopaminergic signaling in the mesocorticolimbic system, and models of addiction posit positive and negative reinforcement as closely linked to dopaminergic reward systems (39). In positive reinforcement, an increase in drinking is associated with increases in the release of dopamine in the brain, producing a feeling of pleasure. Whereas in negative reinforcement, alcohol is taken to alleviate a negative emotional state, and is associated with decreased dopamine in the striatum. And it is widely accepted that, in patients with chronic AUD, reward thresholds are increased and dopamine function is decreased, leading to a general “dopamine-impoverished” brain (40). The enduring reduction of dopaminergic systems activity in patients’ brains logically implies that drugs used in the treatment of AUD should enhance dopamine function to restore brain circuits disrupted by alcohol use. The idea of a strict hypo-dopaminergic state in the brain of AUD patients remains controversial (41). However, dopamine antagonists have never shown eﬀectiveness in the treatment of AUD (42). Furthermore, preclinical studies have shown that baclofen inhibits dopamine transmission (43–48). Such dopamine antagonist properties do not a priori posit baclofen as a good candidate for the treatment of subjects who have a dopamine- impoverished brain. However, some studies have shown that baclofen could have dose-dependent eﬀects on dopamine systems, with low doses increasing dopamine transmission and high doses inhibiting it. This has been demonstrated in animals (49), in in vitro cell preparations (50), and in humans (51). From a clinical standpoint, this hypothesis is not entirely satisfying because the clinical eﬀects of baclofen in AUD patients are not really those of an antidopaminergic eﬀect. Indeed, acute administration of baclofen may produce sedation, and sedation could be related to an inhibition of dopaminergic systems, but baclofen often produces a behavioral disinhibition, and quite frequently an evident hypomania (approximately 15% of patients). Disinhibition could be possibly linked to a dose-related eﬀect of baclofen on dopaminergic neurons, where a low dose of baclofen activates VTA neurons and a high dose inhibits them (50, 51). However, the experience of baclofen treatment in patients with AUD shows that most often the stimulant eﬀect is not dose-dependent; it appears at any dose, and sometimes at high doses. And when a disinhibitory eﬀect occurs at a low dose, it is almost never followed by a state of inhibition when doses are increased, as should be the case if baclofen had a dose-dependent biphasic eﬀect. Citation: These eﬀects have been shown not only for alcohol consumption, but also for the consumption of cocaine (67), amphetamine (68), and even of non-drug reinforcers such as sucrose, saccharin, or regular food pellets, suggesting that baclofen produces a generalized suppression of reward- motivated behaviors (2). The second is that microinjection of baclofen directly into the VTA blocks the behavioral response to cues and the cue-evoked ﬁring of subpopulations of NAc neurons that respond to predictive cues (69). Baclofen and other GABA- B agonists microinjected into the VTA suppress alcohol-seeking behavior (70), alcohol consumption (71), and alcohol-induced conditioned place preference (72). Microinjection of baclofen in the nucleus accumbens also decreases binge-like alcohol drinking (73). Given that the microinjection of baclofen in the VTA dampens the activity of dopaminergic neurons, it has been hypothesized that baclofen may exert its anti-addictive properties by means of its ability to reduce the activity of dopaminergic neurons (74). cortex, thalamus, hippocampus and amygdala for DARPP-32. These regions are not primarily dopaminergic, but nevertheless receive dopaminergic projections, and, for many of them, they are part of the reward network (38). In summary, chronic baclofen produces G-protein desensitization in a certain number of structures, and alterations in signaling via kinase cascades, with resistance to desensitization, in a set of structures that are closely related to the dopamine network. It may be hypothesized that structures showing desensitization could be involved in the adverse eﬀects of baclofen, which all tend to vanish over time, while structures showing no desensitization are involved in the therapeutic eﬀect on baclofen in AUD—these last structures being possibly involved in the indiﬀerence toward alcohol. Indeed, when baclofen produces a complete indiﬀerence toward alcohol, there is no tolerance to this eﬀect [no requirement for “markedly increased dose [...] to achieve the desired eﬀect,” as tolerance is deﬁned in the DSM-5 (16)]; on the contrary baclofen can be progressively decreased over time in patients indiﬀerent to alcohol, until, for many patients, a possible discontinuation of the treatment after a more or less lengthy period of time. Frontiers in Psychiatry \| www.frontiersin.org THE ANTI-DOPAMINERGIC HYPOTHESIS In baclofen-treated patients, a disinhibitory eﬀect is generally accompanied by a sensation of well-being, or euphoria, or even by a hypomanic state; and it is well established that these feelings or symptoms are associated with an increase in striatal dopamine activity (75–77). On the other hand, inhibition of dopaminergic systems has been consistently related to apathy, anhedonia and depression (78). Baclofen’s ability of to produce major depression during treatment of AUD is a subject of discussion [see (79)]; if this were the case, it is certainly very rare. However, baclofen makes patients sometimes feel dull, apathetic, and joyless (approximately 15% of patients), Studies investigating the eﬀect of baclofen in animal models of addiction show that systemic administration of baclofen reduces the acquisition and maintenance of alcohol consumption (52–55), motivation to drink (56), binge-like drinking (57), relapse-like drinking (58), severity of alcohol October 2018 \| Volume 9 \| Article 506 4 Baclofen Mechanism of Action de Beaurepaire anterior, posterior and dorsal cingulate cortex; the orbitofrontal cortex; the dorso-medial prefrontal cortex; the amygdala, the hippocampus and para-hippocampus; and the cerebellum (93– 95). These diﬀerent structures are interconnected through complex networks. Preclinical models have demonstrated that a direct inactivation of some of these structures can suppress craving or the reinstatement of drinking. For instance, it has been shown that inactivation of the prelimbic cortex inhibits ethanol self-administration (80); that the inactivation of the baso-lateral amygdala attenuates context-induced alcohol- seeking (81); or that the inactivation of the ventral subiculum decreases context-induced relapse to alcohol seeking (82). Therefore, inactivation of localized parts of the reward network may globally inhibit craving or compulsive drinking. Studies in AUD patients have shown the same kind of results. Transcranial Magnetic Stimulation (TMS) targeted to the dorsal anterior cingulate cortex (dACC) has been shown to reliably suppress craving (83, 96); a similar eﬀect has been found with Deep Brain Stimulation (DBS) targeted in the nucleus accumbens (84). It is well established that TMS and DBS eﬃcacy are related to their ability to change network connectivity (97). suggesting, rather, a state of atypical depression that does not meet the criteria of a major depressive episode. This state is possibly related to a hypo-dopaminergic state. Thus, baclofen can produce states of disinhibition/hypomania or of apathy, in about 30% of patients. The large majority of patients never experience these symptoms. THE ANTI-DOPAMINERGIC HYPOTHESIS This shows that baclofen can have an eﬀect on dopaminergic systems during treatment of AUD patients, but this eﬀect can be that of a hyper- or a hypo- dopaminergic eﬀect, and that it occurs in a minority of patients. More importantly, the clinical experience shows that the states of sedation, apathy, disinhibition, or hypomania have no relation to the therapeutic eﬀect of baclofen; they occur independently of an anti-craving eﬀect, and these symptoms are commonly considered as side-eﬀects of baclofen. As a whole, these elements are not compatible with a general hypothesis that would posit an inhibition of dopaminergic systems as a central mechanism for the anti-addictive action of baclofen (Table 1). Another reason why the therapeutic eﬀect of baclofen in AUD is likely not to be related to a global anti-dopaminergic eﬀect is that it is not compatible with the phenomenology of the state of indiﬀerence toward alcohol. Patients indiﬀerent to alcohol are no longer interested in alcohol, but they experience normal enjoyment for the other aspects of their life. People who enjoy life normally necessarily have intact reward systems. The state of indiﬀerence is always reached after treatment has lasted for a certain amount of time, which is likely a period of remodeling brain circuits. It cannot be excluded that that an initial blockage of the dopaminergic systems participates in the remodeling of brain circuits. The above-mentioned study by Keegan et al. shows that chronic baclofen treatment induces plastic changes in a number of structures and systems, most of which are part of, or are closely linked with, the brain’s reward network. Many studies have shown that AUD is associated with abnormal brain connectivity. It seems, in a simpliﬁed way, that in AUD patients connectivity is increased in regions that are involved in appetitive drive and reduced in regions that mediate executive control, while in long-term abstinent patients activity is decreased in reward circuitry and increased in executive control regions (98–102). This is however a simpliﬁed view of the question, which is certainly far more complex (103– 108). But the important point in the context of the present article is to highlight that there are dysfunctional networks in the brains of AUD patients, and to try to understand how eﬀective pharmacological treatments used in AUD can normalize these dysfunctional networks. The literature dealing with the eﬀects of pharmacological treatments for AUD on brain connectivity is scarce. THE ANTI-DOPAMINERGIC HYPOTHESIS A study by Morris et al. (85) showed that AUD patients have heightened local eﬃciency of neural networks, indicating disturbances of information processing—more isolation and clustering of functionally related regions, stronger processing in certain regions, with less cross-talk between distinct functional processes—, and that naltrexone, a commonly used treatment of AUD, can normalize these abnormalities. Gamma-hydroxybutyrate (GHB), a medication used in the treatment of AUD that activates GABA-B receptors, has been shown to markedly alter functional connectivity in healthy volunteers (109). Generally speaking, it is very likely that all eﬀective AUD treatments, whether pharmacological, psychological, or using local stimulation, do so by normalizing functional connectivity, possibly leading to a decrease in the strength of appetitive networks and to an increase of that of executive control regions, with a recovery of balanced cross-talk between the diﬀerent local functions. Frontiers in Psychiatry \| www.frontiersin.org LONG-TERM NETWORK ALTERATIONS The concept of indiﬀerence is not a scientiﬁc concept, while those of craving and compulsive drinking are such concepts. Craving/compulsive consumption of alcohol occurs after repeated consumption of alcohol, likely in relation with complex adaptations in brain circuits. The challenge is to explain how a chronic treatment with a GABA-B agonist is able to overcome these numerous and complex brain adaptations, and to lead to a state of indiﬀerence toward alcohol. The diﬀerent components of the progressive set-up of the compulsive drinking behavior involve neural substrates that belong to the dopaminergic reward network. Schematically, the reward network has three major components (38): a VTA-ventral striatum/nucleus accumbens system (VTA-NAc) that encodes stimuli valence; a VTA-amygdala/hippocampus system (which in the amygdala includes the basolateral and central nuclei) that forms associative related memories; And a VTA-medial prefrontal cortex system (VTA-mPFC) that regulates executive control. Studies have shown that craving occurring in response to alcohol cues is associated with the activation of structures, which, for the most part, belong the reward system. These structures are the nucleus accumbens/ventral striatum; the It is therefore hypothesized that chronic baclofen treatment produces a state of indiﬀerence through a normalization of brain network connectivity. Chronic baclofen produces many October 2018 \| Volume 9 \| Article 506 5 Baclofen Mechanism of Action de Beaurepaire TABLE 1 \| Correspondence between potential mechanisms, symptoms and neurobiological substrates. Mechanism Symptom Neurobiological substrate Method References Dopamine Anticraving effect Sedation/apathy Hypomania/mania Indifference ց dopamine in VTA-NAc ց dopamine in VTA-NAc ր dopamine in striatum Long-term remodeling of dopamine circuits? Intra-VTA B inject Peripheral B inject Brain imaging (2) (review) (44) (75) Connectivity Inhibits reinstatement Eth seeking decrease Eth relapse Anticraving effect Anticraving effect AUD treatment Indifference Inactivation of infralimbic cortex in rats (similar to anterior cingulate in humans) Inactivation of baso-lateral amygdala Inactivation of ventral subiculum Stimulation of dorsal anterior cingulate Stimulation of the nucleus accumbens Multiple networks Gaba tone in the amygdala? Gaba inhibition Gaba inhibition Gaba inhibition TMS DBS Naltrexone Baclofen (80) (81) (82) (83) (84) (review) (85) Substitution Similar effects of Eth and GABA-B activation General symptoms Anxiolysis Withdrawal GHB deﬁciency hypothesis Multiple brain structures Brain GHB receptors Eth and GABA-B agonists studies GHB studies (86); (87) (16); (88) (89); (90) (16); (91) (92) B, baclofen; Eth, ethanol; VTA-NAc, Ventral Tegmental Area-Nucleus Accumbens; Gaba inhibition, local injection of baclofen+muscimol; TMS, Transcranial Magnetic Stimulation; DBS, Deep Brain Stimulation. LONG-TERM NETWORK ALTERATIONS References, only the ﬁrst author is cited; Two references on the substitution line, the ﬁrst refers to alcohol, the second to baclofen or other GABA-B agonists. B, baclofen; Eth, ethanol; VTA-NAc, Ventral Tegmental Area-Nucleus Accumbens; Gaba inhibition, local injection of baclofen+muscimol; TMS, Transcranial Magnetic Stimulation; DBS, Deep Brain Stimulation. References, only the ﬁrst author is cited; Two references on the substitution line, the ﬁrst refers to alcohol, the second to baclofen or other GABA-B agonists. increased the dose of baclofen up to 270 mg/day, and became completely indiﬀerent to alcohol at that dose. At the dose of 260 mg/day, he was not indiﬀerent at all. It is the addition of 10 mg that abruptly and completely changed his attitude toward alcohol. The long experience of baclofen prescription in AUD shows that this abrupt occurrence of a state of indiﬀerence at a given dose is common. Patients call it “my threshold.” The threshold of indiﬀerence is unique to each patient. Some reach it at moderate doses, some at high or very high doses. The passage from a state of extreme vulnerability to compulsive drinking to a state of indiﬀerence is however not always as abrupt as in the case of Olivier Ameisen. Instead, it is often preceded by a period of a slow decrease of craving; but almost all patients who reach a state of indiﬀerence say that at a certain dose they felt a complete change in their attitude toward alcohol. For the majority of patients, baclofen treatment is a quest to reach the threshold of indiﬀerence. In a recently published guidance for baclofen treatment of AUD, primarily written by expert patients, the quest for the threshold of indiﬀerence was clearly described (79). In this guidance article, the authors present what they call the “Ameisen test:” “One of the best ways to conﬁrm that the eﬀective treatment dose has been reached is to ask the patient to go to the shop where s/he used to buy alcohol. If the desire to drink alcohol is ignited by the sight of wine and spirits, the baclofen dose should continue to be increased progressively. If the sight of alcohol has no more eﬀect than looking at nappies or washing powder, the eﬀective dose of baclofen has been reached.” Patients indiﬀerent to alcohol are no longer concerned or stressed by the sight of alcohol. Alcohol has become devoid of meaning. Frontiers in Psychiatry \| www.frontiersin.org LONG-TERM NETWORK ALTERATIONS It is well established that craving and the subsequent sequence of compulsive drinking are triggered by feelings of stress that can themselves be triggered by the exposition to alcohol cues (118). The analysis of the mechanisms potentially involved in the dose-dependent, and often abrupt, passage from a state of extreme vulnerability to compulsive drinking to a state changes in the brain that could impact connectivity. We have previously mentioned that chronic baclofen produces plastic changes in regions of the reward system, including desensitization in G-protein-dependent systems and alterations in signaling of several kinase cascades (FAK, GSK3ß, DARPP- 32) that are resistant to desensitization (37). In addition, AUD is associated with marked brain neuro-immune alterations (110); and studies have shown that baclofen has anti-inﬂammatory and neuroprotective eﬀects on the brain. Baclofen attenuates neuro- inﬂammation (111) and inﬂammatory signaling (112); inhibits the release of pro-inﬂammatory cytokines from microglia (113) and from astrocytes (114); and decreases oxidative stress (111); interestingly, baclofen is an allosteric modulator of CXCR4, a receptor for the chemokine CXCL12, which has been causally involved in several neurological disorders, including stroke, brain tumors, HIV encephalopathy and multiple sclerosis (115). GABA-B-receptor activation alters also the activity of dopamine, serotonin, norepinephrine, GABA and glutamate, which are prominent neurotransmitters implicated in alcohol dependence and are involved in the modulation of brain networks. It is not known whether these eﬀects of baclofen on neurotransmitter or neuroimmune factors can alter functional connectivity in AUD, but it has been shown that neuroimmune/neurotransmitter dysregulation in other psychiatric disorders, such as bipolar disorder, disrupt local brain network connectivity and have deleterious eﬀects on the brain, and that these eﬀects can be treated with appropriate pharmacological treatments (116). Abnormal glutamate release and function have been found in the brains of AUD patients and glutamate and/or GABA neurotransmission may underlie resting-state functional deﬁcits in drug addiction (117). Therefore, the eﬀects of baclofen on neuroimmune/neurotransmitter systems may participate in a normalization of functional connectivity in patients with AUD. I diﬀ t l h l i i l h Th f Indiﬀerence to alcohol is a special phenomenon. The case of Olivier Ameisen is very illustrative (3, 14). Ameisen progressively October 2018 \| Volume 9 \| Article 506 Frontiers in Psychiatry \| www.frontiersin.org 6 Baclofen Mechanism of Action de Beaurepaire the strength of a long-lasting associative learning “carved” in the limbic memory. LONG-TERM NETWORK ALTERATIONS This could be in accordance with studies that show that higher doses of baclofen are correlated with a higher severity of addiction (8, 132). Clinical experience also shows that when the eﬀective dose is reached—the threshold dose producing a state of indiﬀerence—the treatment has to be continued for several months at the same dose before it can be reduced. It is proposed that this delay is necessary to completely suppress the Pavlovian association between the cue and the reward. It has been highlighted that, in patients indiﬀerent to alcohol, those who remain completely free of alcohol for many months are those who will be able to stop taking baclofen, while those who continue to drink, even at moderate levels and without any real desire for alcohol but who do so out of habit or on certain social occasions, will have greater diﬃculty in stopping baclofen (79). Indeed, in terms of connectivity and synaptic strength, it is likely that the continuation of regular drinking reactivates the Pavlovian association between the cue and the reward every time, making it impossible to suppress, and paving the way for relapse if baclofen is stopped. of complete indiﬀerence is critical in addressing the question of the mechanism of action of baclofen in the treatment of AUD. From a clinical/phenomenological standpoint, craving and compulsive drinking can be assimilated to a Pavlovian reaction where stress or the activation of an alcohol-related cue or mental imagery is associated with the memory of a reward, and triggers an irrepressible drinking behavioral sequence. The learning of cue-reward associations is a slow process causing long-lasting synaptic plasticity changes in cortico-limbic-striatal circuitry, via multiple gene and protein expression. It is well established that there is a very important relation between stress and alcohol use (34, 35). The biological bases of craving and drinking in response to stress or alcohol cues have been extensively studied in preclinical models of alcohol addiction and in AUD patients themselves. Interestingly, endogenous substances like dopamine and corticotropin-releasing factor and exogenous acute and chronic ethanol act in brain regions that regulate stress and anxiety-related behaviors (119), the most important region being the amygdala. The amygdala is a critical part of the reward network, involved in the way cues associated with rewards gain access to regions attributing incentive salience (120). LONG-TERM NETWORK ALTERATIONS The way stress and cues associated with rewards are processed in the amygdala may therefore determine subsequent behaviors such as compulsive drinking. Baclofen can interfere with these processes. Baclofen aﬀects memory processes in rodent addiction models, impairing consolidation of memory (121), facilitating extinction learning (122), and interfering with fear extinction (123). Amygdala CREB is known to be involved in the modulation of fear memory (124); and baclofen suppresses stimulant-induced increases in pCREB levels in the amygdala (125). Progressive increase in the reinforcing eﬀects of drug cues is associated with the increases in BDNF and extracellular signal regulated kinase (ERK) activity in the central nucleus of the amygdala (126–128); and GABA-B receptors are involved in the regulation of BDNF release (129), and of the ERK pathway (130). Baclofen also has important neuromodulatory eﬀects in the amygdala, through its inhibitory action on neurotransmitters and complex eﬀects on second-messenger signaling (37). It reduces the strength of excitatory (glutamate) and inhibitory (GABA) transmission in the amygdala by a presynaptic mechanism (131). Furthermore, as mentioned previously, alcohol addiction is associated with impaired GABA clearance and increased GABA tone in the amygdala, associated, in turn, with higher anxiety-like behavior (30). GABA-B receptor stimulation, which inhibits GABA transmission, should therefore be useful in the treatment of alcohol dependence and associated anxiety (Table 1). Frontiers in Psychiatry \| www.frontiersin.org THE SUBSTITUTION HYPOTHESIS In other words, the dose of baclofen would be that which is necessary to overcome October 2018 \| Volume 9 \| Article 506 7 Baclofen Mechanism of Action de Beaurepaire GABA-B agonist, and alcohol has no direct action on GABA- B receptors. However, it is very likely that the two substances indirectly act on the same systems, especially the glutamatergic and GABAergic systems. Even though alcohol does not directly act on GABA-B, it increases GABA(B1) and GABA(B2) receptor expression in diﬀerent parts of the brain, while baclofen partially reverses these eﬀects (87). Besides, it has been shown that stimulation of presynaptic GABA-B receptors decreases the GABAergic eﬀects of alcohol (86), demonstrating that GABA- B activation can moderate the behavioral sensitivity to alcohol. This has led Clapp et al. (135) to propose that treatment with a GABA-B agonist could substitute for the anxiolytic eﬀect of ethanol, leading to its reduced consumption. Similarly, GABA- B stimulation may substitute the GABA transporter GAT-3 deﬁciency in the brains of alcoholics, leading to a normalized GABA function in the amygdala (30). More generally, chronic alcohol consumption alters many brain substances, receptors, and pathways (136), including several that interact with GABA- B receptors, such as, among others, the PKA and PKC pathways (137), the Akt pathway (138), the mTORC-1 pathway (139), the ERK1/2 pathway (130), BDNF release (129), or CREB (125): in all these systems, GABA-B stimulation could, in some ways, substitute for the eﬀects of alcohol. The deﬁnition of substitution, limited to the notion that substitution should strictly involve substances that act on the same receptors, has been questioned. For instance, Chick and Nutt proposed much more broad and unspeciﬁc criteria to deﬁne substitution (140). It remains that chronic alcohol consumption impacts many brain systems and that GABA-B may interfere with some of these eﬀects with potential clinical advantages, assimilated to substitution or not. Baclofen and GHB share several common neurobiological eﬀects, including GABA-B activation; among alcohol, baclofen and GHB, only GHB occurs naturally in the brain and has brain speciﬁc receptors. According to Ameisen, a deﬁcit in brain GHB could cause dysphoria, itself promoting alcohol misuse. Baclofen could be able to compensate the deﬁcit in GHB, thereby suppressing dysphoria and possible dependence (Table 1). REFERENCES randomised, double-blind controlled study. Lancet (2007) 370:1915–22. doi: 10.1016/S0140-6736(07)61814-5 randomised, double-blind controlled study. Lancet (2007) 370:1915–22. doi: 10.1016/S0140-6736(07)61814-5 randomised, double-blind controlled study. Lancet (2007) 370:1915–22. doi: 10.1016/S0140-6736(07)61814-5 1. Hudgson P, Weightman D. Baclofen in the treatment of spasticity. Br Med J. (1971) 4:15–7. 7. Rigal L, Alexandre-Dubroeucq C, de Beaurepaire R, Le Jeunne C, Jaury P. Abstinence and “low-risk” consumption 1 year after the Initiation of high-dose baclofen: a retrospective study among “high- risk” drinkers. Alcohol Alcohol. (2012) 47:439–42. doi: 10.1093/alcalc/ ags028 2. Agabio R, Leite-Morris KA, Addolorato G, Colombo G. Targeting the GABAB receptor for the treatment of alcohol use disorder. In: Colombo G, editor. GABAB Receptor. The Receptors Vol. 29. Springer International Publishing (2016). doi: 10.1007/978-3-319-46044-4_15 8. de Beaurepaire R. Suppression of alcohol dependence using baclofen: a 2-year observational study of 100 patients. Front Psychiatry (2012) 3:103. doi: 10.3389/fpsyt.2012.00103 3. Ameisen O. 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THE SUBSTITUTION HYPOTHESIS Alcohol and baclofen produce many similar symptoms or behavioral eﬀects in patients. Both can produce unsteady gait, dizziness, feelings of drunkenness, mood alterations, sensory alterations, confusion, impairment in attention and memory, and sleep disorders, among others (16, 88). Both can also reduce anxiety (89, 90). Patients taking baclofen often spontaneously notice these similarities. Abrupt withdrawal from alcohol and high-dose baclofen may also produce similar symptoms, including confusion, hallucinations, delirium, and seizures (16, 91) (Table 1). The main diﬀerence between alcohol and baclofen is that alcohol progressively produces a state of dependence, while this is not the case with baclofen (although a few cases have been reported (133)—likely because these are exceptional). Besides, chronic alcohol consumption induces tolerance, whereas tolerance with baclofen is equivocal: as previously mentioned, there is no tolerance to the clinical eﬀectiveness of baclofen in AUD, but there is tolerance to most of its adverse eﬀects (e.g., its eﬀects on locomotor activity) (134). These clinical similarities raise the possibility that baclofen and alcohol act on the same brain systems, and that baclofen could be a substitution treatment for alcohol dependence. In addictology, a substitution substance is a substance that acts on the same receptors as the targeted substance of abuse. The two substances share many similar eﬀects, but the substitution substance is less prone than the substance of abuse to induce dependence, or not prone at all to do so. Substitution treatments appeared in addictology about 50 years ago for the treatment of opiate addiction. Buprenorphine and other substitution substances used in the treatment of opiate addiction indeed act directly as full agonists, or as agonist/antagonists, on opiate receptors. The problem with baclofen and alcohol is that they do not act on the same receptors. Baclofen is a selective Chronic neuromodulatory eﬀects of baclofen in the amygdala may change the processing of stress and cues, and ultimately alter the functional connectivity within the reward network, in such a way that cues associated with rewards lose their meaning. The dose of baclofen needed to achieve this eﬀect could be very variable from one subject to another in relation to each individual’s variable strength of the Pavlovian association between the cue and the reward. AUTHOR CONTRIBUTIONS Another approach to the substitution hypothesis has been proposed by Ameisen (92, 141), who hypothesized that there is a deﬁcit of GHB in the brain of AUD patients. He highlighted the similar behavioral eﬀects of alcohol, baclofen and GHB. The author conﬁrms being the sole contributor of this work and has approved it for publication. CONCLUSION This review on the mode of action of baclofen from a clinical standpoint and with a biological perspective highlighted three potential modes of action of baclofen; namely on dopamine, functional connectivity, and as a substitution drug. It is tempting to hypothesize that these approaches are complementary, and that they could be synthesized in the proposition that baclofen may suppress the Pavlovian association between cues and rewards through an action in a critical part of the dopaminergic network (the amygdala), thereby normalizing the functional connectivity in the reward network. It is also proposed that this action is made possible by the fact that baclofen and alcohol act on similar brain systems in certain regions of the brain. 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https://openalex.org/W3092361376	https://www.econjournals.com/index.php/ijeep/article/download/10003/5524	English	null	ENERGY CONSUMPTION AND ECONOMIC GROWTH: EVIDENCE FROM POST-COMMUNIST COUNTRIES	International journal of energy economics and policy	2,020	cc-by	6,063	International Journal of Energy Economics and Policy International Journal of Energy Economics and Policy, 2020, 10(6), 59-65. Uktam Umurzakov1, Bakhodir Mirzaev1, Raufhon Salahodjaev1,2, Arletta Is 1Tashkent Institute of Irrigation and Agricultural Mechanization Engineers, Uzbekistan, 2Westminster International University in Tashkent, Uzbekistan. 3ERGO Analytics, Uzbekistan. Email: salahodjaev@gmail.com DOI: https://doi.org/10.32479/ijeep.10003 Accepted: 01 September 2020 Received: 30 May 2020 ABSTRACT Current study aimed to explore the nexus between energy and economic growth in post-communist nations during 1995-2014. As an empirical model, the growth model is employed including economic complexity indicator as a measure of capabilities for exporting sophisticated products. We employ Pedroni’s (1999) panel cointegration tests and Panel Dynamic OLS estimation to assess the long-term link between the variables. At 1% significance level, we confirm cointegration between energy consumption and economic growth. Panel Dynamic OLS results revealed that economic growth positively influences energy consumption in Post-Communist states. The Dumitrescu and Hurlin (2012) panel Granger causality test results discovered unidirectional causality running from economic growth to energy consumption. Economic growth causes energy consumption in post-communistic countries, confirming the conservation hypothesis. Keywords: Energy, Growth, Post-Communist JEL Classifications: C23, O47, Q40 Keywords: Energy, Growth, Post-Communist JEL Classifications: C23, O47, Q40 This Journal is licensed under a Creative Commons Attribution 4.0 International License Energy Consumption and Economic Growth: Evidence from Post-Communist Countries Uktam Umurzakov1, Bakhodir Mirzaev1, Raufhon Salahodjaev1,2, Arletta Isaeva3, Shakhnoza Tosheva3 1T hk I i f I i i d A i l l M h i i E i U b ki 2W i I i l U i i i Uktam Umurzakov1, Bakhodir Mirzaev1, Raufhon Salahodjaev1,2, Arletta Isaeva3, Shakhnoza Tosheva3 1Tashkent Institute of Irrigation and Agricultural Mechanization Engineers, Uzbekistan, 2Westminster International University in Tashkent, Uzbekistan. 3ERGO Analytics, Uzbekistan. Email: salahodjaev@gmail.com 1. INTRODUCTION 2012; Zhixin and Xin, 2011). Any policies aimed at reduction in energy consumption will have deteriorating implications for economic growth, which in turn will further decrease energy consumption. The neutrality hypothesis assumes that there is no causal link between energy consumption and economic growth. Consequently, understanding the effect of energy consumption on GDP growth is crucial for governments to adopt energy policies aimed at sustainable development. Related studies on the nexus between energy and growth is grounded on several different premises: the growth hypothesis, the conservation hypothesis, the feedback hypothesis, and the neutrality hypothesis. The growth hypothesis assumes that energy consumption is an instrumental determinant of economic growth complementary to other antecedents such as capital or labor. This implies that any measures leading to a decrease in energy consumption will have harmful effects on GDP growth. As a result, the direction of causality runs from energy consumption to economic growth. The conservation hypothesis suggests there is unidirectional causality from GDP growth to energy consumption (Gozgor et al., 2018). Therefore, any measures associated with reducing energy consumption will have no effect on economic output. The feedback hypothesis indicates existence of bi- directional causality between energy consumption and economic growth (Dagher and Yacoubian, 2012; Wesseh and Zoumara, Turning to the empirical evidence that investigates the link between energy and growth, the findings are, at best, mixed. A number studies document that energy use is a good determinant of economic growth (Chaudhry et al., 2012), while other studies find insignificant (Ozturk et al., 2010), bi-directional, or even negative effects (Narayan, 2016; Yildirim et al., 2014). Wang et al. (2011) explore the causal relationship between energy and economic growth in China for the period 1972-2006. The study applies the autoregressive distributed lag (ARDL) bounds testing This Journal is licensed under a Creative Commons Attribution 4.0 International License International Journal of Energy Economics and Policy \| Vol 10 • Issue 6 • 2020 Umurzakov, et al.: Energy Consumption and Economic Growth: Evidence from Post-Communist Countries approach. The study reports that both in the short- and long-term, economic growth is driven by rise in energy consumption and, “that restrictions on energy use may significantly hamper economic growth” (p. 4405). Esen and Bayrak (2017) revisit the energy- growth nexus for a sample of 75 net energy-importing countries for the period 1990 to 2012. 1. INTRODUCTION The authors apply a panel cointegration and error correction model to assess the causal relationship. The results suggest energy has a causal effect on economic development in both the short- and long-term. Rezitis and Ahammad (2015) use panel data of nine South and Southeast Asian countries including Malaysia, Pakistan, Bangladesh, Brunei Darussalam, India, Indonesia, the Philippines, Sri Lanka, and Thailand. Authors identify bi-directional causality between economic growth and energy consumption in observed countries based on Granger causality test results. The cases of individual countries prove the results of panel analysis, which states that economies depend on energy consumption. The relationships between GDP growth and energy usage, which is found in the cases of Bangladesh, Brunei Darussalam, India, and Thailand, support the growth hypothesis. Malaysia and the Philippines show results in favor of the feedback hypothesis while Sri Lanka tends toward the conservation hypothesis. Support of the neutrality hypothesis is found in the cases of Indonesia and Pakistan. Guo (2018) analyzes the nexus between economic growth and energy consumption in China. Using the time division of two periods, 1978-1992 and 1991-2016, the authors identify a strong dependence of the Chinese economy on energy consumption in favor of the growth hypothesis. These findings are proved by a co-integration test and error correction model. However, this relationship decreases year by year because China understands the unsustainability of relying only on energy input. Apergis and Ozturk (2015) tested the Environmental Kuznets Curve (EKC) among 14 Asian countries. The EKC hypothesis states a concave graphical representation of the relationship between level of emissions and economic growth. According to the hypothesis, economic growth and emission levels increase at the same rate at the early stages and start to decline after an economy reaches a threshold level. Using the GMM model, the authors identified the unidirectional causal relationship between economic growth and emission level under the EKC hypothesis. Vidyarthi (2015) empirically investigates the link between energy consumption and economic growth for a panel of five South Asian economies, namely India, Pakistan, Bangladesh, Sri Lanka, and Nepal, over the period from 1971 to 2010. The study relies on Pedroni cointegration and the Granger causality test based on panel vector error correction to assess long-term evidence. The results suggest that in the long-term, there is bi-directional causality between energy consumption and GDP per capita, while in the short-term conservation policies may harm economic growth in these developing markets. 1. INTRODUCTION The study, using cointegration tests, dynamic ordinary least squares (DOLS), and fully modified ordinary least squares (FMOLS) estimators, finds that overall energy consumption has pro-growth effects in energy-importing nations. Raza et al. (2016) investigates the effect of energy consumption, measured by electricity consumption, on economic growth in four South Asian countries for the years 1980-2010, namely Pakistan, India, Bangladesh, and Sri Lanka. Applying the random effects model, Pedroni cointegration technique, and Granger causality, the study finds that energy consumption has a unidirectional causal effect on GDP growth. the neutrality hypothesis. Similarly, Huang et al. (2008) studied the nexus between economic growth and energy consumption using a sample of 82 countries between 1972 and 2002. These countries were divided into four categories, including low income, lower middle income, upper middle income, and high-income countries. According to their findings, low-income countries are under the neutrality hypothesis of connection between economic growth and energy consumption. Other groups of countries have unidirectional causality of energy-growth the nexus which is described by the conservation hypothesis. Authors apply a Generalized Method of Moments (GMM) to estimate the panel VAR model in each category of countries. Al-mulali et al. (2013) explored the relationship between energy and economic growth for Latin American and Caribbean countries. The authors apply a canonical cointegrating regression (CCR) for the period 1980-2008. The results suggest that, “56% of the countries have a long run bi-directional positive relationship between energy consumption and economic growth, 6% of the countries have a long run bi-directional negative relationship between energy consumption and economic growth, 16% of the countries were found to have a positive one way long run relationship between energy consumption and economic growth, 16% of the countries have a long run positive relationship between economic growth and energy, and 6% of the countries have no relationship between energy consumption and economic growth” (p. 46). Shahbaz et al. (2018) assesses the link between growth and energy consumption for 10 energy-consuming countries using data from 1960 to 2015. The results, based on the Quantile-on-Quantile (QQ) approach, suggest that although energy consumption increases economic growth, the magnitude of this impact differs across countries depending on the relative importance of energy or technological development. In a similar vein, Apergis and Payne (2010) explore the link between energy consumption and GDP growth for 9 South American nations for the period 1980-2005. 1. INTRODUCTION In contrast, the results of the Granger causality and panel cointegration test, conducted by Narayan and Smyth (2008) within the G7 countries, identify short- and long-term nexuses between economic growth and energy consumption supporting the growth hypothesis. The same trend is found by Ciarreta and Zarraga (2008) who conduct analysis of 12 European countries using panel causality and cointegration models. The research doesn’t identify the connection between economic growth and electricity consumption in the short-term, which proves the neutrality hypothesis, but cointegration between observed variables is found in the long-term. According to their findings, there is evidence in favor of the growth hypothesis. The 1% increase in electricity consumption causes a 0.3% increase in GDP in the case of observed European countries. The feedback hypothesis is also proved by Bildirici and Kayıkçı (2012) based on the sample of Commonwealth Independent States (CIS) using data of the period from 1990 to 2009. The sample consists of 11 countries including Armenia, Azerbaijan, Georgia, Kazakhstan, Kyrgyzstan, Moldova, Belarus, Russian Federation, Tajikistan, Ukraine, and Uzbekistan. These countries are divided by 3 groups with accordance to income. The authors use the Pedroni cointegration, Fully Modified Ordinary Least Squares (FMOLS), and Panel ARDL methods in their work. According to the results of cointegration tests, economic growth and electricity consumption are cointegrated in all 3 groups. The causality tests show bi-directional relationships between economic growth and electricity consumption. The results of FMOLS and ARDL show the negative impact of electricity usage on economic performance in the second group and a positive effect in the first group in favor of the growth hypothesis. This work is closely related to the current study, although it differs in terms of sample size and main independent variable. To the best of our knowledge, this is the first study to assess the relationship between energy consumption and economic growth for a sample of post-communist countries. The results of our paper show that, overall there are unidirectional relationships between economic growth and energy consumption in favor of the conservation hypothesis in 26 countries for the period from 1995 to 2014. In a similar vein, Ozturk et al. (2010) investigates the relationship between energy consumption and economic growth for a sample of 51 low- and middle-income countries from 1971 to 2005. The results suggest that, overall, there is a long-term effect of energy consumption on economic growth. 1. INTRODUCTION Similarly, an analysis of the Indian economy is conducted by Ozturk and Uddin (2012) and studies the energy-growth carbon emission nexus using the Johansen–Juselius maximum likelihood procedure. The results prove the feedback hypothesis and identify strong causal relationships between energy consumption and carbon emissions in India. The conservation hypothesis is also proved by Yildirim et al. (2014) using a sample of ASEAN countries, including Indonesia, Malaysia, the Philippines, Singapore, and Thailand. An analysis of data from the period of 1971–2009 shows a unidirectional nexus from economic growth to energy consumption in the cases of Indonesia, Malaysia, the Philippines, and Thailand. According to the results, Singapore doesn’t demonstrate any relationship between energy and economic performance, thereby supporting 60 International Journal of Energy Economics and Policy \| Vol 10 • Issue 6 • 2020 Umurzakov, et al.: Energy Consumption and Economic Growth: Evidence from Post-Communist Countries Umurzakov, et al.: Energy Consumption and Economic Growth: Evidence from Post-Communist Countries Eggoh et al. (2011) assess the relationship between energy consumption and economic growth in a sample of 21 African economies over the period from 1970-2006 using cointegration techniques and OLS, DOLS, and PMG estimators. The study finds a bivariate causal relationship between energy consumption and economic growth for both net energy importing and exporting countries. Eggoh et al. (2011) assess the relationship between energy consumption and economic growth in a sample of 21 African economies over the period from 1970-2006 using cointegration techniques and OLS, DOLS, and PMG estimators. The study finds a bivariate causal relationship between energy consumption and economic growth for both net energy importing and exporting countries. 20 countries which export and import energy. Similar results are found by Lee and Chang (2007), whose work estimates the VAR model by GMM technique using a sample of 22 developed and 18 developing countries. The results show bidirectional relationships between economic growth and energy consumption in developed countries under the feedback hypothesis. However, this growth- energy nexus is indicated as unidirectional in developing countries, in favor of the conservation hypothesis. Similarly, Lee and Chang (2008) state that panel cointegration and panel-based error correction models prove the neutrality hypothesis in the short-term within a sample of 16 Asian countries. The results of long-term analysis show the unidirectional dependence of economic growth on energy consumption and prove the growth hypothesis. 1. INTRODUCTION However, when the authors disaggregate countries by income group, their results confirm the conservation hypothesis for low income countries and feedback hypothesis for middle income countries. Gorus et al. (2019) further investigates the link between energy consumption and economic growth for Middle East and North Africa (MENA) countries for the period 1975-2014. Their results suggest that, in the short-and intermediate-term, economic growth has a causal effect on energy usage, while in the long-term energy consumption has a positive impact on economic growth. Therefore, while energy conservation policies may be implemented in the short-term and intermediate- term, these policies are harmful for economic progress in the long- term. These findings agree with Ouedraogo (2013), who assesses the relationship between energy consumption and economic growth for the economic community of West African States (ECOWAS) over the period 1980-2008. The study relies on panel unit root, Granger causality, and panel cointegration techniques to explore the causal relationships. The results suggest that in the short-term, GDP growth has an effect on energy consumption, while energy consumption is causal to economic growth in the long-term. The rest of this study is structured as follows: Section 2 discusses data and methods; Section 3 offers empirical results; Section 4 concludes the manuscript. 2. DATA AND EMPIRICAL MODEL To the best of our knowledge, previous studies exploring the nexus between energy and economic growth are based on traditional growth models (Yuan et al., 2008; Apergis and Payne, 2010a; Shahbaz and Dube, 2012; Maji and Sulaiman, 2019). This model can be represented as: Y=f (K, L) (1) (1) Dividing the equation by labor (L), we obtain the following: Dividing the equation by labor (L), we obtain the following: Y/L=f (K/L,1) (2) (2) According to Mahadevan and Asafu-Adjaye (2007), there is a unidirectional growth-energy nexus under the growth hypothesis among developing countries and a bidirectional relationship among developed countries under the feedback hypothesis. Authors use the panel error correction model with a sample of where Y denotes real GDP per capita (constant 2010 US$). where Y denotes real GDP per capita (constant 2010 US$). Energy consumption is measured as energy use (kg of oil equivalent) and ECI is an economic complexity index computed using Standard International Trade Classification. The summary statistics and variables description is depicted in Table 1. where K/L is productivity measure, L is labor. where K/L is productivity measure, L is labor. In many empirical studies, gross fixed capital formation is used as a measure of capital (Yuan et al., 2008; Apergis and Payne, Umurzakov, et al.: Energy Consumption and Economic Growth: Evidence from Post-Communist Countries Following Apergis and Payne (2010b), Inglesi-Lotz (2016), and Maji and Sulaiman (2019), we run the panel unit root test proposed by Im et al. (2003). The specification of the test is depicted below: 2010; Inglesi-Lotz, 2016; Maji and Sulaiman, 2019). However, for several post-communistic countries, the data on gross fixed capital formation is not available. Gozgor et al. (2018) stated that Economic Complexity Indicator (ECI) provides a good measure for productivity and economic structure. After replacing K/L and incorporating energy consumption, the model can be depicted as: ( 1) ρ σ ε − = + + it i i t i it it y y x (5) (5) where xit represents the combination of all the explanatory variables in the model; ρi denotes the autoregressive elasticities, ϵit is the residual term, i=1,…N for each country and t=1,…T is the time period. Y/L=f (ECI, EC) (3) (3) where ECI is an economic complexity indicator and EC is energy consumption. Table 2: Correlation matrix Variables ln Y ln EC ECI ln Y 1 ln EC 0.764 1 ECI 0.646 0.420 1 Equation (3) considers the relationship between energy consumption, ECI, and economic growth, shedding light on the nexus between consumption of conventional energy and economic growth employing secondary data on post-communistic countries during 1990 and 2018, available from the World Bank’s World Development Indicators. After balancing the data, our sample includes 26 post-communist countries – Armenia, Azerbaijan, Bulgaria, Bosnia and Herzegovina, Belarus, Czech Republic, Estonia, Georgia, Croatia, Hungary, Kazakhstan, Kyrgyz Republic, Lithuania, Latvia, Moldova, Poland, Romania, Russian Federation, Serbia, Slovak Republic, Slovenia, Montenegro, Tajikistan, Turkmenistan, Ukraine, and Uzbekistan between 1995and 2015. Furthermore, we transformed the model (3) into the logarithmic form, obtaining the following model: ntional energy and economic post-communistic countries m the World Bank’s World ncing the data, our sample ies – Armenia, Azerbaijan, Belarus, Czech Republic, azakhstan, Kyrgyz Republic, Romania, Russian Federation, a, Montenegro, Tajikistan, tan between 1995and 2015. odel (3) into the logarithmic : ECI 2 (4) (constant 2010 US$). as energy use (kg of oil complexity index computed Classification. The summary depicted in Table 1. where K/L is productivity measure, L is labor. D RESULTS employ Pedroni’s (1999) Dynamic OLS estimation 2) panel Granger causality and causality, we report the esults (Tables 2 and 3). no worrisome correlation ollinearity. ln Y 1 ln EC 0.764 1 ECI 0.646 0.420 1 Table 3: Panel unit root test results Variable Form Method Statistic P-value Conclusion ln GDP per capita Level LLC −2.546 0.005 Stationary Breitung 10.25 1.000 Non stationary IPS 3.52 0.999 Non stationary Fisher- ADF 23.50 0.999 Non stationary Fisher-PP 74.27 0.009 Non stationary 1st difference LLC −4.57 0.000 Stationary Breitung −3.63 0.000 Stationary IPS −7.43 0.000 Stationary Fisher- ADF 151.68 0.000 Stationary Fisher-PP 303.16 0.000 Stationary ln Energy Level LLC −1.63 0.052 Stationary Breitung 0.1291 0.551 Non stationary IPS 0.1344 0.554 Non stationary Fisher- ADF 55.563 0.211 Non stationary Fisher-PP 63.303 0.068 Stationary 1st difference LLC −8.454 0.000 Stationary Breitung −8.411 0.000 Stationary IPS −9.844 0.000 Stationary Fisher- ADF 190.124 0.000 Stationary Fisher-PP 357.411 0.000 Stationary ECI Level LLC −2.160 0.015 Stationary Breitung −0.7 0.242 Non stationary IPS −1.937 0.026 Stationary Fisher- ADF 50.026 0.393 Non stationary Fisher-PP 92.536 0.000 Stationary 1st difference LLC −5.988 0.000 Stationary Breitung −8.826 0.000 Stationary IPS −12.485 0.000 Stationary Fisher- ADF 220.120 0.000 Stationary Fisher-PP 773.547 0.000 Stationary ln EC ECI nergy use (kg of oil auivalent) Economic complexity indicator (SITC) WDI Atlas MIT 7.569 0.419 0.689 0.749 5.647 −1.591 8.550 1.787 480 480 Table 3: Panel unit root test results ln ln Y EC ECI 1 2 (4) (4) International Journal of Energy Economics and Policy \| Vol 10 • Issue 6 • 2020 3. METHODS AND RESULTS For a small number of observations (N) and time period (T), Lopez and Weber (2017) suggest testing the null hypothesis based on z bar tilde statistics. Dumitrescu and Hurlin (2012) provide no guidance for lag order selection. However, it is recommended to select the number of lags based on an information criterion (AIC/BIC/HQIC) (Chang et al., 2013; Lopez and Weber, 2017). The test is based on the individual Wald statistics of Granger non-causality averaged across the cross-section units. The null hypothesis claims no causal relationship between the variables. The testing procedure of the null is based on z-bar and z bar tilde. For a small number of observations (N) and time period (T), Lopez and Weber (2017) suggest testing the null hypothesis based on z bar tilde statistics. Dumitrescu and Hurlin (2012) provide no guidance for lag order selection. However, it is recommended to select the number of lags based on an information criterion (AIC/BIC/HQIC) (Chang et al., 2013; Lopez and Weber, 2017). 1 ( 1) 1 1 ρ σ θ ∈ ε = − − = = + + + ∑ n it i i t i it ij it it j y y x (7) (7) where ρi shows the number of lags in the ADF regression. where ρi shows the number of lags in the ADF regression. IPS tests the null hypothesis of non-stationarity in every individual panel series over the alternative of no unit-root in at least one panel. For the robustness check, we additionally run panel unit root tests proposed by Levin et al. (2002) and Breitung and Das (2005), as well as the Fisher-type test. In general, the null hypotheses of the tests claim non-stationarity, while alternative assume that some or at least one panel is stationary. Results of the panel data unit root test are reported in Table 3. In the level form, GDP per capita does not contain unit-root only under the LLC test. Both energy consumption and ECI are stationary under LLC and Fisher PP-type tests. As some test statistics report non-stationarity, we reject the null hypothesis and take the first difference of the variables to re-test the model for unit- root. As a result, all variables are stationary at the 1% significance level. Stationarity enables further estimation of the model. Table 6 reports the results of the Dumotrescu and Hurlin’s panel Granger causality test results. 3. METHODS AND RESULTS We first test whether economic growth does not Granger-cause energy consumption. At the 1% significance level, we reject the null hypothesis as z-bar tilde Table 4: Pedroni and Kao panel cointegration tests results Test statistics Statistics P-value V t t 0 43 0 332 Table 4: Pedroni and Kao panel cointegration tests results Table 4: Pedroni and Kao panel cointegration tests results Test statistics Statistics P-value V-stat 0.43 0.332 Panel rho-stat −3.32 0.000 Panel PP-stat −7.54 0.000* Panel ADF-stat −7.54 0.004*** Group rho stat −1.31 0.095* Group PP stat −8.91 0.000* Group ADF stat −2.22 0.013 Kao −7.99 0.000* , * and * indicate significance at 1%, 5% and 10% levels, respectively Furthermore, we conduct the Pedroni and Kao (1999) cointegration tests reported in Table 4. Pedroni panel cointegration is a one-tailed test that tests the presence of a long-term relationship using seven test statistics. The test statistics can be divided into two categories: group statistics that average the results of the individual country, and test statistics and panel statistics that pool the statistics along the within- dimension (Neal, 2014). The null hypothesis assumes no cointegration and it can be rejected when the majority of test statistics reject it. Table 5: Impact of renewable energy on economic growth Table 5: Impact of renewable energy on economic growth Variables Pedroni DOLS DOLS FMOLS OLS ln energy 1.599* 0.66* 0.37* 0.349* 111 17.86 19.57 5.31 ECI 0.0323* 0.02 0.06* −0.006 40.06 1.39 6.86 0.77 *, and * indicate significance at 1%, 5% and 10% levels, respectively. While figures in parentheses are t-statistic According to Table 4, 6 out of 7 test statistics reject the null hypothesis, revealing a long-term relationship between energy consumption and economic growth. Kao tests for panel cointegration support the results of Pedroni as well at a 1% significance level. Since cointegration between the variables is confirmed, we estimate the model using the Pedroni Panel Dynamic OLS (DOLS). Panel DOLS is an extension of time series DOLS, estimating the cointegrating vector that exhibits a cointegrating relationship between variables (Neal, 2014). To verify the consistency of results, we employ time series cointegrated regression estimators (DOLS and FMOLS) adapted to panel data by Khodzhimatov (2018), as well as an OLS estimator. 3. METHODS AND RESULTS Following Yuan et al. (2008), we employ Pedroni’s (1999) panel cointegration tests and Panel Dynamic OLS estimation and Dumitrescu and Hurlin’s (2012) panel Granger causality test. Before estimating panel DOLS and causality, we report the correlation matrix and unit root test results (Tables 2 and 3). According to Table 2, there are no worrisome correlation coefficients which may cause multicollinearity. y ECI Level LLC −2.160 0.015 Stationary Breitung −0.7 0.242 Non stationary IPS −1.937 0.026 Stationary Fisher- ADF 50.026 0.393 Non stationary Fisher-PP 92.536 0.000 Stationary 1st difference LLC −5.988 0.000 Stationary Breitung −8.826 0.000 Stationary IPS −12.485 0.000 Stationary Fisher- ADF 220.120 0.000 Stationary Fisher-PP 773.547 0.000 Stationary Table 1: Descriptive statistics Variables ln Y ln EC ECI Indicator GDP per capita (constant 2010 US$) Energy use (kg of oil eauivalent) Economic complexity indicator (SITC) Source WDI WDI Atlas MIT Mean 8.451 7.569 0.419 Std. dev 1.034 0.689 0.749 Min 5.905 5.647 −1.591 Max 10.144 8.550 1.787 N. of observations 480 480 480 62 Energy Consumption and Economic Growth: Evidence from Post-Communist Countries Once the causal relationship is established, we proceed to identify the direction of causality by using the panel Granger non-causality test for heterogeneous panel data models proposed by Dumitrescu and Hurlin (2012). The benchmark model is presented below, where xi t, and yi t, are observations of two stationary variables for individual i in period t. 1 1 1 ∈ θ ∈ ε = − = = + ∑ n it ij it it j (6) (6) The IPS test is based on heterogeneity of the autoregressive parameter (Maddala and Wu, 1999). It allows for different orders of serial correlation (Inglesi-Lotz, 2016) and follows the normal averaging Dickey Fuller (ADF) (Apergis and Payne, 2010b): y y x i t i ik i t k k K ik i t k i t k K , , , , 1 1 (8) (8) If eq. 6 is substituted into eq. 5: If eq. 6 is substituted into eq. 5: The test is based on the individual Wald statistics of Granger non-causality averaged across the cross-section units. The null hypothesis claims no causal relationship between the variables. The testing procedure of the null is based on z-bar and z bar tilde. REFERENCES Huang, B.N., Hwang, M.J., Yang, C.W. (2008), Causal relationship between energy consumption and GDP growth revisited: A dynamic panel data approach. 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Journal of Academic Research in Economics, 4(2), 149-166. The causal link between energy consumption and economic growth is explored for a group of post-communist countries covering the period 1995 and 2014 and utilizing the Pedroni cointegration and panel Granger causality test. Our findings confirm the cointegration between energy consumption and economic growth. Panel Dynamic OLS results revealed a positive and significant impact of energy consumption on economic growth. The Dumitrescu and Hurlin’s (2012) panel Granger causality test results discovered unidirectional causality running from economic growth to energy consumption. Economic growth causes energy consumption in post-communistic countries, confirming the conservation hypothesis. Dumitrescu, E.I., Hurlin, C. (2012), Testing for Granger non-causality in heterogeneous panels. Economic Modelling, 29(4), 1450-1460. Eggoh, J.C., Bangaké, C., Rault, C. 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(2018), An analysis of energy consumption and economic growth of Cobb-Douglas production function based on ECM. In: IOP Conference Series: Earth and Environmental Science. Vol. 113. Bristol: IOP Publishing. p012071. 3. METHODS AND RESULTS Table 6: DH granger non-causality test results Table 6: DH granger non-causality test results Economic growth does not granger-cause energy consumption W-bar 2.694 Z-bar 5.869* (0.000) Z-bar tilde 4.099* (0.000) Optimal number of lags (AIC) 4 Lags tested 1-4 Energy consumption does not granger-cause economic growth W-bar 1.175 Z-bar 0.608 (0.544) Z-bar tilde 0.057 (0.954) Optimal number of lags (AIC) 4 Lags tested 1-4 *, and * indicate significance at 1%, 5% and 10% levels, respectively. While figures in parentheses are P-values Economic growth does not granger-cause energy consumption W-bar 2.694 Table 5 describes the results of panel cointegrated regressions. Our results revealed a positive and significant impact of energy consumption on economic growth. The results of the robustness check from panel DOLS, FMOLS, and OLS yield the same positive and significant coefficients of energy consumption. Such results are in line with the findings of Rezitis and Ahammad (2015) and Gozgor et al. (2018). 63 Umurzakov, et al.: Energy Consumption and Economic Growth: Evidence from Post-Communist Countries Chaudhry, I.S., Safdar, N., Farooq, F. (2012), Energy consumption and economic growth: Empirical evidence from Pakistan. Pakistan Journal of Social Sciences, 32(2), 371-382. statistics are higher than critical values. Furthermore, we check for the presence of bidirectional causality. Due to the large probability value of z-bar tilde statistics (0.9543), we fail to reject the null hypothesis. Our estimates revealed unidirectional causality running from economic growth to energy consumption. 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https://openalex.org/W2981654133	https://www.ajol.info/index.php/as/article/download/190216/179436	English	null	Determinants of adoption of risk management strategies in electronic banking among agro-entrepreneurs in Ughelli North Local Government Area of Delta State, Nigeria	Deleted Journal	2,019	cc-by	5,081	Please cite as: Ugwuja V.C. and Onavwie O. (2019). Determinants of adoption of risk management strategies in electronic banking among agro-entrepreneurs in Ughelli North Local Government Area of Delta State, Nigeria Agro-Science, 18 (3), 19-24. DOI: https://dx.doi.org/10.4314/as.v18i3.4 DETERMINANTS OF ADOPTION OF RISK MANAGEMENT STRATEGIES IN ELECTRONIC BANKING AMONG AGRO-ENTREPRENEURS IN UGHELLI NORTH LOCAL GOVERNMENT AREA OF DELTA STATE, NIGERIA Ugwuja V.C. and Onavwie O. Department of Agricultural Economics & Extension, University of Port Harcourt, Nigeria Corresponding author’s email: vivian.ugwuja@uniport.edu.ng Corresponding author’s email: vivian.ugwuja@uniport.edu.ng ABSTRACT This study examined the determinants of adoption of risk management strategies in electronic banking (digital finance) among agro entrepreneurs in Ughelli North local Government area of delta state, Nigeria. Data were collected with the aid of structured questionnaire, administered on 79 respondents using two-stage sampling procedure. The data were analyzed with the use of descriptive statistics and regression analysis. This study showed that 34.0% of the respondents were male while 57.0% of the respondents were female, 39.2% of the respondents were in the age bracket of 41-50 years. The regression result shows that gender, age, marital status, level of education, farming status were all significant factors that influence risk management strategies in electronic banking. Household size, business experience, income and savings were not significant. Also, it was found that constraints faced by agro entrepreneur in adopting these risk management strategies includes the length of time it takes for unsuccessful funds transfer to be returned, the delayed response of banks to issues relating to electronic banking risks, ATM are not readily available and the ones that are, are usually congested resulting to agro entrepreneurs using the ATM at odd hours, amongst others. Proper awareness on these risk management strategies should be given to these rural entrepreneurs, and government should charge banks with the sole responsibility of providing a more secure and coordinated methods of carrying out these activities. Key words: digital finance, electronic banking risks, ATM and cyber security 19 19 Agro-Science Journal of Tropical Agriculture, Food, Environment and Extension Volume 18 Number 3 (September 2019) pp. 19-24 ISSN 1119-7455 Objectives of the Study j y The main objective of the study is to analyze the determinants of adoption of risk management strategy in electronic banking (e-banking) among agro entrepreneur in Ughelli North Local Government Area (LGA), Delta State. The specific objectives were to (i) describe the socio-economic characteristics of agro-entrepreneurs in Ughelli North LGA (ii) identify the various sources of risk faced by agro-entrepreneurs in accessing e-banking products and services in the study area, (iii) identify electronic banking risk management strategies adopted by agro entrepreneurs in the study area, (iv) examine the socio-economic factors that influence adoption of e-banking risk management strategies among agro-entrepreneurs in the study area, and (v) examine constraints to adoption of e banking risk management strategies among agro-entrepreneur in the study area. Exponential Function Exponential Function Y = B0 + B1LogX1 + B2LogX2 + B3LogX3 + B4LogX4 + B5LogX5 + B6LogX6 + B7LogX7 + B8LogX8 + B9 X9 + e Determinants of E-Banking Risk Management by Agro-Entrepreneurs in Delta State, Nigeria 20 Afiesere, Ekuigbo, Ekrejebor, Eruemukobwarien, Iwhremaro, Iwhreneneiwhaeorie, Ododegho and Odovie. The people in this area mostly involve in agricultural activities such as farming, processing of agro-products, buying and selling of agro- products. Two-stage sampling was used to select respondents. Stage one was a purposive selection of four communities who have access to banking facilities. Stage two involved the purposive selection of 20 respondents who access electronic banking products from each community, which made it a total 80 respondents for the study, but 79 copies of questionnaire were used for the analysis. The data for this study were collected from primary source with the aid of questionnaire which was objective oriented. Objectives i,ii, iii and v were achieved using descriptive statistics such as frequencies and percentages, while objective iv was aachieved using regression analysis. excessive, developing a strategy to ameliorate appropriate individual risks until the overall level is reduced to an acceptable level. Risk management approaches differ from one firm to the next, which partly reflects different risk management goals. The rapid spread of Internet banking all over Nigeria is its acceptance as an extremely cost effective delivery channel of banking services as compared to other existing channels. However, the introduction of electronic banking is not an unmixed blessing to agro entrepreneur, along reduction in cost of transactions, it has also brought about the exposure of these gullible agro entrepreneurs to risks and even new forms of risks to which banks conducting e-banking expose these entrepreneurs to. Regulators and supervisors all over the world are concerned that while banks should remain efficient and cost effective, they must be conscious of different types of risks this form of banking entails and have systems in place to manage the same (Solanki, 2012). This study is aimed at looking at those factors that can be adopted by agro entrepreneurs to reduce or combat these risks associated with electronic banking. Model Specification p Regression analysis was used to test for significant relationship between socioeconomic characteristics of the agro entrepreneurs and the level of adoption of risk management strategies. The implicit form of the linear regression is: INTRODUCTION especially the card technology, is presently enjoying the highest popularity in Nigerian banking market. To be competitive in the internet business, financial institutions need to harness the power of the internet successfully (Adewuyi, 2011). Hence it is important to understand the benefits, barriers and impediments as it relates to electronic banking services marketing in developing economies such as Nigeria. Despite the fact that this technology is a good one, it comes with so many risks. These risks caused by this phenomenon include hacking of servers and security breach, agro entrepreneurs, employees and customers’ unfamiliarity with new technology, lack of infrastructures necessary to provide E-services, not taking an appropriate strategic approach by bank managers and by governmental top managers are challenges facing banks and financial institutions. One of the most important responsibilities of agro entrepreneurs is to understand these risks and the risk managements strategies associated to electronic banking. Risk management can be described as the process of determining the maximum acceptable level of overall risk of engaging in a proposed activity. It involves using risk assessment techniques to determine the initial level of risk and, if it is Electronic Banking also known as digital finance is defined as “the use of technology to communicate instructions and receive information from a financial institution where an account is held. This service includes the system that enables customers of financial institution, individuals or businesses to access accounts, transact business, or obtain information on financial products and services through a public or private network” (Abdul and Muhammad, 2013). It has experienced tremendous growth in many countries especially Africa and today, it has transformed the traditional banking practice in Nigeria. Electronic banking in Nigeria has changed the way services are delivered by the banking sectors to their customers. Electronic banking services, has lower operating costs, improves customer services delivery, retains customers, reduces branch traffics, and downsize the number of branch staff (Parisa, 2006). Essentially, through the use of Information and Communication Technology banks now employ different channels such as online banking, mobile banking and Automated Teller Machine to deliver their services. Report on Electronic banking system in Nigeria reveals that e-payment machinery, Determinants of E-Banking Risk Management by Agro-Entrepreneurs in Delta State, Nigeria Y-F (X1, X2, X3, X4, X5, X6, X7, X8, X9 ) + e, where Y is the level of adoption of risk management strategies, Y was derived as U/V × 100, U is number of risk management strategies adopted by an agro entrepreneur, V is total number of risk management strategies X1 is age of the agro entrepreneur (years), X2 is educational level (Years), X3 is marital status (0 = single, 1 = married), X4 is gender (0 = male, 1 = female), X5 is business experience of the agro entrepreneur (years), X6 is personal annual income of the agro entrepreneurs (₦), X8 is annual savings (₦), and e is error term. The relationship between the dependent and each of the independent variable was examined using the four functional forms, linear, semi-log exponential and double log. Linear Function Y = B0 + 1B1XB1 + B2X2 + B3X3 + B4X4 + B5X5 + B6X6 + B7X7 + B8X8 + B9X9 + e Linear Function Y = B0 + 1B1XB1 + B2X2 + B3X3 + B4X4 + B5X5 + B6X6 + B7X7 + B8X8 + B9X9 + e Linear Function Y = B0 + 1B1XB1 + B2X2 + B3X3 + B4X4 + B5X5 + B6X6 + B7X7 + B8X8 + B9X9 + e MATERIALS AND METHODS The study was conducted in Ughelli North LGA in Delta State, located at coordinates latitude 9˚45N and longitude 8˚43E, with headquarters in the city of Ughelli. This LGA occupies a geographical area of 818 km2. According to the National Population Census of Nigeria (NPC, 2006), it has a population of 321,028, the LGA has a total number of 17 communities which includes among others Semi –Log Function: Log Y = B0 + B1X1 + B2X2 + B3X3 + B4X4 + B5X5 + B6X6 + B7X7 + B8X8 + e Hypothesis of the Study H0: There is no significance relationship between socio-economic characteristics of agro entrepreneur and adoption of electronic banking risk management strategies. B6X6 + B7X7 + B8X8 + B9X9 + e Exponential Function Y = B0 + B1LogX1 + B2LogX2 + B3LogX3 + B4LogX4 + B5LogX5 + B6LogX6 + B7LogX7 + B8LogX8 + B9 X9 + e Semi –Log Function: Log Y = B0 + B1X1 + B2X2 + B3X3 + B4X4 + B5X5 + B6X6 + B7X7 + B8X8 + e Double–Log Function LogY=B0+B1LogB1X1+B2LogB2X2+B3LogB3X3+B 4LogB4X4+B5LogB5X5+B6LogB6X6+B7LogB7X7+ B8LogB8X8 + e 6 6 7 7 8 8 9 9 Exponential Function Y = B0 + B1LogX1 + B2LogX2 + B3LogX3 + B4LogX4 + B5LogX5 + B6LogX6 + B7LogX7 + B8LogX8 + B9 X9 + e Semi –Log Function: Log Y = B0 + B1X1 + B2X2 + B3X3 + B4X4 + B5X5 + B6X6 + B7X7 + B8X8 + e Double–Log Function LogY=B0+B1LogB1X1+B2LogB2X2+B3LogB3X3+B 4LogB4X4+B5LogB5X5+B6LogB6X6+B7LogB7X7+ B8LogB8X8 + e Exponential Function Y = B0 + B1LogX1 + B2LogX2 + B3LogX3 + B4LogX4 + B5LogX5 + B6LogX6 + B7LogX7 + B8LogX8 + B9 X9 + e Semi –Log Function: Log Y = B0 + B1X1 + B2X2 + B3X3 + B4X4 + B5X5 + B6X6 + B7X7 + B8X8 + e Double–Log Function LogY=B0+B1LogB1X1+B2LogB2X2+B3LogB3X3+B 4LogB4X4+B5LogB5X5+B6LogB6X6+B7LogB7X7+ B8LogB8X8 + e Entrepreneurs The socio-economic characteristics of the respondents were summarized in Table 1. The results show a slightly higher ratio for females than males. This showed that 43.0% of the respondents were male while 57.0% of the respondents were females. The study also revealed that 39.2% of the Table 1: Distribution of farmers’ socio-economic characteristics Socio-economic characteristics Frequency (n = 79) Percentage (%) Gender Male 34 43.0 Female 45 57.0 21-30 12 15.2 31-40 19 24.1 41-50 31 39.2 51-60 13 16.5 61-70 4 5.1 Marital status Single 19 24.1 Married 51 64.6 Widowed 7 8.9 Divorced 2 2.6 Level of educational status No formal education 2 2.5 Primary 11 13.9 Secondary 29 36.7 Tertiary 35 44.3 Others 2 2.5 Family size 1-3 18 22.8 4-6 51 64.6 7-10 10 12.7 Farming status Full time 46 58.2 Part time 33 41.8 Enterprise type Livestock 18 22.8 Crop farming 17 21.5 Agro processing 2 2.5 Fish farming 22 27.8 Marketing 16 20.3 Others 4 5.1 Business experience 1-10 62 78.5 11-20 14 17.7 21-30 1 1.3 31-40 2 2.5 Average monthly income (₦) 10,000-50,000 3 3.8 60,000-500,000 50 63.3 600,000-1,000,000 12 15.2 1,100,000-1,500,000 2 7.6 1,600,000-2,000,000 6 7.6 2,100,000-2,500,000 2 2.5 2,600,000-3,000,000 4 5.1 Annual savings (₦) 10,000-200,000 65 82.3 210,000-400,000 7 8.9 410,000-800,000 4 5.1 801,000 – 1.000.000 2 2.5 Field survey, 2018 Table 1: Distribution of farmers’ socio-economic Table 1: Distribution of farmers’ socio-economic characteristics Sources of Risk Faced by Agro-Entrepreneurs in Accessing e-banking Products and Services The sources of risks faced by the agro entrepreneurs in Ughelli north local government are summarized in Table 2. The result showed that 67.1% of the respondent agreed that they have faced risk in the form of text message requesting for their bank details, which they sent and from which their accounts were hacked and money withdrawn. This is one of the most prominent forms of risk because hackers now send phishing messaging at random for people awaiting those who fall victims. About 63.3% of the respondents accepted that they were accosted by criminals immediately after withdrawal from automated teller machines (ATM).This is as a result of the customers using ATM at odd hours in a bid to avoid long queue, thereby making them susceptible to hoodlums and criminals. RESULTS AND DISCUSSION Socio-Economic Characteristics of Agro-Based Entrepreneurs Socio-Economic Characteristics of Agro-Based Entrepreneurs Entrepreneurs About 51.9% of the agro entrepreneurs in the study area further stated that they had fallen victim to text messages from criminals who claimed that their ATM card is expired or due for renewal, requesting for the sensitive security numbers on the card from which their money was withdrawn. The same percentage of people also said they had suffered from failed transactions when using their bank application, these transactions were thought to have failed due to Double–Log Function Double–Log Function LogY=B0+B1LogB1X1+B2LogB2X2+B3LogB3X3+B 4LogB4X4+B5LogB5X5+B6LogB6X6+B7LogB7X7+ B8LogB8X8 + e 21 Ugwuja V.C. and Onavwie O. Ugwuja V.C. and Onavwie O. Ugwuja V.C. and Onavwie O. respondents were in the age bracket of 41-50 and 24.1% were observed to be in the age bracket of 31-40, which indicates that the respondents were in the middle and active ages of production. Married individuals were the highest among the respondents with the percentage of 64.6%, followed by single with the percentage 24.1% while 8.9% were widowed, and 2.6% divorced. A breakdown of the education statistics indicates that 2.5% had no formal education, 13.9% had attained the primary education, and 44.3% of the respondents had attained tertiary education and this was the highest level of education attained by my respondents, followed closely by 36.7% of the respondents who had attained secondary education, which is quite appropriate because a good level of education should be attained in other to be able to operate or use most of the electronic products and services. About 64.6% had a family size of 4-6, 22.8% had a family size of 1-3, and family size of 7-10having a percentage of 12.7% which showed that the family size of 4-6 were the most dominant in the area of study hence they had the highest frequency. The respondents which were into agribusiness fulltime were the most dominant in the study with a percentage of 58.2% while 41.8% were part-time agro entrepreneurs. About 27.8% of these respondents were into fish farming, 22.8% were into livestock production, 21.5% were into crop farming, 20.3% were into marketing of agricultural products while 2.5% focused on agro processing. where B0 is intercept, B1 to B8 are estimated coefficients. The criteria used in selecting the functional equation that will best fit for regression were highest R2 value, highest number of significant variables, highest F-value and conformity to apriority expectations of the coefficient. Risk Management Strategies Adopted by Agro Entrepreneurs network issues but money was debited from their account. About 41.8% said that their ATM card was accessed by third party, this was corroborated by findings of Yousafzai et al. (2003) who reported that lack of security in protecting personal information is a problem in using internet banking according. About 29.1% of the respondent said that the stress faced in the bank in other to get back their funds which was lost due to using one or two banking services platform is their problem, whereas 25.5% suffered deduction of money on transactions not approved while using POINT ON SALES (POS). About 22.8% claimed to have received calls from unknown people claiming to be their account manager requesting for some sensitive details which lead to the compromising of their accounts. Risk management strategies adopted by the agro entrepreneurs are shown (Table 3). Majority (92.4%) of the respondents block and ignore irregular email and text messages that have to do with their bank details thereby preventing risk due to phishing, 87.5% said they no longer use lonely ATMs or go to the ATM at odd hours. About 73.4% indicated that they keep contact with their banks in relation to information got online or via SMS relating to their bank accounts. About 53.2% indicated that since their experience with these risks they are now careful whenever they login to their online banking platform to prevent preying eyes. Whereas 50.6% indicated they call their banks to deactivate their ATM card whenever lost, 45.6% said that they periodically check with their banks if everything is okay with their accounts in order to detect any move to hack their accounts. About 44.3% of the agro entrepreneurs agreed that they do not use birthdates and address that would be easy for attackers to find out. Also 44.3% indicated that not using the same passwords for all their bank account are both strategies they used in combating security risks. Whereas 30.4% of the respondents indicated that they no longer click on links from suspicious looking emails, 25.3% of them revealed they now use reliable mobile security software to protect their devices. About 22.8% of the respondent agreed that they download mobile apps only from official stores, while 11.4% reported that they always make use of https:// (which is more secured) instead of http:// while logging into websites. Sources of Risk Faced by Agro-Entrepreneurs in Accessing e-banking Products and Services 22 Table 2: Sources of risk faced by agro-entrepreneurs in accessing e-banking products and services Sources of risks Frequency Percentage Similar email address with that of the bank that requested for customers details from which accounts were hacked 9 11.8 Text message requesting for customers bank details, from which account was hacked and money withdrawn 53 67.1 Fake website which when accessed leads to hacking of my device and exposure of my bank details ATM card and security number was accessed by 3rd party Accosted by criminals immediately after withdrawal from the automated teller machine (ATM) 7 33 50 8.9 41.8 63.3 Text messages saying ATM CARD had expired, requesting for the security numbers on the card 41 51.9 Failed transactions done with banks mobile application due to network issues, but money was debited from my account 41 51.9 Bad and negatives comments from my friends and family hindered me from continuing with these platforms 7 8.9 Stress passed through in the bank in other to get back money lost due to transfer failures 23 29.1 Error message every time I try to make transactions using my banks short code 12 15.2 Similar email address with that of the bank requesting for change of online banking password, which led to hacking of my account 5 6.3 Stolen phone resulting to loss of access to my online banking platform 8 10.1 Fake online shopping site, where details of debit card entered resulted to loss of my money 6 7.6 Payments made on point on sales (POS) which wasn’t approved, yet money was deducted from my account 20 25.5 Unknown call from someone claiming to be account manager, requesting for some details, which compromised my account 18 22.8 Field survey, 2018 Determinants of E-Banking Risk Management by Agro-Entrepreneurs in Delta State, Nigeria 22 nants of E-Banking Risk Management by Agro-Entrepreneurs in Delta State, Nigeria 22 Determinants of E-Banking Risk Management by Agro-Entrepreneurs in Delta State, Nigeria 22 Socio-Economic Factors in Level of Adoption of Risk Management Strategies in e-Banking Socio-Economic Factors in Level of Adoption of Risk Management Strategies in e-Banking electronic banking. The negative effects of age may be due to younger farmers being technology savvy which helps to adopt to different risk management strategies. This is in line with the findings of Ugwuja et al. (2017) who reported that younger farmers adopted more risk management strategies in fish farming. Marital status (X3) was significant at 5% and has positive coefficient. g g g Results of the regression analysis for the socio- economic characteristics for all the four functional forms are shown in Table 4. Double-log form was chosen as the lead equation based on the highest value of coefficient of multiple determination (R2), more significant coefficients, highest F-value and conformity to theoretical expectations of the R2. The R2 value of 0.691 indicates that about 69% of the variation in the dependent variation was explained by the independent variable included in the regression while the remaining 31% was due to other factors not specified in the model; F-ratio with 5.693 value in the regression is significant at 1%. It implies that all the variables have significant or joint effect on the dependent variables. This implied that being married increased the probability of level of adoption of risk management strategies. Level of education (X4) was significant at 1% and it related positively to level of adoption of risk management strategies in electronic banking. This shows that the more literate a farmer is the more chances of adopting to many risk management strategies. Farming status (X6) was significant at 5% and had a negative coefficient. This shows that being a full time farmer reduces the chances of adopting more of risk management strategies. This may be due to part-time agro- entrepreneurs getting income from other sources and increasing their chances of having more resources that will enable them to adopt to more risk management strategies. Household size, business experience, income and savings were not significant. Gender (X1) was significant at 5% and related positively to level of adoption of risk management strategies in electronic banking. This shows that men were more likely to adopt to many risk management strategies. Age (X2) was significant and had a negative coefficient. Socio-Economic Factors in Level of Adoption of Risk Management Strategies in e-Banking This implied that being a younger farmer increased the probability of adopting more risk management strategies in Table 3: Risk management strategies adopted by agro entrepreneurs Risk managements strategies Frequency Percentage I take special care whenever I want to login to my online banking platform to prevent preying eyes from getting my details 42 53.2 I ignore irregular email and Text messages that has anything to do with my bank details 73 92.4 I no longer click on links in suspicious looking email no matter how enticing they look 24 30.4 I contact my bank to crosscheck my information receive online or via text message 58 73.4 I periodically check with my bank to confirm if everything is okay 36 45.6 I no longer use birthdates, address and other words or numbers that would be easier for attackers to find out or guess my passwords 35 44.3 I don’t use the same passwords for all my bank accounts because guessing of one password can compromise the other accounts 35 44.3 I don’t use lonely ATMs or go to the ATM during late hours 69 87.5 I now use a reliable mobile security software to protect my device 20 25.3 I now use a two factor authentication(pin and OTP) process for my mobile banking 18 22.8 I called my bank immediately to deactivate my misplaced ATM card 40 50.6 I installed antivirus to prevent third party from accessing my computer or phone 18 22.8 I don’t use public Wi-Fi for my online transactions 8 10.1 I always use https:// instead of http: to log in my account I download apps only from reputable 29 11.4 Vendors 18 22.8 Table 4: Regression results on level of adoption of risk management strategies in e-banking by agro-entrepreneurs Variables Linear Exponential Semi-log Double log Constant 57.688(2.936) 202.462 (3.814)* 4.096(8.805)* 7.204 (5.760)* Gender(X1) 0.146 (1.358) 0.203(1.908)* 0.164(1.541) 0.226(2.163) Age(X2) -0.518(-3.428)* -0.586 (-3.620)* -0.480(-3.205)* -0.575 (-0.608)* Marital status (X3) 0.269(2.309) 0.266 (2.053) 0.241(2.091) 0269(2.112) Level of education (X4) 0.402(3.684)* 0.386(3.496)* 0.394 (3.651)* 0.392(3.611)*** Household size(X5) 0.010 (0.090) 0.042(0.385) -0.028 (-0.256) 0.029(0.273) Farming status(X6) -0.181(-1.796)* -0.183(-1.804)* -0.217 (-2.168) -0.207 (-2.068) Business 0.046 0.072 0.034 0.066 Experience(X7) (0.355) (0.537) (0.262) (0.499) Income(X8) 0.047(0.301) 0.100(0.528) 0.106 (0.689) 0.173(0.930) Savings(X9) -0.045(-0.300) -0.154(-0.847) -0.107 (-0.718) -0.208 (-1.160) R2 0.667 0.656 0.688 0.691 F-value 5.431* 4.780* 5.204* 5.693* Field Survey (2018); * Significant at 1% level, Significant at 5% level, *Significant at 10% level. Risk Management Strategies Adopted by Agro Entrepreneurs Finally 10.1% reported that they do not use public Wi-Fi for online transactions. p g Again 15.2% indicated that the error messages they get when trying to access their bank short code is not encouraging, this is also due to network issues. Also 11.8% of the respondent reported that similar email address with that of their banks emailed them requesting for their banking details in other to perform upgrades from which their accounts were hacked. About 10.1% mentioned that loss of their phones which led to lack of access to their online banking platforms. About 8.9% agreed that fake websites which were accessed compromises their devices and leads to hacking of their accounts, same percentage also agreed that negative comments from friends and family about electronic banking was the issue they faced which lead to total mistrust in electronic banking. Finally 7.6% of the respondents indicated that fake online shopping site, where details of debit card were entered resulted to loss of their money. 23 Ugwuja V.C. and Onavwie O. Ugwuja V.C. and Onavwie O. Ugwuja V.C. and Onavwie O. Socio-Economic Factors in Level of Adoption of Risk Management Strategies in e-Banking Constraints Faced by Agro Entrepreneurs in Adopting Risk Management Strategies Constraints Faced by Agro Entrepreneurs in Adopting Risk Management Strategies charge banks with the responsibility of educating the customers, proper dispersal of knowledge on what these programs are, the risk involved and what methods can be used to combat them would be a bold step. It is also important for CBN toadopt a clear and relevant regulation that addresses electronic banking which will specify the need for banks to urgently attend to customers who are experiencing these risks. Adopting Risk Management Strategies The constraints faced by agro entrepreneurs is summarized in Table 5, with 79.7% of the respondents revealing that the length it takes for unsuccessful transfer to be returned back to their account is too long. About 63.3% said that ATMs are not really available in their locality and the ones available are always crowded during the day so they still result to going to the ATM when it’s lonely making them susceptible to this criminal. Whereas 49.9% of the respondent indicated that lack of information on what to do in times of risk is their issues, this is due to the lack of awareness banks do not create for their customers. Only 12.7% of the respondents said that there’s no protection of their privacy in the network services, that’s why hackers can get access to their details which they use in sending spam messages. REFERENCES Abdul G.W. and Muhammad R. (2013). An opportunities and constraints in expending e-banking in developing countries. Interdisciplinary Journal of Contemporary Research in Business March, 4 (11), 11-12 Adewuyi I.D. (2011). Electronic banking in Nigeria: challenges of the regulatory authorities and the way forward. International Journal of Economic Development Research and Investment, 2 (1), 149-156 National Population Commission (2006). Nigeria 2006 Population Census Arranged by State. National Population Commission, Abuja, Nigeria Adewuyi I.D. (2011). Electronic banking in Nigeria: challenges of the regulatory authorities and the way forward. International Journal of Economic Development Research and Investment, 2 (1), 149-156 p , National Population Commission (2006). Nigeria 2006 Population Census Arranged by State. National Population Commission, Abuja, Nigeria Socio-Economic Factors in Level of Adoption of Risk Management Strategies in e-Banking Values in parenthesis are the t-values 24 Table 5: Constraints faced by agro-entrepreneurs in adopting risk management strategies in electronic banking Constraints Frequency Percentage My bank doesn’t always respond on time to issues concerning these risk 55 69.6 I do not have proper information from the bank or any other medium on how to go about these risks 39 49.9 The ATM are not really available in my locality and the ones that are available are usually crowded during the day, so I still result to waiting for odd hours before making withdrawal 50 63.3 I cannot operate most of these devices, making me always at risk since I call on people to help me use them 26 32.9 I can barely differentiate my banks email fromthese hackers because the bank do not have any uniqueness distinguishing theirs 11 13.9 There’s no protection of customers privacy in our network providers because I think most of the time my details are gotten by their negligence not mine 10 12.7 Most of these fake websites are linked to my bank websites, which makes me always trust and fall for them without knowing 10 12.7 The security breach are usually as a result of the banks fault thereby exposing my details 10 12.7 It takes long for unsuccessful fund transfer to be returned 63 79.7 Field Survey (2018) Determinants of E-Banking Risk Management by Agro-Entrepreneurs in Delta State, Nigeria 24 nants of E-Banking Risk Management by Agro-Entrepreneurs in Delta State, Nigeria CONCLUSION Electronic banking though very convenient has so much associated risks. Managing these risks is pertinent to effective use of electronic banking products. Findings from the study showed that that gender, age, marital status, level of education, farming status are the main factors determining level of adoption of risk management strategies in electronic banking among agro entrepreneur in my study area. Constraints faced by agro entrepreneur in adopting these risk management strategies includes the length it takes for unsuccessful funds transfer to be returned, the delayed response of banks to issues relating to electronic banking risks, the crowdedness of ATM available leading to their inability to access as at when due. The study recommends that Central Bank of Nigeria should Parisa A. (2006). Adoption of e-Banking Services by Iranian Customers. Unpublished MSc Thesis. Lulea University of Technology, Division of Industrial Marketing and E-Commerce Solanki V.S. (2012). Risks in e-banking and their management. International Journal of Marketing, Financial Services & Management Research, 1 (3), 20-23 Ugwuja V.C., Dickson-Johnson P.M. and Familusi L.C (2017). Determinants of adoption level of risk management strategies by fish farmers in Degema Local Government Area of Rivers State, Nigeria. Agro-Science, 16 (2), 31-37 Yousafzai S., Pallister J. and Foxall G. (2003). A proposed model of e-trust for electronic banking. Technovation, 23 (11), 847-860
https://openalex.org/W1576964147	http://uu.diva-portal.org/smash/get/diva2:1508378/FULLTEXT01	English	null	It is time to classify biological aging as a disease	Frontiers in genetics	2,015	cc-by	3,916	Edited by: Alex Zhavoronkov, The Biogerontology Research Foundation, UK Reviewed by: Joao Pedro De Magalhaes, University of Liverpool, UK Correspondence: Sven Bulterijs, sven.bulterijs@heales.org Reviewed by: Joao Pedro De Magalhaes, University of Liverpool, UK Correspondence: Sven Bulterijs, sven.bulterijs@heales.org Reviewed by: Joao Pedro De Magalhaes, University of Liverpool, UK History of Disease Deﬁnitions What is considered to be normal and what is considered to be diseased is strongly inﬂuenced by historical context (Moody, 2001/2002). Matters once considered to be diseases are no longer classiﬁed as such. For example, when black slaves ran away from plantations they were labeled to suﬀer from drapetomania and medical treatment was used to try to “cure” them (Reznek, 1987). Similarly, masturbation was seen as a disease and treated with treatments such as cutting away the clitoris or cauterizing it (Reznek, 1987). Finally, homosexuality was considered a disease as recently as 1974 (Reznek, 1987). In addition to the social and cultural inﬂuence on disease deﬁnition, new scientiﬁc and medical discoveries lead to the revision of what is a disease and what is not (Butler, 2008). For example, fever was once seen as a disease in its own right but the realization that diﬀerent underlying causes would lead to the appearance of fever changed its status from disease to symptom (Reznek, 1987). Conversely, several currently recognized diseases, such as osteoporosis, isolated systolic hypertension, and senile Alzheimer’s disease, were in the past ascribed to normal aging (Izaks and Westendorp, 2003; Gems, 2011). Osteoporosis was only oﬃcially recognized as a disease in 1994 by the World Health Organization (WHO, 1994). It is time to classify biological aging as a disease 1 Faculty of Science, Ghent University, Ghent, Belgium, 2 Heales vzw, Brussels, Belgium, 3 Biochemistry Department, University of Oxford, Oxford, UK, 4 The Biogerontology Research Foundation, London, UK, 5 Institutionen för Biologisk Grundutbildning, Uppsala University, Uppsala, Sweden, 6 Institute for Translational Medicine, School of Science, University of Buckingham, Buckingham, UK Keywords: aging, nosology, disease, medicine, philosophy of medicine, multisystemic disease, disease complex Current Deﬁnitions of Disease Disease is a complex phenomenon and a current deﬁnition must consider both a biological and social explanation. The medical deﬁnition of disease is any abnormality of bodily structure or function, other than those arising directly from physical injury; the latter, however, may open the way for disease (Marcovitch, 2009). The disorder has a speciﬁc cause and recognizable signs and symptoms, and can aﬀect humans, other animals, and plants (Martin, 2010). The social aspect of disease is signiﬁcant when trying to divide a line between a healthy and a pathological state. This is a highly context and value driven process and, considering the WHO deﬁnition of health as a “state of complete physical, mental, and social well-being and not merely the absence of disease or inﬁrmity,” it is not as simple as classifying disease as the opposite of health (WHO, 1946). “Someone starving to death is not taken to have a disease, but is still not considered healthy” (Reznek, 1987). Specialty section: This article was submitted to Genetics of Aging, a section of the journal Frontiers in Genetics Specialty section: This article was submitted to Genetics of Aging, a section of the journal Frontiers in Genetics Received: 02 April 2015 Accepted: 25 May 2015 Published: 18 June 2015 Received: 02 April 2015 Accepted: 25 May 2015 Published: 18 June 2015 Reznek (1987) shows that diseases do not constitute a “natural kind.” If diseases were to share a particular real essence in virtue of which they are all diseases, an unidentiﬁed disease could be classiﬁed simply by comparing it to an already known pathology and seeing if it fell into the same natural kind as, for example, tuberculosis or cancer. What is a disease and what is not is thus something we invent to create a classiﬁcation of medically-treatable conditions. In many cases, diseases also seem to lack a nominal sense, as there are no necessary and suﬃcient conditions OPINION published: 18 June 2015 doi: 10.3389/fgene.2015.00205 Is Aging a Disease? The vast majority of animal species undergo the process of aging. Whilst aging is a nearly universal occurrence, it should be noted that other medical problems such as muscle wastage leading to sarcopenia, reduction in bone mass and density leading to osteoporosis, increased arterial hardening resulting in hypertension, atherosclerosis, and brain tissue atrophy resulting in dementia, all of which are nearly universal in humans, are classiﬁed as diseases in need of medical interventions (Bierman, 1985; WHO, 1994; Izaks and Westendorp, 2003; Gems, 2011). Also, autopsy studies indicate that amyloidosis may be almost universal in elderly people (Blumenthal, 2002) and, in autopsies performed by the Supercentenarian Research Foundation (SRF), amyloidosis has been identiﬁed as the cause of death in about 70% of people over 110 years of age (Coles and Young, 2012). Should we remove amyloidosis from medical textbooks as an age-related disease just because it happens to occur in almost every elderly subject? It should be noted that some have indeed raised the question if senile amyloidosis should be seen as a disease (Blumenthal, 1995). David Gems notes that while aging is universal this fact does not exclude aging from being a disease but rather means that aging is a “special form of disease” (Gems, 2011). Even if one is to continue with the reasoning that aging is a natural process, this does not mean that because disease is seen as a transgression from a natural, normal state, aging should fall outside the scope of medicine. Other natural processes such as pregnancy, cosmetic issues and the like, although not deﬁned as diseases, are accepted targets for medical intervention such as contraception, in vitro fertilization and cosmetic surgery (Boorse, 1975). Traditionally, aging has been viewed as a natural process and consequently not a disease (Callahan and Topinkova, 1998; Hayﬂick, 2007). This division may have, in part, originated as a way of establishing aging as an independent discipline of research (Blumenthal, 2003). Some authors go as far as to create a division between intrinsic aging processes (termed primary aging) and diseases of old age (termed secondary aging) (Hazzard, 2001). For example, photoaging, the accelerated deterioration of skin as a result of UV rays during one’s lifetime, is considered by dermatologists as a condition leading to pathology (Rabe et al., 2006). In contrast, chronological skin aging is accepted as the norm. Citation: Bulterijs S, Hull RS, Björk VCE and Roy AG (2015) It is time to classify biological aging as a disease. Front. Genet. 6:205. doi: 10.3389/fgene.2015.00205 June 2015 \| Volume 6 \| Article 205 Frontiers in Genetics \| www.frontiersin.org 1 Biological aging is a disease Bulterijs et al. or below 85/55 is abnormal and a sign of disease (WHO, 2015). The stratiﬁcation of reference ranges for age is needed to distinguish fully developed adults from still developing children. This avoids, for example, the lack of sperm production in human male neonates being classiﬁed as a disease (Boorse, 1977). In contrast, stratifying elderly from younger aged adults is not based on any good biological argument but instead masks aging as separate from disease, despite it being apparent that aging represents a deviation of the more desired state of youthful physical and mental capacities (Callahan and Topinkova, 1998). Whilst a statement such as this could be considered ageist, such a conception is based on the misunderstanding of what is meant by youthful. Aging as the passage of time and the accumulation of wisdom is not undesirable; the physiological decline that accompanies the process, however, most certainly is (Mackey, 2003). that they must have to qualify as diseases, rather than as separate pathological conditions such as injuries, disabilities, and deformities. Despite it being diﬃcult to deﬁne disease as a whole, we can simplify our deﬁnitions of disease and our approaches to their treatment by grouping particular pathologies together. For example, cholera, tuberculosis, and pneumonia are all separate diseases in their own right but can be collectively treated as bacterial diseases. The case is similar for HIV, inﬂuenza, and measles—all individual impairments to a normal state of health, yet grouped together as viral diseases. Having these classes of disease makes initial strategies for treatment far simpler, which, above all, is the main reason for having a deﬁnition(s) of disease in the ﬁrst place. Could we not take the same approach and add in a further group to treat the aging process as a disease in itself? Frontiers in Genetics \| www.frontiersin.org Is Aging a Disease? Thus, someone with a blood pressure of below 120/80 is seen as normal while a blood pressure above 140/90 June 2015 \| Volume 6 \| Article 205 Frontiers in Genetics \| www.frontiersin.org 2 Biological aging is a disease Bulterijs et al. health and lifespan of model organisms, from worms and ﬂies, to rodents and ﬁsh. We can now consistently improve the lifespan of C. elegans by more than ten-fold (Ayyadevara et al., 2008), more than double the lifespan of ﬂies and mice (Bartke et al., 2001; Sun et al., 2002), and improve the lifespan of rats and killiﬁsh by 30 and 59%, respectively (Valenzano et al., 2006; Zha et al., 2008) (see Figure 1 and Supplementary Table 1). Currently, our treatment options for the underlying processes of aging in humans are limited. However, with current progress in the development of geroprotective drugs, regenerative medicine, and precision medicine interventions, we will soon have the potential to slow down aging (Bulterijs, 2011, 2012). Finally, we should note that recognizing aging as a disease would shift anti-aging therapies from the Federal Drug Administration’s (FDA) regulations for cosmetic medicine to the more rigorous regulations for disease treatment and prevention (Gems, 2011). The ﬂuidity of what constitutes a disease and what is normal has recently been illustrated by the declaration by the American Medical Association House of Delegates that obesity is a disease (American Medical Association, 2013). Obesity, just like aging, does not comply with the traditional deﬁning characteristics of disease. But yet to target an issue such as obesity, categorizing it as a disease makes the road to developing treatment a much easier one to take. As aging appropriately ﬁts the deﬁnition of disease, there is a shifting consensus that aging should be seen as a disease process in itself, and not a benign progression of age that increases the risk of disease. Is Aging a Disease? As well as being seen as separate from disease, aging is looked at as a risk factor for developing disease (Hayﬂick, 2002; Collier et al., 2011; Niccoli and Partridge, 2012). Interestingly, the so-called “accelerated aging diseases” such as Hutchinson- Gilford Progeria Syndrome, Werner syndrome, and Dyskeratosis Congenita are considered diseases. Progeria is considered a disease but yet when the same changes happen to an individual 80 years older they are considered normal and unworthy of medical attention (Caplan, 2005). The reason commonly cited against classifying aging as a disease is that it constitutes a natural and universal process, while diseases are seen as deviations from the normal state (Caplan, 1992). The distinction between natural and unnatural may depend on the notion of design, purpose, and function (Hausman and Kennedy, 1975; Becker and Becker, 2001). Evolutionary theory of aging teaches us that aging is caused by the decrease in the force of selection against alleles with deleterious eﬀects later in life (Williams, 1957). Aging is thus the consequence of evolutionary neglect, not evolutionary intent (Olshansky et al., 2002). If aging serves no purpose then the notion of aging as a natural process might be mistaken (Caplan, 2005). While most still seem to consider aging not to be a disease others have started to question this position. Some have argued that aging should be considered a disease (Caplan, 1992; Gems, 2003, 2011), a syndrome (Esser and Keller, 1976) or a “disease complex” (Perlman, 1953, 2003). Whilst many aging researchers have openly declared that the universality of the aging process means it is not a disease, aging ﬁts the given medical deﬁnition of a disease. There is no disputing the fact that aging is a “harmful abnormality of bodily structure and function.” What is becoming increasingly clear is that aging also has speciﬁc causes, each of which can be reduced to a cellular and molecular level, and recognizable signs and symptoms (López-Otín et al., 2013). Additionally, normal in a medical context is generally deﬁned as no deviation outside of the normal reference range for that age and sex, whilst diseases are seen as deviation from this normal condition for that age and sex (Boorse, 1975; Assaf et al., 2010). The Beneﬁts of Labeling Aging as a Disease Callahan and Topinkova (1998) write: “In short, not only does aging lend itself to be characterized as a disease, but the advantage of doing so is that, by rejecting the seeming fatalism of the label “natural,” it better legitimizes medical eﬀorts to either eliminate it or get rid of those undesirable conditions associated with it.” The goal of biomedical research is to allow people to be “as healthy as possible for as long as possible” (de Magalhães, 2014). Having aging recognized as a disease would stimulate grant-awarding bodies to increase funding for aging research and develop biomedical procedures to slow the aging process (Kelland, 2010). Indeed, Engelhardt states that calling something a disease involves the commitment to medical intervention (Engelhardt, 1975). Furthermore, having a condition recognized as a disease is important to have treatment refunded by health insurance providers (Reznek, 1987). References 7.M433 López-Otín, C., Blasco, M. A., Partridge, L., Serrano, M., and Kroemer, G. (2013). The hallmarks of aging. Cell 153, 1194–1217. doi: 10.1016/j.cell.2013. 05.039 Blumenthal, H. T. (2003). The aging-disease dichotomy: true or false. J. Gerontol. Med. Sci. 58A, 138–145. doi: 10.1093/gerona/58.2.M138 Mackey, T. (2003). An ethical assessment of anti-aging medicine. J. Anti Aging Med. 6, 187–204. doi: 10.1089/109454503322733045 Marcovitch, H. (2009). Black’s Medical Dictionary, 42nd Edn. London: A&C Black. Boorse, C. (1975). On the distinction between illness and disease. Philos. Public Aﬀ. 5, 49–68. Marcovitch, H. (2009). Black’s Medical Dictionary, 42nd E Martin, E. A. (2010). Concise Medical Dictionary, 8th Edn. Oxford: Oxford University Press. Boorse, C. (1977). Health as a theoretical concept. Philos. Sci. 44, 542–573. Moody, H. R. (2001/2002). Who’s afraid of life extension? dy, H. R. (2001/2002). Who’s afraid of life extension? Generations Bulterijs, S. (2011). Metformin as a geroprotector. Rejuvenation Res. 14, 469–482. doi: 10.1089/rej.2011.1153 Neuberg, G. W. (2009). The cost of end-of-life care: a new eﬃciency measure falls short of AHA/ACC standards. Circ. Cardiovasc. Qual. Outcomes 2, 127–133. doi: 10.1161/CIRCOUTCOMES.108.829960 Bulterijs,S. (2012). The future of longevity. Pan Eur. Netw. 5, 116–117. Butler, R. (2008). The Longevity Revolution: The Beneﬁts and Challenges of Living a Long Life. New York, NY: PublicAﬀairs. Niccoli, T., and Partridge, L. (2012). Ageing as a risk factor for disease. Curr. Biol. 22, R741–R752. doi: 10.1016/j.cub.2012.07.024 Callahan, D., and Topinkova, E. (1998). Is aging a preventable or curable disease. Drugs Aging 13, 93–97. doi: 10.2165/00002512-199813020-00001 Olshansky, S. J., Hayﬂick, L., and Carnes, B. A. (2002). Position statement on human aging. J Gerontol. A. Biol. Sci. Med. Sci. 57, B292–B297. doi: 10.1093/gerona/57.8.B292 Caplan, A. L. (1992). If I Were a Rich Man could I Buy a Pancreas? and Other Essays on the Ethics of Health Care. Bloomington, IL: Indiana University Press. Perlman, R. M. (1953). The aging syndrome. J. Am. Geriatr. Soc. 2, 123–129. Caplan, A. L. (2005). Death as an unnatural process. Why is it wrong to seek a cure for ageing? EMBO Rep. 5, S72–S75. doi: 10.1038/sj.embor. 7400435 Perlman, R. M. (2003). Commemorating the 50th anniversary of JAGS: the aging syndrome. 1953. J. Am. Geriatr. Soc. 51, 558–561. doi: 10.1046/j.1532- 5415.2003.51167.x Coles, L. S., and Young, R. D. (2012). Supercentenarians and transthyretin amyloidosis: the next frontier of human life extension. Prev. Med. 54, S9–S11. doi: 10.1016/j.ypmed.2012.03.003 Rabe, J. H., Mamelak, A. 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We would like to thank Adam Summerﬁeld, Alex Zerox, Ethan Sarnoski, and Conclusion We believe that aging should be seen as a disease, albeit as a disease that is a universal and multisystemic process. Our current healthcare system doesn’t recognize the aging process as the underlying cause for the chronic diseases aﬀecting the elderly. As such, the system is setup to be reactionary and therefore about 32% of total Medicare spending in the United States goes to the last 2 years of life of patients with chronic illnesses, without any signiﬁcant improvement to their quality of life (Cooper, 1996; Neuberg, 2009). Our current healthcare system is untenable both from a ﬁnancial and health and well-being prospective. Even minimal attenuation of the aging process by accelerating research on aging, and development of geroprotective drugs and During the last 25 years, by targeting the underlying processes of aging biomedical scientists have been able to improve the FIGURE 1 \| The methuselahs in lab. The increase in maximum lifespan in the laboratory was obtained in 5 animal species, both without any interventions, and by dietary, chemical, or genetic interventions. For each organism the impact of increase in maximum lifespan through intervention is indicated in the graph using fold change. interventions. For each organism the impact of increase in maximum lifespan through intervention is indicated in the graph using fold change. FIGURE 1 \| The methuselahs in lab. The increase in maximum lifespan in the laboratory was obtained in 5 animal species, both without any interventions, and by dietary, chemical, or genetic June 2015 \| Volume 6 \| Article 205 Frontiers in Genetics \| www.frontiersin.org 3 Biological aging is a disease Bulterijs et al. Mallory E. McLaren for their critical reading of the manuscript and useful suggestions. regenerative medicines, can greatly improve the health and well- being of older individuals, and rescue our failing healthcare system. Mallory E. McLaren for their critical reading of the manuscript and useful suggestions. regenerative medicines, can greatly improve the health and well- being of older individuals, and rescue our failing healthcare system. References I., and Sauder, D. N. (2006). Photoaging: mechanisms and repair. J. Am. Acad. Dermatol. 55, 1–19. doi: 10.1016/j.jaad.2005.05.010 Collier, T. J., Kanaan, N. M., and Kordower, J. H. (2011). Ageing as a primary risk factor for Parkinson’s disease: evidence from studies of non-human primates. Nat. Rev. Neurosci. 12, 359–366. doi: 10.1038/nrn3039 Reznek, L. (1987). The Nature of Disease. London: Routledge & Kegan Paul. Sun, J., Folk, D., Bradley, T. J., and Tower, J. (2002). Induced overexpression of mitochondrial Mn-superoxide dismutase extends the life span of adult Drosophila melanogaster. Genetics 161, 661–672. Cooper, M. M. (1996). The Dartmouth Atlas of Health Care. Chicago, IL: American Hospital Publishing. de Magalhães, J. P. (2014). The scientiﬁc quest for lasting youth: prospects for curing aging. Rejuvenation Res. 17, 458–467. doi: 10.1089/rej. 2014.1580 Valenzano, D. R., Terzibasi, E., Genade, T., Cattaneo, A., Domenici, L., and Cellerino, A. (2006). Resveratrol prolongs lifespan and retards the onset of age-related markers in a short-lived vertebrate. Curr. Biol. 16, 296–300. doi: 10.1016/j.cub.2005.12.038 Engelhardt, H. T. Jr. (1975). “The concepts of health and disease,” in Evaluation and Explanation in the Biomedical Sciences, eds H. T. Jr. Engelhardt and T. S. F. Spicker (Dordrecht: Reidel), 125–141. WHO. (1946). Preamble to the Constitution of the World Health Organization. New York, NY: WHO. June 2015 \| Volume 6 \| Article 205 4 Frontiers in Genetics \| www.frontiersin.org Bulterijs et al. Biological aging is a disease WHO. (1994). Assessment of Fracture Risk and its Application to Screening for Postmenopausal Osteoporosis. WHO Technical Report Series 843. Geneva: WHO. Conﬂict of Interest Statement: The Editor Zhavoronkov declares that, despite being aﬃliated to the same institution as the authors Raphaella S. Hull and Avi G. Roy, the review process was handled objectively and no conﬂict of interest exists. The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. WHO. (2015). Blood Pressure. Available online at: http://www.who.int/gho/ncd/ risk_factors/blood_pressure_prevalence/en/ Williams, G. C. (1957). Pleiotropy, natural selection and the evolution of senescence. Evolution 11, 398–411. doi: 10.2307/ 2406060 Copyright © 2015 Bulterijs, Hull, Björk and Roy. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Genetics \| www.frontiersin.org WHO. (1994). Assessment of Fracture Risk and its Application to Screening for Postmenopausal Osteoporosis. WHO Technical Report Series 843. Geneva: WHO. June 2015 \| Volume 6 \| Article 205 WHO. (2015). Blood Pressure. Available online at: http://www.who.int/gho/ncd/ risk_factors/blood_pressure_prevalence/en/ References The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Zha, Y., Taguchi, T., Nazneen, A., Shimokawa, I., Higami, Y., and Razzaque, M. S. (2008). Genetic suppression of GH-IGF-1 activity, combined with lifelong caloric restriction, prevents age-related renal damage and prolongs the life span in rats. Am. J. Nephrol. 28, 755–764. doi: 10.1159/0001 28607 June 2015 \| Volume 6 \| Article 205 Frontiers in Genetics \| www.frontiersin.org 5
https://openalex.org/W4388691595	https://www.researchsquare.com/article/rs-3600790/latest.pdf	English	null	Gastric microecology changes in sepsis patients with gastrointestinal bleeding: a report of 2 cases and literature review	Research Square (Research Square)	2,023	cc-by	3,661	Gastric microecology changes in sepsis patients with gastrointestinal bleeding: a report of 2 cases and literature review Fangjie Fu West China Hospital of Sichuan University Chen Zhang West China Hospital of Sichuan University Zhengying Xu West China Hospital of Sichuan University Peng Ji West China Hospital of Sichuan University Zhongwei Zhang (  716461751@qq.com ) West China Hospital of Sichuan University Case Report Posted Date: November 15th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3600790/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Page 1/9 Page 1/9 Abstract Sepsis, characterized by life-threatening organ dysfunction resulting from an uncontrolled response to infection, can impact various systems of the body, including the digestive system. Prior research has identified sepsis as a significant risk factor for gastrointestinal bleeding. However, there is limited reporting on the gastric microecology of individuals with sepsis complicated by gastrointestinal bleeding. This paper presents the cases of two patients, shedding light on this issue. The first case was a 29-year- old female who developed sepsis during perioperative liver transplantation, while the second case features a 34-year-old female with acute pancreatitis complicated by septic shock. Both patients underwent gastroscopy following gastrointestinal bleeding, revealing evident gastric mucosal injuries. Notably, the second patient exhibited suppurative gastritis. Metagenomic Next-Generation Sequencing (NGS) of gastric juice from these two patients unveiled microecological alterations in the stomach. The sequencing results indicated a substantial presence of pathogenic sequences, underscoring the role of direct gastric mucosal injury due to infection as a significant contributor to gastrointestinal bleeding. This study not only introduces a novel approach to pinpoint the causes of gastrointestinal bleeding in sepsis but also provides valuable insights for clinical diagnosis and treatment. Background Sepsis is a critical medical condition characterized by life-threatening organ dysfunction resulting from the uncontrolled immune response to infection. It can lead to multiple organ dysfunction syndrome, posing a substantial risk to the lives of critically ill patients [1, 2]. In individuals with sepsis, the combination of inflammatory reactions and oxidative stress often leads to gastrointestinal mucosal ischemia, thereby impacting the functionality of the digestive system. Extensive research has established sepsis as a significant risk factor for gastrointestinal bleeding. Nevertheless, there is a notable scarcity of reports focusing on the alterations in gastric microecology among septic patients who develop gastrointestinal bleeding. This paper present detailed insights into the gastric microecology and the diagnostic and treatment procedures applied to two patients afflicted by sepsis complicated with gastrointestinal bleeding. This contribution aims to enhance our understanding of the underlying causes of gastrointestinal bleeding in such cases and offers valuable guidance for the adjustment of treatment strategies. Case 1 A 34-year-old female was admitted to the hospital due to a history of "abnormal liver function for over nine years." The elevated liver transaminase levels had been detected more than nine years ago, and a liver biopsy had confirmed the presence of autoimmune cirrhosis. The patient had previously undergone esophageal variceal ligation, splenectomy, and pericardial devascularization. Upon admission, the patient was conscious, exhibiting moderate jaundice in the skin and sclera, along with slight abdominal distension. No other significant abnormalities were noted during the physical Page 2/9 examination. Initial laboratory results showed a hemoglobin level of 91 g/L, a total bilirubin concentration of 263 µmol/L, a prothrombin time of 16.2s, and an activated partial thromboplastin time of 39s. Abdominal contrast-enhanced CT scans revealed the presence of liver cirrhosis, ascites, and signs of portal hypertension with collateral circulation. Furthermore, a liver biopsy confirmed the diagnosis of nodular cirrhosis with intrahepatic cholestasis. Gastroscopy upon admission revealed mild esophageal varices, along with gastric mucosal congestion and edema (Fig. 1A). The primary admission diagnosis: 1. Decompensated stage of liver cirrhosis (Child-Pugh B); 2. Portal hypertension with esophageal varices. The patient underwent an allogeneic liver transplantation on September 4, 2022, following which she was transferred to the ICU. After surgery, the patient received mycophenolate mofetil and tacrolimus to modulate immune function. Despite these interventions, the bilirubin levels remained elevated after the procedure. Subsequently, percutaneous transhepatic biliary drainage was performed. Eleven days post-operation, the patient had a high fever (39 ℃) with abdominal distension. Blood cultures identified the presence of Enterococcus faecium and Candida albicans, leading to a diagnosis of sepsis. The anti-infective regimen was promptly adjusted to vancomycin, cefoperazone sodium, and sulbactam sodium. Fifteen days following the transplantation, the patient experienced acute gastrointestinal bleeding, characterized by a profusion of dark stools and decreased blood pressure. Immediate interventions included fluid resuscitation, blood transfusion, and intravenous use of proton pump inhibitors. A subsequent gastroscopy revealed multiple extensive ulcers in the gastric body and pronounced exfoliation-like alterations in the gastric mucosa (Fig. 1B). Unfortunately, the patient's gastroscopy showed that the lesions of the stomach body were significantly worse than those on admission. Gastric fluid analysis reported a pH level of 6.0. Both next-generation sequencing (NGS) and culture analysis of the gastric fluid disclosed the presence of Klebsiella aerogenes and Pseudomonas aeruginosa, which correlated with the findings from blood cultures. Case 1 In response to these results, the anti-infection regimen was further adjusted to colistin sulfate, cefoperazone sodium sulbactam sodium, caspofungin, and hemostatic therapy involving the administration of human fibrinogen, human prothrombin complex, fresh frozen plasma, and transfusion of red blood cell suspension. Tragically, two days later, the patient's condition deteriorated drastically, resulting in severe gastrointestinal hemorrhage, hypovolemic shock, disseminated intravascular coagulation (DIC), and ultimately, respiratory and cardiac arrest. Despite extensive rescue efforts, the patient succumbed to her condition. Case 2 P 3 9 A 29-year-old female patient was admitted to the hospital with a complaint of "abdominal pain persisting for 20 days, dyspnea for one day." The patient had experienced sustained epigastric colic for the preceding 20 days, starting at the 39 + 1 week of her gestation period, which was accompanied by bouts of vomiting. Upon examination, an emergency ultrasound indicated pancreatic enlargement, and laboratory tests revealed a significant elevation in blood amylase and lipase levels. Consequently, a diagnosis of acute severe pancreatitis was established, leading to the performance of an emergency Page 3/9 Page 3/9 cesarean section. Postoperative blood culture and abdominal drainage identified the presence of carbapenem-resistant Acinetobacter baumannii. Despite initial medical management, the patient's condition worsened, manifesting as aggravated dyspnea. She was subsequently transferred to our hospital following endotracheal intubation. Importantly, the patient had no history of digestive tract disorders. cesarean section. Postoperative blood culture and abdominal drainage identified the presence of carbapenem-resistant Acinetobacter baumannii. Despite initial medical management, the patient's condition worsened, manifesting as aggravated dyspnea. She was subsequently transferred to our hospital following endotracheal intubation. Importantly, the patient had no history of digestive tract disorders. Upon admission, the patient was in a state of analgesia and sedation. Wet rales could be heard in both lungs. The whole abdomen is distended and tender, and 6 drainage tubes can be seen in the abdomen. Laboratory tests were as follows: hemoglobin 70 g/L, platelet count 18*109/L, prothrombin time 17.7 s, activated partial thrombin time 48.7 s, creatinine 316 µmol/L, lipase 399 IU/L, and pancreatic amylase 92 IU/L. Abdominal enhanced CT: Pancreatic swelling, extensive peripancreatic exudation, peritonitis signs, omentum and mesangial swelling, gastrointestinal wall swelling, stratified enhancement (Fig. 2A). The primary diagnoses upon admission included acute pancreatitis, septic shock, and multiple organ dysfunction syndrome, which encompassed the heart, liver, and coagulation systems. After admission, the patient was subjected to a regimen that included the administration of tigecycline and colistin sulfate for anti-infective purposes, intravenous noradrenaline to raise blood pressure and other supportive treatments. Over the course of her hospitalization, the patient underwent blood culture and abdominal drainage culture assessments. It was also observed that a small amount of brown drainage fluid could be seen in the gastrointestinal decompression unit. Case 2 On the third day following her admission, gastroscopy revealed ulcers and numerous dark-brown protuberances in the middle curvature of the gastric body and across the mucosa from the anterior wall to the antrum, indicative of ulcerated gastric mucosa and purulent secretions. This presentation was consistent with suppurative gastritis (Fig. 2B). Gastric fluid analysis reported a pH level of 6.5. Gastric fluid samples were sent to NGS and culture testing. Subsequent findings indicated the presence of Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Acinetobacter baumannii, Saccharomyces cerevisiae, Aspergillus fumigatus, and Candida albicans. On the sixth day, culture results from the abdominal drainage fluid indicated Candida albicans, while blood cultures identified Pseudomonas aeruginosa. Simultaneously, gastric juice cultures revealed the presence of Stenotrophomonas maltophilia and Candida albicans. In response to these findings, voriconazole antifungal therapy was initiated, accompanied by component blood transfusions. Gastric fluid analysis reported a pH level of 6.5. Gastric fluid samples were sent to NGS and culture testing. Subsequent findings indicated the presence of Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Acinetobacter baumannii, Saccharomyces cerevisiae, Aspergillus fumigatus, and Candida albicans. On the sixth day, culture results from the abdominal drainage fluid indicated Candida albicans, while blood cultures identified Pseudomonas aeruginosa. Simultaneously, gastric juice cultures revealed the presence of Stenotrophomonas maltophilia and Candida albicans. In response to these findings, voriconazole antifungal therapy was initiated, accompanied by component blood transfusions. Unfortunately, despite these efforts, there was no significant improvement in the patient's hemodynamic status. Her condition continued to deteriorate, characterized by progressive gastrointestinal bleeding and multiorgan dysfunction. Tragically, the patient's clinical state failed to respond to rescue efforts and was ultimately declared deceased Unfortunately, despite these efforts, there was no significant improvement in the patient's hemodynamic status. Her condition continued to deteriorate, characterized by progressive gastrointestinal bleeding and multiorgan dysfunction. Tragically, the patient's clinical state failed to respond to rescue efforts and was ultimately declared deceased. Discussion Sepsis is a complex condition that can affect multiple systems in the body, often leading to the development of multiple organ dysfunction syndrome. The gastrointestinal tract, encompassing the Page 4/9 Page 4/9 esophagus, stomach, duodenum, small intestine, and colon, plays a crucial role in maintaining a dynamic balance between epithelial tissue, the microbiota, and the immune system. This balance contributes to various essential functions, including nutrient absorption, immune regulation, and defence against pathogen invasion [3]. Although previous research has established sepsis as a high-risk factor for gastrointestinal stress ulcer bleeding, there is a paucity of studies examining changes in gastric microecology in patients experiencing sepsis complicated by gastrointestinal bleeding. In a healthy physiological state, the stomach undertakes the initial steps of food digestion, creating a unique internal environment and microecology. Conventionally, the acidic nature of gastric juice and the presence of digestive enzymes were thought to be inhospitable to most microorganisms entering the digestive system, with only specific microorganisms able to survive [4]. Studies have identified common gastric bacteria, including thick-walled bacteria, Bacteroides, Proteus, Lactobacillus, Streptococcus, which show significant correlation with gastric acid secretion [5]. Dietary and environmental factors further influence the composition of gastric microorganisms [6]. In a healthy physiological state, the stomach undertakes the initial steps of food digestion, creating a unique internal environment and microecology. Conventionally, the acidic nature of gastric juice and the presence of digestive enzymes were thought to be inhospitable to most microorganisms entering the digestive system, with only specific microorganisms able to survive [4]. Studies have identified common gastric bacteria, including thick-walled bacteria, Bacteroides, Proteus, Lactobacillus, Streptococcus, which show significant correlation with gastric acid secretion [5]. Dietary and environmental factors further influence the composition of gastric microorganisms [6]. Patients with sepsis frequently experience excessive systemic inflammatory responses and oxidative stress, leading to gastrointestinal mucosal ischemia, which in turn impairs secretory and immune barrier functions. Notably, the prophylactic use of proton pump inhibitors (PPIs) in the intensive care unit (ICU) has gained prominence in recent years. However, recent research has shown no significant clinical differences in gastrointestinal bleeding incidence between sepsis patients who received prophylactic PPIs and those who did not [7]. Furthermore, the significance of gastric acid in immune responses and nutrient absorption has become more apparent, underlining the need for a judicious evaluation of PPI use, considering both indications and treatment duration for patients [8]. Discussion Both patients in this case report were administered proton pump inhibitors, which may further disrupt the acidic gastric environment and lead to alterations in the gastric microecology. Animal studies have indicated that in a mouse model with impaired gastric acid secretion and increased gastric juice pH, the colonization and copy number of pathogens like Yersinia and Salmonella significantly increased in the stomach [9]. In vitro experiments have demonstrated that low pH could effectively inhibit the accumulation and invasion of Klebsiella pneumoniae in intestinal mucosal epithelial cells [10]. However, methods for assessing gastric microecological composition remain limited, and traditional gastric juice cultures have constraints in terms of sensitivity, specificity, timeliness, and depth of information. In the case of our first patient, who developed sepsis complicated by gastrointestinal bleeding, gastroscopy revealed multiple large ulcers and extensive mucosal exfoliation, markedly different from the typical erosion seen in stress ulcers. Moreover, gastric juice pH was elevated beyond normal levels. As a response, we conducted a gastric juice NGS test to explore infection-related factors, which yielded a substantial quantity of pathogen sequences. Some of these pathogens corresponded with the findings of blood culture, suggesting the possibility of blood-borne transmission leading to gastric mucosal infection. This highlights that gastric mucosal damage directly caused by changes in the gastric environment and infection can be a contributory factor to gastrointestinal bleeding in sepsis patients. Page 5/9 The second patient, suffering from acute pancreatitis and septic shock, had no prior history of digestive tract disease. Abdominal CT scans revealed pronounced swelling and stratified enhancement of the gastrointestinal wall, while gastroscopy revealed ulcers of varying sizes and numerous dark-brown protuberances, indicative of suppurative gastritis. Suppurative gastritis is a rare occurrence in patients with septic gastrointestinal bleeding, with the capacity to infiltrate the gastric mucosa, submucosal structures, and even the lamina propria, carrying a mortality rate as high as 27% [11]. The treatment of suppurative gastritis hinges on prompt diagnosis and the administration of broad-spectrum antibiotics [12]. In this case, the pronounced gastric mucosal injury resulted from infection. The pathological changes associated with gastric mucosal infection in this patient might stem from blood-borne transmission or direct invasion related to abdominal infection. Gastrointestinal bleeding in sepsis patients can arise from various sources. One source could be blood- borne transmission, as evidenced by the NGS results aligning with blood culture outcomes. Another potential origin may be gastrointestinal colonization or translocation of intestinal flora. Ethics approval and Consent to Participate Ethics approval and Consent to Participate Not applicable. Acknowledgment None. Availability of Data and Materials The datasets generated and/or analysed during the current study are not publicly available due to concerns regarding patient privacy, but are available from the corresponding author on reasonable request. Competing Interests The authors declare that they have no competing interests. Discussion Moreover, invasive procedures, such as the indwelling of gastric tubes during hospitalization, could serve as vectors for pathogen transmission. Although this study did not engage in a comparative analysis of pathogenic microorganisms in different regions, the findings from blood culture and gastric juice NGS in the two patients indicate that blood infection might contribute to gastric mucosal lesions in these cases. In the study of gastrointestinal dysfunction caused by sepsis, most investigations center on intestinal mucosal dysfunction. The mechanisms underlying intestinal dysfunction entail intestinal cell apoptosis, an exaggerated inflammatory response, oxidative stress, and mitochondrial dysfunction [13, 14]. This interplay leads to the rupture of the intestinal epithelial cell membrane, intercellular junction damage, and compromised mucosal barrier function [15]. Whether these mechanisms similarly apply to gastric mucosal injury in sepsis needs further examination. In this study, we have innovatively employed gastric juice NGS to comprehensively elucidate the stomach's microecology, confirming that infection-induced gastric mucosal injury plays a significant role in gastrointestinal bleeding. This approach provides a novel methodology for determining the causes of septic gastrointestinal bleeding. In clinical practice, patients presenting with septic gastrointestinal bleeding can benefit from gastric juice pH monitoring, gastric juice culture, and gastric juice NGS examination. These tools can be instrumental in characterizing alterations in gastric microecology, helping to find the causes of bleeding and enabling timely treatment adjustments. As a result, this study offers a vital reference for comprehending the origins of gastrointestinal bleeding in these patients. Figure 1 Gastroscopic results of the first case. A. gastric mucosal congestion and edema. B. multiple huge ulcers in the gastric body, extensive exfoliation-like changes in the gastric mucosa. Figure 2 Results of CT and gastroscopy in the second patient. A. gastrointestinal wall swelling, with stratified enhancement. B. ulcers and a large number of dark-brown protuberances in the middle curvature of the gastric body. Page 6/9 Page 6/9 Funding This case report did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authors' Contribution Study concept and design: FJF, ZWZ (corresponding author); Acquisition of data: CZ, ZYX, FJF, PJ; Analysis and interpretation of data: ZWZ (corresponding author); Drafting and editing of the manuscript: FJF; Critical revision of the manuscript for important intellectual content: PJ, ZWZ; All authors read and approved the final manuscript. Consent for Publication Written informed consent to publish the report was obtained from the patient’s legal guardians. Funding References Influence of gastric acid on susceptibility to infection with ingested bacterial pathogens. Infect Immun. 2008;76(2):639–45. 10. Liu J, Zhang S, Pei H, et al. Klebsiella pneumoniae activates the TGF-β signaling pathway to adhere to and invade intestinal epithelial cells via enhancing TLL1 expression. Int J Med Microbiol. 2022;312(6):151561. 11. Dayu Shi J, He M, Lv, et al. Phlegmonous gastritis in a patient with mixed-phenotype acute leukemia in the neutropenia phase during chemotherapy: A case report. Med (Baltim). 2019;98(45):e17777. 12. Kato K, Tominaga K, Sugimori S, et al. Successful Treatment of Early-Diagnosed Primary Phlegmonous Gastritis. Intern Med. 2015;54(22):2863–6. 12. Kato K, Tominaga K, Sugimori S, et al. Successful Treatment of Early-Diagnosed Primary Phlegmonous Gastritis. Intern Med. 2015;54(22):2863–6. 13. Patra MC, Shah M, Choi S. Toll-like receptor-induced cytokines as immunotherapeutic targets in cancers and autoimmune diseases. Semin Cancer Biol 64, 61–82. 13. Patra MC, Shah M, Choi S. Toll-like receptor-induced cytokines as immunotherapeutic targets in cancers and autoimmune diseases. Semin Cancer Biol 64, 61–82. 14. Chakraborty FHS, Halbgebauer R, et al. Challenge to the intestinal mucosa during sepsis. Front Immunol. 2019;10:891. 14. Chakraborty FHS, Halbgebauer R, et al. Challenge to the intestinal mucosa during sepsis. Front Immunol. 2019;10:891. 15. Susana Lechuga, Andrei I, Ivanov. Disruption of the epithelial barrier during intestinal inflammation: Quest for new molecules and mechanisms. Biochim Biophys Acta Mol Cell Res. . 2017;1864(7):1183–94. 15. Susana Lechuga, Andrei I, Ivanov. Disruption of the epithelial barrier during intestinal inflammation: Quest for new molecules and mechanisms. Biochim Biophys Acta Mol Cell Res. . 2017;1864(7):1183–94. Figures References 1. Phillips MS-HGS, Levy ML, et al. Developing a New Definition and Assessing New Clinical Criteria for Septic Shock: For the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis- 3). JAMA. 2016;315(8):775–87. 1. Phillips MS-HGS, Levy ML, et al. Developing a New Definition and Assessing New Clinical Criteria for Septic Shock: For the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis- 3). JAMA. 2016;315(8):775–87. 2. Rudd KE, Johnson SC, Agesa KM, et al. Global, regional, and national sepsis incidence and mortality, 1990–2017: analysis for the Global Burden of Disease Study. Lancet. 2020;395:200–11. 2. Rudd KE, Johnson SC, Agesa KM, et al. Global, regional, and national sepsis incidence and mortality, 1990–2017: analysis for the Global Burden of Disease Study. Lancet. 2020;395:200–11. Page 7/9 3. Katherine T, Fay ML, Ford, Craig M, Coopersmith. The intestinal microenvironment in sepsis. Acta Mol basis Dis. 2017;1863(10):2574–83. 4. Zhang C, Powell SE, Betel D, et al. The Gastric Microbiome and Its Influence on Gastric Carcinogenesis: Current Knowledge and Ongoing Research. Hematol Oncol Clin North Am. 2017;31(3):389–408. 5. Nardone G, Compare D, Rocco A. A microbiota-centric view of diseases of the upper gastrointestinal tract [J]. Lancet Gastroenterol Hepatol. 2017;2(4):298–312. 5. Nardone G, Compare D, Rocco A. A microbiota-centric view of diseases of the upper gastrointestinal tract [J]. Lancet Gastroenterol Hepatol. 2017;2(4):298–312. 6. Ruijing Y, Guo Y, Gong Q, et al. Microbiological evidences for gastric cardiac microflora dysbiosis inducing the progression of inflammation. J Gastroenterol Hepatol. 2020;35(6):1032–41. 6. Ruijing Y, Guo Y, Gong Q, et al. Microbiological evidences for gastric cardiac microflora dysbiosis inducing the progression of inflammation. J Gastroenterol Hepatol. 2020;35(6):1032–41. 7. Huang M, Han M, Song Z, et al. Stress ulcer prophylaxis in critically ill adult patients with sepsis at risk of gastrointestinal bleeding: a retrospective cohort study. Intern Med J. 2023;53(3):389–96. 7. Huang M, Han M, Song Z, et al. Stress ulcer prophylaxis in critically ill adult patients with sepsis at risk of gastrointestinal bleeding: a retrospective cohort study. Intern Med J. 2023;53(3):389–96. 8. Andrew T, Costarino D, Dai R, Feng, et al. Gastric Acid Suppressant Prophylaxis in Pediatric Intensive Care: Current Practice as Reflected in a Large Administrative Database. Pediatr Crit Care Med. 2015;16(7):605–12. 9. Sharon M, Tennant EL, Hartland T, Phumoonna, et al. Influence of gastric acid on susceptibility to infection with ingested bacterial pathogens. Infect Immun. 2008;76(2):639–45. 9. Sharon M, Tennant EL, Hartland T, Phumoonna, et al. Figures Page 8/9 Figure 1 Gastroscopic results of the first case. A. gastric mucosal congestion and edema. B. multiple huge ulcers in the gastric body, extensive exfoliation-like changes in the gastric mucosa. Gastroscopic results of the first case. A. gastric mucosal congestion and edema. B. multiple huge ulcers in the gastric body, extensive exfoliation-like changes in the gastric mucosa. Figure 2 Results of CT and gastroscopy in the second patient. A. gastrointestinal wall swelling, with stratified enhancement. B. ulcers and a large number of dark-brown protuberances in the middle curvature of the gastric body. Figure 2 Figure 2 Results of CT and gastroscopy in the second patient. A. gastrointestinal wall swelling, with stratified enhancement. B. ulcers and a large number of dark-brown protuberances in the middle curvature of the gastric body. Page 9/9 Page 9/9
https://openalex.org/W2365254742	https://www.scienceopen.com/document_file/a63da52f-7cf1-4af3-87fe-70e0042027fe/ScienceOpen/statecrime.2.1.0030.web.pdf	English	null	Crowdsourcing Research: A Methodology for Investigating State Crime	State crime	2,013	cc-by	7,051	CROWDSOURCING RESEARCH: A METHODOLOGY FOR INVESTIGATING STATE CRIME Christopher Williams Abstract: “Crowdsourcing” now gets 10 million hits on Google, and is being applied to research in commercial, media, academic, civil society and state spheres. Although appearing recent and technology-based, there are also relevant but overlooked manual precursors which embody the fundamentals of using large groups for research. This analytical review provides the bases for developing initiatives further, by assessing: What are the strategies, strengths and weaknesses of crowdsourcing research? What are the questions that should be asked when planning a research design? How is crowdsourcing being applied in relation to state crime, and why? What might be the implications for justice systems, and for criminal and international courts? crowdsourcing research? What are the questions that should be asked when planning a research design? How is crowdsourcing being applied in relation to state crime, and why? What might be the implications for justice systems, and for criminal and international courts? Keywords: crowdsourcing; research; methodology; state crime; accountability; abuse of power <RXDUHWKH:HDUHWKHELOOLRQ (Protest slogan, Washington, 2008) &KULVWRSKHU:LOOLDPVLVD9LVLWLQJ)HOORZDWWKH,QVWLWXWHRI(GXFDWLRQ8QLYHUVLW\RI/RQGRQ Introduction – Crowds +LVWRULFDOO\WKHFURZGZDVSHUFHLYHGE\HOLWHVDVDWKUHDW:LOOLDPV But the internet revealed the potential of the crowd as a resource, and Surowiecki’s The Wisdom of Crowds argued the value of crowd perspectives (2004). The word ³FURZGVRXUFLQJ´ZDVFRLQHGE\-HII+RZHDZULWHUIRUWiredPDJD]LQHLQ DQGFRPPHUFLDOUHVHDUFKLVDQDVSHFWD'H¿QLWLRQVDUHHYROYLQJDQGIURP DUHYLHZ(VWHOOpV$URODVDQGRQ]iOH]/DGUyQGHXHYDUDSURSRVHWKDW Crowdsourcing is a type of participative online activity in which an individual, an institution, a non-proﬁt organization, or company proposes to a group of individuals of varying knowledge, heterogeneity, and number, via a ﬂexible open call, the voluntary undertaking of a task… The media, academics, civil society organizations, and the state also started to deploy crowds to do research on a scale that was previously impossible. Terms STATE CRIME 2.1 SPRING 2013 CROWDSOURCING RESEARCH 31 VXFKDV³GLVWULEXWHGKXPDQFRPSXWDWLRQ'+&´³VRFLDOFRPSXWLQJ´³FROOHFWLYH intelligence”, and “citizen science” were related to crowdsourcing (Quinn and Bederson 2011). In this article, “crowdsourcing research” is assumed to entail an organizing research entity that outsources research tasks, which that entity could not achieve alone, to large groups of self-selected people (lay and expert). URXSVPLJKWFRQWULEXWHWKURXJKXQGHUWDNLQJUHVHDUFKWDVNVGDWDFROOHFWLRQGDWD management, evidence testing, analysis), the development of relevant technologies, being a participant in a mass study (experiments, opinion polls, user surveys), or E\WKHGLVVHPLQDWLRQRI¿QGLQJV&URZGVRXUFLQJUHSUHVHQWVZKDWPHWKRGRORJ\ books term a “framework” (Beissel-Durrant 2004), within which any appropriate SULPDU\RUVHFRQGDU\GDWDFROOHFWLRQPHWKRGVFDQEHXVHG:LOOLDPV The purpose of this analytical review is to assess examples of crowdsourcing research, consolidate an academic perspective, and consider how it can be applied WRWKHLQYHVWLJDWLRQRIVWDWHFULPHUHHQDQG:DUG6WDWHFULPHUHVHDUFKHUV are likely to face distinct methodological challenges, which crowdsourced research might address. These are primarily about the size and power of the state versus that of traditional researchers. States can GHSOR\ODUJHVFDOHUHVRXUFHV to perpetrate and cover-up crime; GHSOR\ODUJHVFDOHUHVRXUFHV to perpetrate and cover-up crime; DFFHVVDZLGHVSHFWUXPRIWHFKQLFDOH[SHUWLVH to analyse socio-political situations; DFFHVV, or PDNHLQDFFHVVLEOH, dangerous, remote or controversial settings; ZRUN ZLWK VLJQL¿FDQW QDWLRQDO DQG LQWHUQDWLRQDO QHWZRUNV; DFFHVV, or PDNHLQDFFHVVLEOH, dangerous, remote or controversial settings; DFFHVV, or PDNHLQDFFHVVLEOH, dangerous, remote or controversial settings; ZRUNZLWKVLJQL¿FDQWQDWLRQDODQGLQWHUQDWLRQDOQHWZRUNV; ZRUNZLWKVLJQL¿FDQWQDWLRQDODQGLQWHUQDWLRQDOQHWZRUNV; FRPPXQLFDWH on a mass scale. Potentially crowdsourced research can start to match and sometimes surpass state dynamics through Potentially crowdsourced research can start to match and sometimes surpass state dynamics through WKHXVHRIPDVVYROXQWHHUV globally, expert and lay (e.g. professional networks, members of amateur organizations); WHFKQRORJLFDODPSOL¿FDWLRQ from local to global scale (e.g. social networking and mass surveillance); ÀH[LELOLW\, because crowdsourced research is not encumbered by traditional systems and ways of thinking (e.g. Introduction – Crowds crowdsourced research can operate during parliamentary holidays, and on the basis of intellectual or moral arguments QRWMXVWODZSROLF\DQG³DFFHSWHG´SUDFWLFH OLQNLQJ mass methodologies (e.g. testimony, documentary, satellite imagery). But perhaps most important is the degree of public empathy and legitimacy that FURZGVRXUFHGQRQVWDWHDFWRUVFDQQRZFUHDWH(YHQWKHRUHWLFDOO\LOOHJDODFWVVXFK But perhaps most important is the degree of public empathy and legitimacy that FURZGVRXUFHGQRQVWDWHDFWRUVFDQQRZFUHDWH(YHQWKHRUHWLFDOO\LOOHJDODFWVVXFK Produced and distributed by Pluto Journals www.plutojournals.com/scj/ Produced and distributed by Pluto Journals www.plutojournals.com/scj/ CHRISTOPHER WILLIAMS 32 as those behind WikiLeaks, DUHTXLFNO\YLHZHGE\PDQ\DVUHÀHFWLQJDSXEOLFLQWHUHVW that may override traditional state law. 7KHPHWKRGRORJ\RIWKHDUWLFOHLVVWUDLJKWIRUZDUG7KH:LNLSHGLD³/LVWRI FURZGVRXUFLQJSURMHFWV´:LNLZDVXVHGDVWKHEDVLVIRULQGXFWLYHDQDO\VLV WRLGHQWLI\DQGGHYHORSFRQFHSWXDOIUDPHZRUNV6SHFL¿FVHDUFKHVRIWKHPHGLD provided examples of the application of crowdsourcing techniques to state crime UHVHDUFKDQGGHWDLOVZHUHFKHFNHGYLDHPDLOV0DQXDOWHFKQLTXHVZHUHLGHQWL¿HG WKURXJKVWDQGDUGOLEUDU\VHDUFKHV0XFKRIWKHLQIRUPDWLRQXVHGKHUHFRPHVIURP novel sources that are transient, guarded, and sometimes muddled. Therefore inevitably there will be details that later require amendment. The main ethical FRQVLGHUDWLRQVIRUDQ\GLVFXVVLRQRIWKLVQDWXUHDUHWRDYRLGSURYLGLQJVSHFL¿FGHWDLOV that may (i) cause morally-sound initiatives to be subverted, or (ii) cause personal or professional harm to those organizing and participating in these initiatives. Only websites directly relevant to state crime research have been fully referenced. Other sites mentioned can be located by searching on the italicized names. The structure of the discussion is to contrast the differences between manual and technological crowdsourcing, from a non-technical perspective. Past and Present Applications of Crowdsourcing Research Although crowdsourcing appears recent, its precursors predate electricity. These PDQXDOWHFKQLTXHV remain relevant, because they embody the transferrable IXQGDPHQWDOV2QHRIWKHPRVWVLJQL¿FDQWRXWFRPHVRIHDUO\FURZGVRXUFLQJLVWKH 2[IRUG(QJOLVK'LFWLRQDU\2(')URPDURXQGYROXQWHHUVZHUHJLYHQ particular texts which they scan read, and they then copied relevant quotations onto slips. Their endeavours eventually amounted to two tons of paper (Winchester 1998). 0XVHXPVDQGOLEUDULHVSURYLGHGHDUO\PRGHOVIRUFURZGVRXUFLQJUHVHDUFK7KH\ frequently created their collections from donations by countless enthusiasts who WUDYHOOHGFROOHFWLQJREMHFWVVSHFLPHQVDQGERRNV/LVWVRIWKH³¿HOGFROOHFWRUV´ZKR created the Pitt Rivers Museum, in Oxford, described helpers as “army”, “medics”, “religious”, “anthropologists” and “archaeologists”, and many of these were FDWHJRUL]HGDV³DPDWHXU´350&ROOHFWRUVODEHOOHGDQQRWDWHGDQGLQGH[HG WKHREMHFWVDQGH[SHUWFXUDWRUVWKHQV\VWHPL]HGWKHVHFURZG¿QGLQJVWRGHYHORSFRQ- ceptualizations such as chieftaincy, dynasties, or belief systems. In parallel, artists VWDUWHGWRVSHFLDOL]HLQGUDZLQJVFLHQWL¿FLPDJHVRIDQWKURSRORJLFDODQGRWKHUIRUPV of data that could not be transported easily. Subsequently, collectors have compiled VLJQL¿FDQWGHWDLOVDERXWVWDWHFULPHWKURXJKLPDJHV±SURSDJDQGDOHDÀHWV1'68 2012; Bytwerk 2012), political posters (Sarhandi 2001), and photos of propaganda PXUDOV+&/&ROOHFWLRQVZHUHDOVRLQWULQVLFDOO\DPHDQVRIGLVVHPLQDWLQJ UHVHDUFK¿QGLQJV0DGDPH7XVVDXG¶VZDVDQHDUO\H[DPSOH07,QPRGHUQ STATE CRIME 2.1 SPRING 2013 STATE CRIME 2.1 SPRING 2013 CROWDSOURCING RESEARCH Produced and distributed by Pluto Journals www.plutojournals.com/scj/ CROWDSOURCING RESEARCH 33 form, the Not to forget Museum of Saddam Hussein Regime CrimesLQ.XZDLW continues the tradition of using animated tableaus and crowdsourced artefacts to perpetuate awareness of the atrocities (Williams 2012: 4). form, the Not to forget Museum of Saddam Hussein Regime CrimesLQ.XZDLW continues the tradition of using animated tableaus and crowdsourced artefacts to perpetuate awareness of the atrocities (Williams 2012: 4). ,QWHUPVRIVWDWHFULPHDQLQWHUHVWLQJSUHFXUVRULVPXFKHDUOLHU,Q)R[H¶V Book of Martyrs documented “persecutions and horrible troubles” perpetrated by the “Roman Prelates”. Foxe starts with a clear research question: “What force…of princes, kings, monarchs, governors, and rulers of this world…with all their strength DQGFXQQLQJKDYHEHQWWKHPVHOYHVDJDLQVWWKLV>3URWHVWDQW@FKXUFK"´[OLY Foxe included testimony evidence from “both men and wemen, whiche can and do EHDUHZ\WQHV´LQFOXGLQJSULVRQHUV.LQJ6RRQWKH³WHVWLPRQLHV poured in on Foxe…as people sought to exonerate themselves and accuse or eulogise RWKHUV´)UHHPDQ7KHERRNJUHZIURPWRRYHUSDJHVDQGVL[W\ ZRRGFXWVEHFDPHRQHKXQGUHGDQG¿IW\)R[H¶VVWUDWHJ\ZDVWRJHQHUDWHIXUWKHU HYLGHQFHLW³H[HPSOL¿HVWKHFRQWLQXDWLRQRIPDQXVFULSWFLUFXODWLRQDQGVFULEDO publication after the advent of printing… a collective process of writing, copying, and circulation of manuscripts as a means by which prisoners in extremis…attempted WRVXVWDLQDQGFRPIRUWWKHLUFRUHOLJLRQLVWV´.LQJ7KHUHVXOWLVVLPLODU to present-day reports of human rights abuses from organizations such as Amnesty International and Human Rights Watch. The involvement of victims also represents a form of victim agency, which can itself be therapeutic (USDJ 1998). A novel form of international survey was developed by the Scottish anthropologist, James Frazer, for his The Golden Bough (1890), which investigated instances of violent practices from around the world. Yet he had only travelled to Italy and UHHFH+LVPHWKRGZDVVLPSOHEXWHIIHFWLYH,QDGGLWLRQWRGRFXPHQWDU\VRXUFHVKH VHQWDQGJDYHTXHVWLRQQDLUHVWRPLVVLRQDULHVGRFWRUVDQGFRORQLDORI¿FLDOVLQPDQ\ SODFHVLQFOXGLQJ-DSDQ&KLQD$IULFDDQGWKH$PHULFDV)UDVHU+HWDONVRI ³SULQWHGTXHVWLRQVVFDWWHUHGEURDGFDVWRYHU(XURSH´)UD]HUL[/LNH)R[H the work was ongoing, and one volume became thirteen. In modern terminology, he used social networks for a longitudinal snowball survey. Professional networks can also be used to demonstrate state abuse of power. For \HDUV(J\SWLDQSV\FKLDWULVW%DVPD$EGHOUDKPDQZRUNLQJDWWKHEl Nadeem Centre for torture victims, started to put red dots on a map, marking police stations where torture had reportedly happened. She initially thought the problem was caused E\DIHZEDGRI¿FHUV:LWKKHOSIURPFROOHDJXHVKHUPDSTXLFNO\¿OOHGZLWKGRWV DFURVVWKHFRXQWU\UHFRUGLQJWKHW\SHRILQMXULHVDQGWRUWXUH,WEHFDPHFOHDUWKDW the same methods and “appliances” were being used, which she concluded showed ³V\VWHPDWLFSUDFWLFH´DQGD³SROLF\´GLFWDWHGE\VHQLRUVWDWHRI¿FLDOV%%& (O1DGHHP&HQWUH 2EMHFWVRIWHQSURYLGHEHWWHUHYLGHQFHRIDWURFLWLHVWKDQUHFRUGVEXWV\VWHPDWLF collection and annotation is crucial, preferably by neutral parties. Careful collection Produced and distributed by Pluto Journals www.plutojournals.com/scj/ 34 CHRISTOPHER WILLIAMS of spent munitions in a war zone can evidence the use of illegal weapons such as cluster bombs and chemical weapons, and many munitions carry precise details DERXWWHFKQLFDOVSHFL¿FDWLRQVFRXQWU\RIRULJLQGDWHVRIPDQXIDFWXUHDQGEDWFK numbers, which can evidence systematic abuse of power. CROWDSOURCING RESEARCH Robert Fisk (2001) provides an example: Pieces of the US manufacturer’s [Lockheed] Hellﬁre air-to-ground missile lie in the local civil defence headquarters in Bethlehem… The missile engine, fuel pipe and shreds of the wiring system have been sorted into plastic bags by ambulance drivers and paramedics, alongside shrapnel from dozens of US-made fuses for shells ﬁred by Israeli tanks into the Christian village of Beit Jalla. The strength of professional networks is that they often parallel state structures – KHDOWKHGXFDWLRQMXVWLFH±DQGVRHYLGHQFHRIV\VWHPDWLFDEXVHEHFRPHVHDVLHUWR track. They are also likely to be seen as embodying a greater degree of integrity and neutrality than activists. 5HFRQVWUXFWLRQLVDVLJQL¿FDQWUHVHDUFKIUDPHZRUNIRULQYHVWLJDWLQJSRZHUIXO SHRSOHEHFDXVHWKH\RIWHQGHVWUR\HYLGHQFH:LOOLDPV7KH Iranian Revolution provides a striking example. When the Americans evacuated WKHLU(PEDVV\WKH\VKUHGGHGDOOVHQVLWLYHGRFXPHQWV%XWKXQGUHGVRIVWXGHQWV SODFHGWKHVKUHGVRQWKHÀRRUDQGQXPEHUHGDQGLQGH[HGWKHPPDQXDOO\7KH\ then brought in local carpet weavers to reassemble the pieces. The documents were SXEOLVKHGDVDERRN±³'RFXPHQWVIURPWKH86(VSLRQDJH'HQ´±DQGSXWRQVDOHWR WKHSXEOLF06)/,7KH\UHYHDOHGWKHFRQWHQWVRIWHOHJUDPVFRUUHVSRQGHQFH and reports from the American State Department and the CIA. Software is now available to reconstruct shredded documents, and has been used to reconstruct Stasi ¿OHVWKURZQLQWRJDUEDJHEDJVLQ+HLQJDUWQHU (DVW$VLDFRQWULEXWHVDGLPHQVLRQWKDWLVLQFRQWUDVWWRWKHPRUHIDPLOLDU short-term cycles of endeavour – staying-power and longitudinal crowdsourcing. An ongoing initiative in Japan, investigates the pollution from US nuclear testing on the Bikini Atoll. In 1955, the Japanese government colluded with the US to reach a “political settlement” and accept a token “consolation payment” in return for declaring the issue resolved (Namba 2011). But in 1985, a high school teacher, 0DVDWRVKL<DPDVKLWDFUHDWHGDJURXSFDOOHGWKH³+DWD+LJK6FKRRO6HPLQDU´ZKLFK IRUWHQ\HDUVLQWHUYLHZHG¿VKHUPHQZKRKDGEHHQH[SRVHGWRWKHUDGLDWLRQDQG mapped their stories together with family testimonies, secret government documents and evidence of how the US tried to obstruct news of the incident becoming public 0DVDPL$IWHU\HDUV<DPDVKLWDKDGLGHQWL¿HGDIIHFWHG¿VKLQJ boats. With help from the Peace Studies Association of Japan, the study of the ³+LGGHQ+LEDNXVKD´FRQWLQXHVIXQGHGE\DFLWL]HQVFLHQFHIRXQGDWLRQWKHTakagi STATE CRIME 2.1 SPRING 2013 STATE CRIME 2.1 SPRING 2013 CROWDSOURCING RESEARCH Produced and distributed by Pluto Journals www.plutojournals.com/scj/ CROWDSOURCING RESEARCH 35 Fund (2012), which aims to “sow the seeds of hope into the hearts of people, RUJDQL]HSHRSOHDQGJHQHUDWHDÀRZOHDGLQJWRUHYROXWLRQDU\FKDQJH´ Fund (2012), which aims to “sow the seeds of hope into the hearts of people, RUJDQL]HSHRSOHDQGJHQHUDWHDÀRZOHDGLQJWRUHYROXWLRQDU\FKDQJH´ RUJDQL]HSHRSOHDQGJHQHUDWHDÀRZOHDGLQJWRUHYROXWLRQDU\FKDQJH´ The internet has permitted an exponential increase in the scope of WHFKQRORJLFDO WHFKQLTXHV for crowdsourced research. Numerous software tools are becoming available to assist (Crowdsourcing.org 2012). For example, the Amazon platform M h i l T k K E W O W L L + , W OOL 7 N +,7 Mechanical TurkKDVEHHQFHQWUDOWRRUJDQL]LQJ+XPDQ,QWHOOLJHQFH7DVNV+, ±WDVNVWKDWFRPSXWHUVFDQQRWGRHI¿FLHQWO\VXFKDVDQQRWDWLQJSKRWRV7KHZRUG processing interface Soylent permits the use of Turk to slice-and-dice tasks into a series of generation and review stages among large groups of writers and editors (Bernstein et al. 2010). CloudCrowd, chunks and orders tasks for crowd work, and similar software can facilitate user-evaluation surveys, for example about the quality RIDSLHFHRIZULWLQJ.LWWXUHWDO &RPPHUFLDO endeavours provide useful lessons for all forms of crowdsourced research. Crowdsourcing.orgSURYLGHVDOLVWRI³FDWHJRULHV´&ORXG/DERXU &URZG&UHDWLYLW\2SHQ,QQRYDWLRQ'LVWULEXWHG.QRZOHGJHDQG&URZGIXQGLQJ 0RWLYDWLRQLVDNH\FRQFHUQRIFRPPHUFLDOHQGHDYRXUV4XLQQDQG%HGHUVRQ 2011), which entails factors such as: Rewards, which can be monetary, public recognition as an excellent contributor, or through the fun of playing and winning a computer game for free. Enjoyment – Tasks can be intrinsically fun, or can be converted into computer games (below). 7KHWDVNEHLQJintegral to another activity. Quality is also a consideration, and can be achieved in a number of ways (Quinn and Bederson 2009, 2011). A “multilevel review” uses secondary groups to rate the work of primary groups. “Forced agreements” push contributors to agree an answer before submitting it. 0DQ\PHGLD organizations now make use of crowdsourced material. The BBC will often put a few lines of “breaking news” online, and ask people who are near WKHHYHQWWRVHQGLQPDWHULDOZKLFKLVWKHQYHUL¿HGRUSUHVHQWHGDVXQYHUL¿HG)RU established news stories, the public may be asked to post comments, which may KHOSHGLWRUVWRXQGHUVWDQGFRQÀLFWLQJYLHZSRLQWVRUGLVFRYHUQHZOHDGV2QDPRUH sophisticated level, SuperMapper has been used by the BBC for “rapid survey” maps which use postcodes to put data instantly onto a map, for example about SHRSOHUHSRUWLQJVSHFL¿FSUREOHPVLQDQHFRQRPLFFULVLV+XGVRQ6PLWK Some news outlets, such as Google News, are now not mediated by human editors. Produced and distributed by Pluto Journals www.plutojournals.com/scj/ CROWDSOURCING RESEARCH ,QFRQÀLFW]RQHVQHWZRUNVOLNHWKHPHGLDFHQWUHRIWKHRevolutionary Council in Syria, deploy complex networks of volunteers and professionals to get news out quickly through social networking sites such as )DFHERRN0&6<,WFDQ CHRISTOPHER WILLIAMS 36 then be picked up and disseminated by organizations such as Lawyers for Justice, RUJURXSVLQH[LOH(,([SHUWVVWUHVVWKDWLWLVLPSRUWDQWWKDWRULJLQDOYLGHR clips and photos are not edited, because they may eventually become valuable evidence in court cases, and editing will cast doubt on the evidence (ISCI 2012). 7KH8.QHZVSDSHUThe GuardianIRUPXODWHGDVLJQL¿FDQWH[SHULPHQWDERXWWKH XVHRIFURZGVRXUFLQJZKHQLWLQYHVWLJDWHGIUDXGXOHQW03H[SHQVHFODLPVXDUGLDQ 7KURXJK)UHHGRPRI,QIRUPDWLRQ)R,UHTXHVWVMRXUQDOLVWVDFTXLUHGFRSLHV RI03V¶FODLPVIRUPVDQGUHFHLSWV7KH\WKHQXVHGDVLPSOHIHHGEDFNLQWHUIDFH ('MDQGR) to deploy 25,000 people as a human search engine to scan-read 500,000 GRFXPHQWVIRUGXELRXVFODLPV0RUHWKDQGRFXPHQWVZHUHDQDO\VHGZLWKLQ KRXUV$VDUHVXOW03VZHUHFRQYLFWHGRIIUDXGUHVLJQHGRUKDGWRSURYLGH DSRORJLHV7KHVXFFHVVUHÀHFWHGPDNLQJSDUWLFLSDWLRQIXQ±LWZDVDFKDOOHQJH The Guardian¶VKRPHSDJHVWDWHG³:HKDYHSDJHVRIGRFXPHQWV RI\RXKDYHUHYLHZHGRIWKHP2QO\WRJR«´(DFKGRFXPHQW DSSHDUHGZLWKDVLPSOHUDWLQJER[KHDGHG³,VWKLVSDJHLQWHUHVWLQJ"6KRXOGZH LQYHVWLJDWHIXUWKHU"´ 1. 1RWLQWHUHVWLQJ, e.g. a cover sheet or stationery 2. ,QWHUHVWLQJEXWNQRZQ, e.g. it’s a duckhouse 3. ,QWHUHVWLQJ±,W¶VVLJQL¿FDQWH[SHQVHVGDWD 4. ,QYHVWLJDWHWKLV I would like to know more 3KRWRVRIWKH03VZHUHDGGHGWRWKHVLWHZKLFKFUHDWHGDUHDOLW\GLPHQVLRQDQG the best-performing volunteers were listed. Timing was crucial, and the venture ZDVODXQFKHGGXULQJZLGHVSUHDGPHGLDLQWHUHVWLQ03V¶H[SHQVHFODLPV7RFRSH with the surge of responses, the Amazon contact-hosting service EC2 was used (Andersen 2009). Two DFDGHPLF disciplines have shown a lead in crowdsourcing research – science (including medical) and history. Science initiatives assist with relevant technological developments, such as software design and forensic procedures, but also contribute an informed understanding of what crowds can do best. Science crowdsourcing is often framed as “citizen science” and “distributed communities”, and facilitated by initiatives such as the Citizen Science Alliance. Astronomers make prodigious use of crowdsourcing. The Planet HuntersSURMHFWVHDUFKHVLPDJHVIURPWKH.HSOHU space telescope. Although computers can do this, the systems are not error proof. 7KHVFLHQWLVWVFRQFOXGH³WKHKXPDQEUDLQZRXOGEHSDUWLFXODUO\JRRGDW¿QGLQJDQ\ weird or unusual systems, involving variable or double stars or multiple interacting SODQHWV´/LQWRWW³7KHKXPDQEUDLQLVSDUWLFXODUO\JRRGDWGLVFHUQLQJSDWWHUQV or aberrations and experiments have shown that when many people work together, WKHFROOHFWLYHZLVGRPRIWKHFURZGVFDQEHEHWWHUWKDQDQH[SHUW´3+ STATE CRIME 2.1 SPRING 2013 STATE CRIME 2.1 SPRING 2013 CROWDSOURCING RESEARCH CROWDSOURCING RESEARCH 37 Similarly, Galaxy ZooFRIRXQGHU.HYLQ6FKDZLQVNLFODLPV³7KHKXPDQEUDLQ is actually better than a computer at pattern recognition tasks like this” (Catone 7KLVVSHFL¿FDOO\KXPDQVNLOOLVLQFUHDVLQJO\XVHGWRLGHQWLI\UHOHYDQWVKDSHV in war zones (below). Similarly, Galaxy ZooFRIRXQGHU.HYLQ6FKDZLQVNLFODLPV³7KHKXPDQEUDLQ is actually better than a computer at pattern recognition tasks like this” (Catone 7KLVVSHFL¿FDOO\KXPDQVNLOOLVLQFUHDVLQJO\XVHGWRLGHQWLI\UHOHYDQWVKDSHV in war zones (below). +LVWRULDQVGHPRQVWUDWHWKDWVWDWHFRQGXFWSDVWDQGSUHVHQWLVLQFUHDVLQJO\HDV\WR GRFXPHQWDQGFRQWLQXHWKHPXVHXPWUDGLWLRQ7KH+RORFDXVWVHFWLRQRI/RQGRQ¶V Imperial War Museum is a pertinent example. The Great War Archive (OU 2012) created a website for digitized artefacts (photos and stories) relating to World War I collected from the general public. Europeana now houses the archive, together with others, for open source research. Using transcription and editing software such as CAPTCHA, the scale of achievements is remarkable. The &LYLO:DU'LDULHV Letters Transcription Project uses volunteers to transcribe and tag handwritten GRFXPHQWV&:'/73:KHQWKH/LEUDU\RI&RQJUHVVUHFHLYHGWKH/LOMHQTXLVW IDPLO\¶VFROOHFWLRQRISKRWRVRIWKH$PHULFDQ&LYLO:DUPRVWZHUHXQLGHQWL¿DEOH 7KH/LEUDU\SRVWHGWKHLPDJHVRQFlickr and asked the public to identify family members, regiments, or a photographer’s painted studio backdrop, and annotate the photos. The relevance of historical methods to state crime extends beyond academic LQWHUHVW+LVWRULFDOFURZGVRXUFHGUHVHDUFKFDQDOVRUHODWHWRJDWKHULQJHYLGHQFH DQGDFKLHYLQJMXVWLFHRYHUORQJWLPHIUDPHVIRUH[DPSOHWKURXJKDUFKLYDOZRUN IRUHQVLFLQYHVWLJDWLRQRIREMHFWVDQGOLIHKLVWRU\UHVHDUFKDPRQJHOGHUO\SHRSOH ,QFUHDVLQJO\VHQLRUJRYHUQPHQWRI¿FLDOVDUHDSSHDULQJLQFRXUWGHFDGHVDIWHUWKHLU PLVGHHGVZKHQYHU\HOGHUO\DQGHYLGHQFHLVKLVWRULFDO:LOOLDPV,Q DIRUPHUSUHVLGHQWRI7XUNH\.HQDQ(YUHQZDVSXWRQWULDODJHGWKLUW\ \HDUVDIWHUWKHFRXSZLWKZKLFKKHZDVLPSOLFDWHG9HOD &LYLOVRFLHW\ organizations now commonly use crowdsourced research. Ushahidi (Swahili for “testimony” or “witness”) is one of the more established organizations, ZKLFKZDVFUHDWHGIROORZLQJ.HQ\D¶VGLVSXWHGSUHVLGHQWLDOHOHFWLRQ,WFROOHFWHG eyewitness accounts of state violence, which were sent in by email and text-message DQGSODFHGRQDRRJOHPDS7KH86KDKLGLRSHQVRXUFHSODWIRUPKDVEHHQXVHGWR PDSFULVHVLQ+DLWL/LE\DDQG(J\SW,QLWLDWLYHVFDQDOVRUHVHDUFKVWDWHPLVGHHGVRQ DORFDOOHYHO3ROLFHVWRSDQGVHDUFKDFWLRQVLQ/RQGRQFDQEHGRFXPHQWHGWKURXJKD %ODFNEHUU\ app which permits the public to upload location details, police numbers, and what happened instantly on the stopandsearch.org website. 0DQ\LQLWLDWLYHVUHÀHFW³FLWL]HQVFLHQFH´DERYHGreenpeace Polluter Watch 33:JDWKHUHGGRFXPHQWVDERXW%3RLOSROOXWLRQLQWKHXOIRI0H[LFR through Freedom of Information (FoI) requests, and 30,000 emails, draft reports and recorded conversations were eventually obtained and archived, “so that members RIWKHSXEOLFFDQSHUIRUPWKHLURZQUHVHDUFKDQGÀDJDQ\GRFXPHQWVWKDWVKRZ evidence of misconduct”. The public was also assisted to use FoI systems to “Submit \RXURZQUHTXHVW´DQGWKHQWRSRVWQHZGRFXPHQWV(QYLURQPHQWDODFWLYLVWVLQ CHRISTOPHER WILLIAMS 38 China, such as those of the ,QVWLWXWHRI3XEOLFDQG(QYLURQPHQWDO$IIDLUV, have ERXJKWDLUTXDOLW\PRQLWRUVDQGSODFHGWKHPLQSROOXWHGDUHDVRI%HLMLQJ6KDQJKDL XDQ]KRXDQG:HQ]KRX7KHVHLQLWLDWLYHVSDUDOOHOJRYHUQPHQWGDWDFROOHFWLRQ methods, and the citizen data is then put on the internet. This forced the government to make its own data available, which created the chance to analyse discrepancies /D)UDQLHUH%XWLQLWLDWLYHVGRQRWQHHGWREHKLJKWHFKRUUHVWULFWHGWR adults. :RUOG:DWHU0RQLWRULQJ'D\organizes around 340,000 young people in FRXQWULHVWRWHVWWKHTXDOLW\RIZDWHUZD\VDQGVKDUH¿QGLQJV::0' T \ \ J Initiatives concerning child victims may eventually set the most interesting SUHFHGHQWVEHFDXVHWKHLUYLFWLPVWDWXVLVXVXDOO\KDUGWRGLVSXWH7KH¿UVWFRQYLFWLRQ by the International Criminal Court (ICC) concerned child soldiers. Waging Peace has submitted 500 children’s drawings to the ICC as “contextual evidence” of YLROHQFHLQ'DUIXU:3$PHULFDQVXUYLYRUVRIDEXVHE\&DWKROLFSULHVWV work on the site ELVKRSVDFFRXQWDELOLW\RUJ to gather and manage evidence. Applying social network analysis (Williams 2012: 149), volunteers are creating “assignment records” for every priest accused of sexual abuse since 1940. Produced and distributed by Pluto Journals www.plutojournals.com/scj/ CROWDSOURCING RESEARCH Using diverse sources, including obituaries, these list “posts held”, “networks”, “contacts” and details such as “prolonged sick leave”. There is a photo gallery, which volunteers link to annotated reports of abuse. In 2011, on behalf of the Survivors Network of those $EXVHGE\3ULHVWV (SNAP), the Centre for Constitutional Rights¿OHGDFDVHZLWKWKH ,&&DJDLQVW3RSH%HQHGLFW;9,DQGRWKHUVHQLRURI¿FLDOVRIWKH9DWLFDQ6WDWHZKLFK is supported by 20,000 pages of “evidence of the widespread and systematic basis of sexual violence in the Church” (CCR 2011). (This seems strangely reminiscent of Foxe and the “horrible troubles” perpetrated by “Roman Prelates”.) To exploit crowd analysis, sites such as Iraq Body Count (IBC 2012) and WikiLeaks (2012) provide a mass of data which researchers can freely use. :LNL/HDNV¶ content can be downloaded in a single archive, and parts are distributed on printed copies (BBC 2010), which helps to ensure that it would survive attempts to destroy the main site. WikiLeaks proposes a simple methodology for accessing and analysis of its online data. 6HDUFKIRUHYHQWV\RXUHPHPEHUWKDWKDSSHQHGIRUH[DPSOHLQ\RXUFRXQWU\ %URZVHE\GDWHRUVHDUFKIRUDQRULJLQQHDU\RX 3LFNRXWLQWHUHVWLQJHYHQWVDQGWHOORWKHUVDERXWWKHP 8VHWZLWWHUUHGGLWPDLOZKDWHYHUVXLWV\RXUDXGLHQFHEHVW Although :LNL/HDNV¶strategies were seen by some people as unethical and even unlawful, once in the public domain, data have been widely used, for example by D/OR\G¶V,QVXUDQFHV\QGLFDWHIRUDFRXUWFDVH0LOPR2QDORFDOOHYHO LQHRUJLDTransparency International (TI) is trying to make documents publicly STATE CRIME 2.1 SPRING 2013 STATE CRIME 2.1 SPRING 2013 CROWDSOURCING RESEARCH CROWDSOURCING RESEARCH 39 DYDLODEOHIRUVFUXWLQ\DERXWWKHFRVWVRIWKHUHQRYDWLRQRIWKH0D\RU¶VRI¿FH “to provide an environment in which all interested parties might participate and contribute equally in the process of monitoring public funds” (TI 2012a). Satellite-based sites are burgeoning, and software such as &HUEHUXV can be used WRWUDQVODWHSKRWRJUDSKLFVDWHOOLWHGDWDLQWRXVDEOH,6GDWDWKURXJKDJDPHLQ which players need to mark interesting features. Satellite initiatives are being used WRPRQLWRUFRUUXSWLRQ,Q0DOD\VLDTransparency International (TI 2012b) is using 1,000 volunteers for its Forest Watch Project, to monitor illegal activity using Google EarthDQGDHULDOSKRWRJUDSK\6LPLODUSURMHFWVDUHRSHUDWLQJLQ,QGRQHVLD 3DSXD1HZXLQHD&KLQDDQGWKH6RORPRQ,VODQGV2WKHUVLPSOHVLWHVDUHUHYHDOLQJ unknown state activity. Secret Bases (Turnbull 2012), uses media reports, maps, DHULDOSKRWRVDQGRRJOH(DUWKWRVKRZDFWLYLWLHVRIWKH%ULWLVKLQWHOOLJHQFHVHUYLFHV US Amnesty International$,86KDVEHHQRSHUDWLQJVDWHOOLWHVXUYHLOODQFHSURMHFWV concerning Sudan and Somalia. Amnesty Nigeria uses a similar mapping strategy to QRWHKXPDQULJKWVFRQFHUQVDVSDUWRILWV³1LJHULD$PQHVW\'LDORJ´SURMHFW$,1 9ROXQWHHUVWDJODUJHDPRXQWVRIVDWHOOLWHGDWDZKLFKKDVEHHQVOLFHGDQG diced for individual tasks. The Sentinel Project for Genocide Prevention started in 2008 and uses volunteers ZKRFRPPLWWHQKRXUVHDFKZHHN6336LPLODUO\WKHSatellite Sentinel ProjectVHWXSZLWKKHOSIURPHRUJH&ORRQH\LQDLPHGWRGHWHUFLYLOZDU between Northern and Southern Sudan, by recording threats to civilians and human rights abuses. 'LJLWDOOREH satellites are used for commissioning photographs, for example of troop movements, which are then analysed by the Harvard Humanitarian Initiative2QWKHJURXQGUHSRUWVRIERGLHVEHLQJEXULHGKDYHEHHQYHUL¿HGWKURXJK aerial photos, and the area then scanned for similar happenings. The public is invited to improve maps using Google Map Maker, and to add landmarks and vulnerable sites such as schools and hospitals (SSP 2010). The Syria Tracker Crisis Map combined data-mining and crowdsourced human intelligence to map human rights violations and killings (Syria Tracker 2012). Data could be logged on the site, emailed, tweeted with the tag EDVKDUFULPHV, RUSKRQHGLQWRDRRJOHSpeak2Tweet service which automatically posts voice messages on twitter. It is categorized in terms of: refugees, killed, missing/detained, IRRGWDPSHULQJZDWHUWDPSHULQJ9DOLGLW\LVFKHFNHGE\YROXQWHHUVIURPWKH 6WDQGE\7DVN)RUFH(SBTF) who score-up or score-down reports of suspicious activities – including “large military equipment”, “large crowds”, “checkpoints” – and triangulate with other sources (iRevolution 2012). Using Tommod, volunteers tag features that are hard for a computer to recognize, such as: “Burnt and/or GDUNHQHGEXLOGLQJIHDWXUHV´³5RRIVDEVHQW´³%ORFNVRQDFFHVVURDGV´³0LOLWDU\ HTXLSPHQWLQUHVLGHQWLDODUHDV´³(TXLSPHQWSHUVRQVRQWRSRIEXLOGLQJVLQGLFDWLQJ potential sniper positions”, and “Shelters composed of different materials than Produced and distributed by Pluto Journals www.plutojournals.com/scj/ CHRISTOPHER WILLIAMS 40 surrounding structures”. SBTF converted the original Syria Tracker map into layers. )RULQVWDQFHWKH³0LOLWDU\´WDJIRUODUJHPLOLWDU\HTXLSPHQWZDVXSORDGHGDQG others could compare with their own reports. Items such as “tanks” could then be cross-referenced with reports of “shelling” (SBTF 2011). Images permit quick before-and-after comparative analysis. CROWDSOURCING RESEARCH CROWDSOURCING RESEARCH 41 and know what they were doing. But many aspects of crowdsourced research are anonymous, which raises new ethical questions (below). and know what they were doing. But many aspects of crowdsourced research are anonymous, which raises new ethical questions (below). It is likely that states will also use crowdsourcing as a means of intelligence gathering from refugees and immigrants who sympathize with their host nation. Based on CrowdMap, Open Nuclear Iran is “a crowdsourced effort developed anonymously by concerned Iranians and experts from the arms control community to document, assess, and share information on Iran’s nuclear program in an open forum” (ONI 2012). A Miliphotos site invites people “to prevent Iran from developing a nuclear weapons capability” through “the collation of related incidents/events (bombings/assassinations/aviation accidents/industrial accidents/etc).” It provides a meticulous list of questions and instructions, to note, “Incidents/events related to the impairment of Iran’s nuclear weapons development and delivery” (Tumatauenga 2012), and seems to come from New Zealand. But who is this informing – amateurs, ZHVWHUQLQWHOOLJHQFHVHUYLFHVRUWKH,UDQLDQJRYHUQPHQW" Produced and distributed by Pluto Journals www.plutojournals.com/scj/ CROWDSOURCING RESEARCH (YHQ1RUWK.RUHDLVQRWLPPXQHIURPFURZGVRXUFHGVDWHOOLWHDQDO\VLV$PHULFDQ students and volunteers spent two years using their “democratized intelligence” to collect news reports, images, accounts, books and maps, which they used to locate VLJQL¿FDQW³EXLOGLQJVPRQXPHQWVPLVVLOHVWRUDJHIDFLOLWLHVPDVVJUDYHVVHFUHW labour camps, palaces, restaurants, tourist sites, main roads and even the entrance WRWKHFRXQWU\¶VVXEWHUUDQHDQQXFOHDUWHVWEDVH´0RVHV:LWKWKHKHOSRI RRJOH(DUWKWKH\SURGXFHG³1RUWK.RUHD8QFRYHUHG´ZKLFKLVRQHRIWKHEHVW SXEOLFO\DYDLODEOHPDSVRI1RUWK.RUHD1.(:0RUHIRUPDOO\SULVRQHU testimony has been triangulated with commercial quality satellite images to evidence WKHH[LVWHQFHDQGZRUNLQJVRIJXODJV+DZN 7KHH[FLWHPHQWVXUURXQGLQJWKHXVHRIVDWHOOLWHVVKRXOGUHÀHFWWZRFRQVLGHUDWLRQV First, to demonstrate state crime, the evidence must show systematic abuse, lines RIFRPPDQGDQGFRQWURODQGWKHLQYROYHPHQWRIKLJKOHYHORI¿FLDOV:LOOLDPV QRWMXVWLQVWDQFHVRIYLROHQFH6HFRQGVDWHOOLWHHYLGHQFHKDVD bad reputation. Few people will forget when, in 2003, the (then) US Secretary of 6WDWH&ROLQ3RZHOOVKRZHGWKH81KLVVDWHOOLWHHYLGHQFHRI:0'LQ,UDTZKLFK ZHUHQHYHUIRXQG(YHQWXDOO\WKHYDOXHRIDHULDOLPDJHVPD\EHPRUHLQWHUPVRI GHWHUUHQFHDQGFRQWUROWKDQIRUUHVHDUFKUHÀHFWLQJ)RXFDXOW¶VSULQFLSOHVRI “surveillance”. The Enough Project, which made use of satellite images to monitor Sudan, used the slogan, “The world is watching because you are watching.” 6WDWHV increasingly use crowdsourcing as a means of control. In China, blogs critical of the government are monitored and attacked by thousands of individuals SDLGE\WKH&KLQHVHJRYHUQPHQW0RQELRW,Q,UDQThe Information Center of the Islamic Revolutionary Guard Corps for Investigating Organized Crime used a site called HUGDE (“vortex”) to show photos of the protests with the faces of some protesters ringed in red. The public were invited to phone or email their identities, and the images were republished on Islamist blogs (Petrossian 2009). In turn, activists are also using blogs to identify individual abusers in repressive VWDWHV,UDQLDQSURWHVWHUVSRVWHGSLFWXUHVRIDVXVSHFWHG%DVLMDJHQWRQDPRWRUF\FOH drawing a gun. Names and rumours quickly circulated, which may not have been DFFXUDWH,Q7H[DVWKHJRYHUQRUFUHDWHGD³9LUWXDO%RUGHU:DWFK´ZKLFKOLQNHG surveillance cameras to the internet to permit the public to report likely illegal LPPLJUDQWVIURP0H[LFR+RZHE6WDWHVDQGUHYROXWLRQDULHVHQOLVWLQJSXEOLF spies is not new, and there is a degree of legitimacy if the participants are willing STATE CRIME 2.1 SPRING 2013 STATE CRIME 2.1 SPRING 2013 The Relevance to State Crime HQHUDOO\FURZGVRXUFHGUHVHDUFKH[WHQGVWKHVFRSHRIVWDQGDUGPHWKRGRORJLHV rather than representing a completely new research framework. For instance, it permits developments in the triangulation of data, such as refugee testimony with satellite data. But there are areas of more distinct innovation relevant across many forms of research. First, UHVHDUFKGHVLJQ can become less linear and more iterative or cybernetic (see below). New technologies create the possibility that data collection can continue XSWRWKHSRLQWRILQLWLDODQDO\VLVDQGHYHQEH\RQG0DSVRIWZDUHFRXOGSHUPLWWKH RQJRLQJFROOHFWLRQRIGDWDDERXWLOOHJDOORJJLQJJHQHUDWHRQJRLQJXSGDWHG¿QGLQJVLQ the form of maps, graphs and charts, and facilitate continuous crowdsourced analysis WKURXJKZLNLV,QSULQFLSOHWKHWLPHVFDOHRIDVWXG\FRXOGEHFRPHLQ¿QLWHZLWK self-updating online research reports and ongoing comment. The role of the original researchers could reduce to that of simply maintaining the system, and formulating QHZGLUHFWLRQV7KLVUHÀHFWVWKH³RSHQQRWHERRN´HWKRVWKDWLVHPHUJLQJZLWKLQ PHGLFDOUHVHDUFK:LOOLDPV$VPDUWFURZGVRXUFHGUHVHDUFKSURMHFW with a popular aim, simple robust methods, and good freeware, could potentially HYROYHLQWRD³YLUDOUHVHDUFKSURMHFW´ZKLFKLVWDNHQXSLQGLIIHUHQWIRUPVDURXQG the world, beyond the control of its instigators. In effect, crowdsourced research could take the concept of “participatory research” onto a new level (Berdou n.d.). A second innovation is that the potential scale of crowdsourced research data collection could invert traditional approaches to VDPSOLQJ. The methodological norm is to create a sampling frame from a population, and then collect data from the sample. But a massive amount of data, easily collected through crowdsourcing, CHRISTOPHER WILLIAMS 42 could be considered as a “data-population”. Samples, random or otherwise, could be taken from that “data-population”. There may be a degree of bias in a “data- population”, but that is true of any population. For example children, older people, those with intellectual disabilities, homeless people, slum dwellers, and overseas or migrant workers are often wrongly included or excluded from traditional survey populations. With some forms of crowdsourcing research, the population-data may be so large that, after excluding extremes, it can be treated as a representative sample in its own right because biases cancel one-another out. This is assumed within aggregated international indicators and indexes. Public health research is already testing these approaches by using “patient-reported data” rather than sample surveys (Scher 2012), although the validity is questioned (Norcie 2011). 0RUHVSHFL¿FDOO\LQUHODWLRQWRVWDWHFULPHFURZGVRXUFHGUHVHDUFKFUHDWHVWZR key possibilities. One is the use of the VWDWHDVDXQLWRIDQDO\VLV, independently of state information and resources. Currently, much state research is highly dependent on state sources of information. And state controlled resources – state university equipment and staff, and civil servants (which may include teachers and medics) ±RIWHQXQGHUSLQLQWHUQDWLRQDOUHVHDUFK(YHQ81LQIRUPDWLRQLVXVXDOO\UHVWULFWHG WRIRUPVWKDWPHPEHUVWDWHVZLOOWROHUDWHDQGWKHLQWHUQDWLRQDO12VRIWHQUHO\ heavily on this. Crowdsourced research presents a way to collect mass international data independently of states. The Relevance to State Crime For instance, air and water pollution levels can be monitored by volunteers, using an agreed methodology, around the world, and FRPSDUDWLYHWDEOHVSXEOLVKHG/DQGWKDWLVRXWRIERXQGVIRUPLOLWDU\SXUSRVHV FDQEHPDSSHGORFDOO\DQGYHUL¿HGE\VDWHOOLWHREVHUYDWLRQDQGDQDVVHVVPHQW made of the percentage of land that national armies control. Initiatives such as the Iraq Body Count, Corporate Watch, and Transparency International demonstrate that independent evidence-gathering can by-pass state systems. International professional networks, such as health and education, already have relevant networks, for example Médecins Sans Frontières and other “without borders” initiatives. University staff could evidence state complicity in illegal arms development and trade; teachers might record state violence and interference in schools. An independent supra-national crowdsourced research approach, might more readily embrace the non-nation states – the “non-self governing territories” and “stateless nations” – on an equal footing. The claim of the Roma to be a “non-territorial nation” certainly deserves accommodating within state crime research, but non-state communities are often invisible within international studies (Williams 2012: 101). A related potential innovation is the development of VWDWHQHWZRUNDQDO\VLV, to track links and relationships between state actors. Readily available macro data can form the basis for local crowdsourced research initiatives. Air and shipping URXWHVDQGPDSVRILQWHUQHWWUDI¿FFDQUHYHDOWKHREYLRXVOLQNV/RFDOUHVHDUFKHUV might compare data like this with who is building new hotels and why, together STATE CRIME 2.1 SPRING 2013 CROWDSOURCING RESEARCH 43 ZLWKUHSRUWVRIZURQJGRLQJE\SXEOLFRI¿FLDOV2QHH[DPSOHFRQFHUQV&DQFXQ the world’s second longest coral reef, which was inhabited by three people until ,WLVQRZDPDVVLYHWRXULVWUHVRUWWKHSRSXODWLRQLVDURXQGDQGLWV DLUSRUWLVWKHVHFRQGELJJHVWLQ0H[LFR'HWDLOVRIÀLJKWVDQGFDUULHUVDUHUHDGLO\ available on Wiki, which show interesting international links (Wiki-Cancun 2012). Since 1990, Colombian drug cartels have used the town, which was noted by the 86'UXJ(QIRUFHPHQW$GPLQLVWUDWLRQ+DZOH\DQGLQWKH0D\RUZDV FKDUJHGZLWKGUXJWUDI¿FNLQJDQGPRQH\ODXQGHULQJ%RRWK/HVVREYLRXV data, such as oil pipelines can help to understand diplomatic policy that otherwise seems inexplicable (Theodora 2012). Investigating who uses whose satellites, and for what, would be similar. The Relevance to State Crime The application of crowdsourced research can also extend the nature of the familiar UHVHDUFKTXHVWLRQV, principally by permitting a micro-macro “glocal” JOREDOORFDOIUDPLQJRIOLQHVRILQYHVWLJDWLRQ([DPSOHVLQUHODWLRQWRWKHPDLQ IRUPVRIVWDWHFULPHUHHQDQG:DUGPLJKWLQFOXGH &RUUXSWLRQ+RZGRORFDOVXVSLFLRQVRIZURQJGRLQJE\LQGLYLGXDOVOLQN with their presence in overseas tax havens, elite holiday resorts, arms fairs, RUGUXJSURGXFLQJUHJLRQV" 6WDWHFRUSRUDWH+RZGRKDUPIXOORFDOLPSDFWVUHODWHWRVWDWHLQWHUHVWV DEURDG" 3ROLFH+RZGRHVSROLFHWUDLQLQJVXSSRUWHGE\LQWHUQDWLRQDODLGOLQNWR FRPPHUFLDOVXSSOLHVRISROLFHHTXLSPHQW" 2UJDQL]HGDQG³GHHSVWDWH´+RZGRFRQWUDFWVIRUDUPVRUDLUSRUWVHFXULW\ V\VWHPVOLQNWRSROLWLFLDQV¶FRPPHUFLDODI¿OLDWLRQVDQGWKHLUUKHWRULFDERXW UHDORULPDJLQHGWHUURULVWWKUHDWV" 6WDWHWHUURULVP+RZLVORFDOPLOLWDU\GLVFRXUVHDPRQJVROGLHUVRUWKHQDWXUH DQGFRQWHQWRIWKHLUWUDLQLQJUHODWHGWRDEXVHRISRZHUE\WKDWPLOLWDU\DEURDG" 7RUWXUH+RZGRSXEOLFVHUYLFHUHFRUGVDQGWKHWHVWLPRQ\RIKHDOWK SURIHVVLRQDOVHYLGHQFHWKHV\VWHPDWLFXVHRIWRUWXUHE\WKHVWDWH" :DUFULPHV+RZGRHVHYLGHQFHIURPLQQRFHQWYLFWLPVHJFKLOGUHQSHRSOH ZLWKPHQWDOGLVDELOLWLHVKHDOWKSUREOHPVUHODWHWRRI¿FLDODFFXVDWLRQVRIZDU FULPHV" HQRFLGH+RZGROLIHKLVWRULHVIURPROGHUSHRSOHYDOLGDWHKLVWRULFDOUHFRUGV RIZDUFULPHV" The practical considerations when framing this form of crowdsourced research question are to consider KRZORFDOSHRSOHFRXOGFROOHFWLQIRUPDWLRQWKDWLVQRWDYDLODEOHWRRXWVLGHUV Produced and distributed by Pluto Journals www.plutojournals.com/scj/ CHRISTOPHER WILLIAMS 44 LQDZD\WKDWLVQRWGDQJHURXVIRUWKHPVHOYHV LQDZD\WKDWLVQRWGDQJHURXVIRUWKHPVHOYHV LQDZD\WKDWLVQRWGDQJHURXVIRUWKHPVHOYHV LQDIRUPWKDW³FURZGV´RXWVLGHDFRXQWU\RUUHJLRQPD\EHDEOHWRFRPSDUH and analyse, LQDIRUPWKDW³FURZGV´RXWVLGHDFRXQWU\RUUHJLRQPD\EHDEOHWRFRPSDUH and analyse, LQZD\VWKDWORFDOSHRSOHFRXOGQRW Asking local people to photograph details of spent munitions, which can then be compared with macro-data about the arms trade, would be an example. Asking local people to photograph details of spent munitions, which can then be compared with macro-data about the arms trade, would be an example. Three potentially important forms of endeavour seem under-developed at present. One is ORQJLWXGLQDOVWXG\, to assess the so-called “creeping disasters”, “slow HPHUJHQFLHV´RU³ORQJZDYHHYHQWV´/6(IRUH[DPSOHVWDWHLPSOLFDWLRQ in environmental harm or geopolitical destabilization. This is probably due to WKHGLI¿FXOW\RIPRWLYDWLQJSHRSOHWRFRQWULEXWHWROHVVH[FLWLQJORQJWHUPZRUN :HQHHGWRHQYLVDJHD³FURZG´DVORQJDQGWKLQQRWMXVWVKRUWDQGZLGH7KH second is that, although high-tech initiatives are very valuable for documenting that abuses of power are widespread, they seem less good evidencing that abuses are V\VWHPDWLF and based on command and control structures. But if states also deploy crowdsourcing as a means of repression, the systematic nature of their actions may become easier to track through electronic metadata and critical process analysis (Williams 2012: 204). Finally, there is little discussion of the HWKLFV of crowdsourcing research. Norcie (2011) considers low fees and the problem that research participants are a self-selecting sample. Schmidt (2011), raises the problem of children becoming involved anonymously, and provides a useful checklist for researchers and participants who should understand: (a) how and when personally identifying information of a participant is made available to a researcher, (b) who can access task participation records and other potentially sensitive information, (c) whether and how the data collected by researchers is stored securely and is secure during download, and (d) whether and how communications between participants and researchers is secure. But state crime research raises bigger problems. Zittrain questions the ethics when the “crowd” is anonymous, and may unknowingly be helping state intelligence agencies. The Relevance to State Crime Using the example of Iran (above), he comments, “The people making WKHLGHQWL¿FDWLRQVLQ,QGLDRUWKH86LGO\GRLQJWKLVRQWKHLUOXQFKKRXULQVWHDG RI0LQHVZHHSHUZRXOGKDYHQRLGHDRIWKHLPSOLFDWLRQVRIZKDWWKH\DUHGRLQJ´ 0DFUHJRU,QKLVUHSRUWRI,UDQLDQDFWLYLVWVFURZGVRXUFLQJLGHQWLWLHVRI STATE CRIME 2.1 SPRING 2013 CROWDSOURCING RESEARCH 45 violent militia, Petrossian (2009) provides an apposite warning, “Information like this is neither fact-checked nor reliable and can have severe consequences for anyone who shares the man’s supposed name. Online, rumours easily replace facts.” Commercial approaches may sometimes intrinsically lack integrity. BP’s attempt at ³FURZGVRXUFLQJ´WKRXVDQGVRILGHDVWRGHDOZLWKWKH0H[LFR%D\RLOVSLOOVHHPHG more like PR than research. Stephen Overell, of the Work Foundation, commented that assessing “20,000 ideas is unmanageable, you’ll need all sorts of procedures for sorting through them” (de Castella 2010). Conclusion 7KHXVHRIWKHWHUP³FURZGVRXUFLQJ´KDVLQFUHDVHGVLJQL¿FDQWO\VLQFH4XLQQ DQG%HGHUVRQDQGDVLPSOHRRJOHVHDUFKQRZJHWVPRUHWKDQ PLOOLRQKLWV%XWZK\LVWKHUHDVSHFL¿FUHVSRQVHLQUHODWLRQWRVWDWHFULPH"7KH question is important because “motivation” is a central concern of crowdsourcing. HQHUDOLQYROYHPHQWLQDFWLYLVPLVOLNHO\WREHPRWLYDWHGE\DOWUXLVPEXWDOVRIURP DVWURQJHUHYROXWLRQDU\GULYHURXU³FKHDWHUGHWHFWRU´±DQLQQDWHVHQVHRILQMXVWLFH (Cosmides and Tooby 1992). From a broader analysis of trends in the accountability RISRZHUIXOSHRSOH:LOOLDPVIHHGEDFNV\VWHPVDUHDOVRVLJQL¿FDQW7KHUH are six aspects which can help to explain why crowdsourcing seems effective when DGGUHVVLQJDEXVHVRISRZHUZKLFKFDQLQIRUPPRWLYDWLRQDOVWUDWHJLHV7KH¿UVWWZR DUHWHFKQLFDODQGWKHRWKHUIRXUSHUFHSWXDO:LOOLDPV VXUYHLOODQFH±awareness and recognition of wrongdoing VXUYHLOODQFH±awareness and recognition of wrongdoing information management ±HI¿FLHQWUHFRUGLQJDQGH[FKDQJLQJRIHYLGHQFH and analysis ż an instant here-and-now perception of events – time-space compression ż a self-perception of being part of an accountability movement ż a perception of mass immunity from retaliation by powerful people ż a perception of effective sanctions against people who abuse their power. information management ±HI¿FLHQWUHFRUGLQJDQGH[FKDQJLQJRIHYLGHQFH and analysis The dynamics of this cybernetic feedback process are much as Norbert Weiner envisaged in &\EHUQHWLFV2U&RQWURODQG&RPPXQLFDWLRQLQ0DQDQG0DFKLQH (1949). Framed now as “sociocybernetics”, “circular causal” relationships within a (conceptually) closed system assume that an entity can cause a change in its environment, information about that change is fed back, and this enables that entity to PDNHIXUWKHUFKDQJHVWRLWVHQYLURQPHQWH\HUDQGYDQGHU=RXZH± Activists who contribute to exposing local industrial pollution, may respond to information about that success by analysing and developing strategies to scale-up Produced and distributed by Pluto Journals www.plutojournals.com/scj/ CHRISTOPHER WILLIAMS 46 their endeavours nationally and internationally, for example from local wetland pollution to rivers and oceans. The excitement surrounding technological initiatives can distract from awareness WKDWWKH\UHÀHFWDFWLYLWLHVWKDWFDQEHGRQHPDQXDOO\7KHVHLQFOXGHFROOHFWLQJ drawing, recording testimony, sorting and archiving, scan-reading, annotating and labelling (tagging), use of social networks, reconstruction of evidence, and mapping. As Foxe showed, the fundamental characteristic of crowdsourced research is that it is generative – it should intrinsically increase participation. It is probably more helpful to view technologically-based strategies as being technologically-enhanced. A starting point for designing crowdsourced research is therefore to ask, what is WKHRSWLPXPIRUPRIHQKDQFHPHQW":KDWFDQ SHRSOHGRWKDWWHFKQRORJ\FDQQRW"HJGHFLSKHUXQFRQYHQWLRQDOKDQGZULWLQJ use common sense, accommodate unexpected data, notice unpredicted RPLVVLRQV¿QGPHDQLQJLQLPDJHVDQGWH[WV SHRSOHGREHWWHUWKDQWHFKQRORJ\"HJVFDQUHDGLQJFKHFNLQJWUDQVFULSWLRQV tagging, annotation, recognize strange shapes or information, paired comparisons – better/worse) FURZGVGREHWWHUWKDQLQGLYLGXDOVH[SHUWV"HJUHSHWLWLYHWDVNVSOD\JDPHV be quick, deploy global-local knowledge) LQGLYLGXDOVH[SHUWVGREHWWHUWKDQFURZGV"HJUHVHDUFKGHVLJQV\VWHPL]DWLRQ theorization) WHFKQRORJ\GREHWWHUWKDQSHRSOH"HJPDVVGDWDVWRUDJHPDVVDFFHVV computation) WHFKQRORJ\GRWKDWSHRSOHFDQQRW"HJDHULDOVXUYHLOODQFHFRPSUHVVWLPH and space) VHYHUDOWHFKQRORJLHVGREHWWHUWKDQRQH"HJURERWLFVDPSOHFROOHFWLRQ photography) The answers should indicate the synergy between humans and technology – things that can be done better by interaction between the two. The answers should indicate the synergy between humans and technology – things that can be done better by interaction between the two. Conclusion %XWWKHTXHVWLRQPRVWUHOHYDQWWRVWDWHFULPHLVSUREDEO\DUHMXVWLFHV\VWHPV ready to accommodate cases based on crowdsourcing, based on mass defendants, ZLWQHVVHVDQGHYLGHQFH",IFDVHVDUHEURXJKWDJDLQVWVWDWHVIRUFURZGVRXUFHG DJJUHVVLRQRUDEXVHVRISRZHUKRZZLOOWKHFRXUWVGH¿QHWKH³FRQWUROOLQJPLQG´" +RZFDQODUJHQXPEHUVRIFKLOGUHQSDUWLFLSDWH"&ODVVDFWLRQV±PDVVWRUWV±DUH EHFRPLQJEHWWHURUJDQL]HGDQGKDYHUHGUHVVHGVLJQL¿FDQWDEXVHVRISRZHUEXWFDQ WKLVEHDGDSWHGIRUFULPLQDODQGLQWHUQDWLRQDOFRXUWV"7KH(8SODQVIRU³FROOHFWLYH UHGUHVV´GRQRWVHHPWRKHOS,WLVSRVVLEOHWKDWWKH,&&ZLOOEHWKH¿UVWFRXUWWKDW STATE CRIME 2.1 SPRING 2013 STATE CRIME 2.1 SPRING 2013 CROWDSOURCING RESEARCH 47 will need to address this problem, for example if the child abuse case against the 9DWLFDQ6WDWHJRHVIRUZDUG61$3 Perhaps the old elites were right to fear the power of crowds, but the challenge now is to ensure it will be for good reasons. As convergence theory reminds us, D³FURZG´LVQRWDUDQGRPJURXS±DUHSUHVHQWDWLYHVDPSOHGHPRFUDWLFPDMRULW\ RUMXU\3HRSOHFRQYHUJHIRUVSHFL¿FUHDVRQVDURXQGVSHFL¿FDUHDVRIHQGHDYRXU JRRGDQGEDG%HUN7KHJRDORIFURZGVRXUFLQJVKRXOGEHEHWWHUMXVWLFHEXW this will require a sound public understanding of state crime, and legitimization of crowd involvement, through global citizenship education and public awareness initiatives. Otherwise crowdsourced research could simply become the means to create global e-enabled lynch mobs, state and non-state. References AIN (2012) “Nigeria Amnesty Dialog”, Amnesty International Nigeria. Available online at http:// QJDPQHVW\SDUWQHUVLQWHUQDWLRQDORUJDFFHVVHG0DUFK $QGHUVHQ0³)RXU&URZGVRXUFLQJ/HVVRQVIURPWKHXDUGLDQ¶V6SHFWDFXODU([SHQVH6FDQGDO ([SHULPHQW´1LHPDQ-RXUQDOLVP/DE$YDLODEOHRQOLQHDWKWWSZZZQLHPDQODERUJIRXU crowdsourcing-lessons-from-the-guardians-spectacular-expenses-scandal-experiment/ (accessed 29 0DUFK BBC (2010) “Anonymous Wikileaks activists move to analogue tactics”, BBC News. Available online DWKWWSZZZEEFFRXNQHZVWHFKQRORJ\DFFHVVHG0DUFK ²²³2XWORRN7KH(J\SWLDQSV\FKLDWULVWWUHDWLQJWKHFRXQWU\¶VWRUWXUHYLFWLPV´BBC News. $YDLODEOHRQOLQHDWKWWSZZZEEFFRXNSRGFDVWVVHULHVRXWORRNSOD\HSLVRGHDFFHVVHG0DUFK 2012). %HLVVHO'XUUDQW³$7\SRORJ\RI5HVHDUFK0HWKRGVZLWKLQWKH6RFLDO6FLHQFHV´8QSXEOLVKHG Working Paper. ESRC National Centre for Research Methods. Available online at http://eprints.ncrm. 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FFUMXVWLFHRUJLFFYDWLFDQSURVHFXWLRQDFFHVVHG$SULO &RVPLGHV/DQG7RRE\-³&RJQLWLYH$GDSWDWLRQVIRU6RFLDO([FKDQJH´LQ-+%DUNRZ /&RVPLGHVDQG-7RRE\HGVThe Adapted Mind: Evolutionary Psychology and the Generation of Culture. New York: Oxford University Press. Crowdsourcing.org (2012) “Community tools”, Crowdsourcing.org. Available online at http://www. FURZGVRXUFLQJRUJFRPPXQLW\WRROVDFFHVVHG0DUFK Produced and distributed by Pluto Journals www.plutojournals.com/scj/ CHRISTOPHER WILLIAMS 48 &:'/73³&LYLO:DU'LDULHV /HWWHUV7UDQVFULSWLRQ3URMHFW´$YDLODEOHRQOLQHDWKWWSGLJLWDO &:'/73³&LYLO:DU'LDULHV /HWWHUV7UDQVFULSWLRQ3URMHFW´$YDLODEOHRQOLQHDWKWWSGLJLWDO OLEXLRZDHGXFZGWUDQVFULSWVKWPODFFHVVHG0DUFK GH&DVWHOOD7³6KRXOGZHWUXVWWKHZLVGRPRIFURZGV"´BBC News. Available online at http:// QHZVEEFFRXNKLPDJD]LQHVWPDFFHVVHG$SULO J S (,³6\ULD¶VUHYROXWLRQ¿QGVDWHPSRUDU\KRPHLQ&DLUR´Egypt Independent. 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(1990) The Making of The Golden Bough: The Origins and Growth of an Argument/RQGRQ 3DOJUDYH0DFPLOODQ )UD]HU-The Golden Bough: A Study in Magic and Religion/RQGRQ:RUGVZRUWK(GLWLRQV ) 7 6 ³) - K ´ L 2 I G 'L L I 1 L O %L K )UD]HU-The Golden Bough: A Study in Magic and Religion/RQGRQ:RUGVZRUWK(GLWLRQV )UHHPDQ76³)R[H-RKQ±´LQ2[IRUG'LFWLRQDU\RI1DWLRQDO%LRJUDSK\. References )UD]HU-The Golden Bough: A Study in Magic and Religion/RQGRQ:RUGVZRUWK(GLWLRQV )UHHPDQ76³)R[H-RKQ±´LQ2[IRUG'LFWLRQDU\RI1DWLRQDO%LRJUDSK\. Oxford: Oxford University Press. g y g g )UHHPDQ76³)R[H-RKQ±´LQ2[IRUG'LFWLRQDU\RI1DWLRQDO%LRJUDS Oxford: Oxford University Press. H\HU)DQGYDQGHU=RXZH-³6RFLRF\EHUQHWLFV´LQ&91HJRLWDHG+DQGERRN &\EHUQHWLFV1HZ<RUN0DUFHO'HNNHU 33:³3ROOXWHU:DWFK´Greenpeace. Available online at http://www.polluterwatch.com/ bp-documents (accessed 1 April 2012). UHHQ3DQG:DUG7State Crime: Governments, Violence and Corruption/RQGRQ3OXWR3UHVV UHHQ3DQG:DUG7State Crime: Governments, Violence and Corruption/RQGRQ3OXWR3UHVV XDUGLDQ³,QYHVWLJDWH\RXU03¶VH[SHQVHV´The Guardian. Available online at http:// mps-expenses.guardian.co.uk/ (accessed 3 February 2010). XDUGLDQ³,QYHVWLJDWH\RXU03¶VH[SHQVHV´The Guardian. Available online at http:// mps-expenses.guardian.co.uk/ (accessed 3 February 2010). +DZN'7KH+LGGHQXODJ([SRVLQJ1RUWK.RUHD¶V3ULVRQ&DPSV. Washington: US Committee IRU+XPDQ5LJKWVLQ1RUWK.RUHD J +DZOH\&³'UXJVFDVWFORXGRYHU0H[LFDQSDUDGLVH´USA Today0D\ \ J S y \ +&/³+DUYDUG7HKUDQSURSDJDQGDPXUDOV´+DUYDUG&ROOHJH/LEUDU\. Available online at http:// K O K G G OO WL GL LW O OO WL W K O I G 0 K +&/³+DUYDUG7HKUDQSURSDJDQGDPXUDOV´+DUYDUG&ROOHJH/LEUDU\. Available online at http:// KFOKDUYDUGHGXFROOHFWLRQVGLJLWDOBFROOHFWLRQVWHKUDQBPXUDOVFIPDFFHVVHG0DUFK +&/ +DUYDUG7HKUDQSURSDJDQGDPXUDOV +DUYDUG&ROOHJH/LEUDU\. 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https://openalex.org/W4210802220	https://zenodo.org/records/5947193/files/37378.pdf	English	null	Comparative characteristics of the effectiveness of complex therapy in the surgical treatment of acute condyloma of the anogenital region with the use of drugs - nitrogen donors	Journal of Education, Health and Sport	2,021	cc-by	6,332	Slepichko A. N., Deykalo I. M. Comparative characteristics of the effectiveness of complex therapy in the surgical treatment of acute condyloma of the anogenital region with the use of drugs - nitrogen donors. Journal of Education, Health and Sport. 2021;11(09):895-907. eISSN 2391-8306. DOI http://dx.doi.org/10.12775/JEHS.2021.11.09.105 https://apcz umk pl/JEHS/article/view/JEHS 2021 11 09 105 Slepichko A. N., Deykalo I. M. Comparative characteristics of the effectiveness of complex therapy in the surgical treatment of acute condyloma of the anogenital region with the use of drugs - nitrogen donors. Journal of Education, Health and Sport. 2021;11(09):895-907. eISSN 2391-8306. DOI http://dx.doi.org/10.12775/JEHS.2021.11.09.105 https://apcz umk pl/JEHS/article/view/JEHS 2021 11 09 105 Slepichko A. N., Deykalo I. M. Comparative characteristics of the effectiveness of complex therapy in the surgical treatment of acute condyloma of the anogenital region with the use of drugs - nitrogen donors. Journal of Education, Health and Sport. 2021;11(09):895-907. eISSN 2391-8306. DOI http://dx.doi.org/10.12775/JEHS.2021.11.09.105 https://apcz.umk.pl/JEHS/article/view/JEHS.2021.11.09.105 https://zenodo.org/record/5947193 Received: 2 5.08.2021. Revised: 12.09.2021. Accepted: 30.09.2021. UDК: 615.036.8:616.5-089:616-006.52:[617.64+618.16] The journal has had 5 points in Ministry of Science and Higher Education parametric evaluation. § 8. 2) and § 12. 1. 2) 22.02.2019. © The Authors 2021; This article is published with open access at Licensee Open Journal Systems of Nicolaus Copernicus University in Torun, Poland Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author (s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non commercial license Share alike. (http://creativecommons.org/licenses/by-nc-sa/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. The authors declare that there is no conflict of interests regarding the publication of this paper. Received: 2 5 08 2021 Revised: 12 09 2021 Accepted: 30 09 2021 Comparative characteristics of the effectiveness of complex therapy in the surgical treatment of acute condyloma of the anogenital region with the use of drugs - nitrogen donors A. N. Slepichko, I. M. Deykalo Luhansk State Medical University Ivan Horbachevsky Ternopil National Medical University Ivan Horbachevsky Ternopil National Medical University A. N. Slepichko, I. M. Deykalo Luhansk State Medical University Abstract The paper presents comparative data on the effectiveness of complex treatment of acute condyloma of the anogenital region (n = 105). Destruction of genital warts was performed surgically, preceded by immunomodulatory and antiviral systemic therapy and local treatment with antiseptic measures and hyaluronic acid, which continued in the postoperative period. Patients of the first group (n = 70) were added to the complex therapy drug - nitrogen donor L - arginine. Efficacy control consisted of subjective assessment and patient complaints; objective determination of the clinical condition, the course of the postoperative period, assessment of the state of microcirculation; laboratory dynamics of interleukin levels; the presence of recurrence of the disease. The use of nitrogen donor (L- arginine) in complex therapy allows to eliminate subjective symptoms, signs of secondary bacterial contamination in the preoperative period, postoperative pain, post-traumatic stress and to provide a significantly higher rate of epithelization, which optimizes the patient's stay. prognosis for recurrence of AW. The obtained results showed a significantly higher efficiency 895 of complex treatment of acute genital warts of the anogenital region in the group of patients receiving a nitrogen donor L - arginine. In the group of patients receiving L-arginine (n = 70), there were 2 (2,86 %) relapses. In the control (n = 35) group – 3 (8,57 %) (p < 0.05). of complex treatment of acute genital warts of the anogenital region in the group of patients receiving a nitrogen donor L - arginine. In the group of patients receiving L-arginine (n = 70), there were 2 (2,86 %) relapses. In the control (n = 35) group – 3 (8,57 %) (p < 0.05). Key words: acute condyloma of the aogenital region; arginine; interleukin profile; fluometry; microcirculation; L - arginine. At the present hour, there is an increase in the incidence of human papillomavirus infection (HPV) worldwide. Its frequency continues to increase, becoming epidemic [7]. HPV induces a number of diseases of pre-tumor and tumor origin, has various forms and its own link of pathological influence [4]. HPV is highly contagious. According to the literature, the infection, even with one unprotected contact, ranges from 40 - 60%. [2]. In turn, there is evidence of a dissonance between infection and the implementation of clinical manifestations. Thus, according to current epidemiological data, 75,6 % of sexual partners of patients with anogenital warts do not have clinical manifestations of the disease. Abstract [1], which leads to an increase in infection and carrier [17]. Gender differences in the speed of HPV infection are now proven. According to the latest literature, the risk of transmission of any type of HPV from man to woman is lower than from woman to man, which explains the higher elimination of infection, faster reduction of viral load and low seroconversion in men compared to women [2]. According to the type of oncogenicity, the types of HPV, which affecte the anogenital link, are divided into three groups - low, medium and high oncogenic risk. High risk of oncogenicity (hrHPV) includes types 16, 18, 48, 56. Medium - 31, 33, 35, 51, 52, 58. Low risk of oncogenicity (lrHPV) includes types 6, 11, 42, 43, 44. In the future, the level of oncogenic risk of HPV affects the course of the pathological process [8]. More than 90% of acute warts (AW) caused by HPV types 6 and 11 are one of the most common sexually transmitted diseases, and account for 1% of the world's sexually active population [2, 8, 11]. The peculiarity of the pathogenesis of HPV is its high tropism and infection of only proliferating epitheliocytes. Intense replication and release of the virus occur in differentiated keratinocytes located in the upper layers of tissue [8]. There are a number of factors in the pathogenesis of HPV that allow long-term persistence of the virus in human cells. By infecting keratinocytes, HPV causes a minimal inflammatory response due to inhibition of the cellular and humoral response. Under the influence of the virus a decrease in interferon levels α-γ occurs, inhibition of 896 histocompatibility complex, changes in interleukin profile and qualitative composition of lymphocytes in the direction of increasing suppressors, inhibition of maturation of Langerhans cells (immunocompetent cells), which leads to impaired apoptosis of infected cells [11, 15]. In addition to known factors such as immunosuppression, concomitant somatic pathology, the use of cytostatics high infectious index, the presence of sexually transmitted infections [15], the course of the disease is affected by microtrauma, surgical trauma that accompanies the postoperative period. In turn, there is currently no effective method of treatment other than surgical excision of genital warts [8, 10]. There is no data to indicate which treatment is most effective. Abstract This figure depends on the state of arteriolar vascular tone and / or decreased blood flow in the venules. σ («flax») is the standard quadratic deviation (SCR) of the amplitude of blood flow oscillations from the mean value of M, which characterizes the temporal variability of microcirculation, which is provided by neurogenic and mitogenic tone. The decrease in σ indicates the suppression of active vasomotor mechanisms of blood flow modulation. Кv - coefficient of variation - reflects the relationship between perfusion and its variability: Kv = σ / M × 100 % [3, 9, 12]. Кv - coefficient of variation - reflects the relationship between perfusion and its variability: Kv = σ / M × 100 % [3, 9, 12]. Mathematical processing of research results and comparative assessment of the significance of differences between the main and control groups was carried out using programs for calculating the indicators of the variational series and the Student's criterion. The reliability interval was obtained with a probability of 95% (p <0,05) [6]. In designing the treatment, we were guided by modern scientific data on the epidemiology and pathogenesis of AW anogenital area, namely: 1). The main method of infection – contact (sexual, household, artificial), which is clinically realized under conditions of local changes in the skin and mucous membranes, in the form of microtrauma or inflammation on the background of microbial and fungal contamination. 2). The conditions for the manifestation of AW of the anogenital region in infected patients are changes in the immune system. 3). AW is a reservoir of vegetative forms HPV [4], so their surgical removal reduces the viral load. 4). At present, there are no evidence-based treatments for AW, including surgical removal, or other types of destruction [4, 8, 10]. 1). The main method of infection – contact (sexual, household, artificial), which is clinically realized under conditions of local changes in the skin and mucous membranes, in the form of microtrauma or inflammation on the background of microbial and fungal contamination. 2). The conditions for the manifestation of AW of the anogenital region in infected patients are changes in the immune system. 3). AW is a reservoir of vegetative forms HPV [4], so their surgical removal reduces the viral load. 4). At present, there are no evidence-based treatments for AW, including surgical removal, or other types of destruction [4, 8, 10]. Abstract There is no clear understanding of how modern treatments affect the spread or recurrence of the disease, and the physiological process of wound healing, which is based on the proliferation of cells of the basal layer of the epithelium, in itself can provoke recurrence of the disease [10, 18]. The method of treatment is determined taking into account the following factors: the size of the lesion, the number of lesions (single or multiple), the degree of lesion (intense or extensive), anatomical location (internal or external genitalia), patient preferences, ease of treatment, adverse effects of drugs and surgery, as well as the experience of the doctor, among others [18]. The aim of the study. Optimization of the results of treatment of acute genital warts of the anogenital region with different clinical forms of the disease through the determination of somatic and immune status of patients, and their timely correction. Materials and methods. 105 patients with AW of the anogenital region were examined and treated, among which single AW – 36 (34,29 %); multiple AW – 66 (62,86 %); giant Bushke-Levenstein warts – 3 (2,86 %.). The control group consisted of 20 people. In terms of gender, the main group consisted of 63 (60 %) women and 42 (40%) men. In the control – 10 (50 %) women and 10 (50 %) men. The exclusion criteria from the main group were the presence of diabetes, pregnancy, HIV, syphilis, oncological pathology of the skin of the anogenital link, rectum, genitals. The level of cytokines TNFa, IL-4 IL-6 IL-8 was determined using a solid-phase enzyme-linked immunosorbent assay with monoclonal and polyclonal antibodies. The intensity of pain in the preoperative and postoperative period was determined by the Visual Analog Scale (VAS): 0 - absence, 1-2 - weak, 3-4 - insignificant, 5-6 - moderate, 7-8 - strong, 9-10 most pronounced, intolerable [14]. 897 Assessment of local blood circulation was determined by laser Doppler flowmetry (LDF). The examination was performed on a Doppler laser fluometer LAKK-01 (NPP "LAZMA", Russia) with a single-channel sensor with a wavelength of 0.63 μm (in the red range) and three optical fibers using a surface sensor to measure the surface of the skin. for three minutes. The microcirculation index (M), which is the total capillary circulation in real time, was determined. M is the arithmetic mean value of the microcirculation index, which is measured in perfusion units. Abstract Based on this, our proposed treatment design included antiviral / immunomodulatory therapy, local antibacterial and antifungal treatment, surgical treatment. Systemic antibiotic therapy was prescribed according to the indications. AW excision was performed by electrocoagulation using the BOWA device (Germany) with mandatory histological verification of the removed tissues. 898 For antiviral action, we used «Alokin-Alpha» (Aloferon). The standard course of treatment was 12 days and was administered in the preoperative and postoperative periods as subcutaneous injections at a dose of 1 mg every other day (for a total of 6 injections). Alloferon belongs to the group of oligopeptides, is an effective inducer of the synthesis of endogenous alpha- and gamma-interferons and an activator of the natural killer system. The drug stimulates the recognition and lysis of defective cells by cytotoxic lymphocytes and is highly effective against infections caused by influenza A and B viruses, hepatitis B, herpes, HPV [16]. In order to normalize the microcynosis and the functional state of the intestine, all patients were prescribed a biologically active supplement «Subalin-forte» (capsules) (FZ "Biopharma" Ltd., Ukraine), which is a source of active culture of Bacillus subtilis. Also live bacteria Bacillus subtilis have antiviral activity due to stimulation of α2-interferon synthesis [13]. «Subalin-forte» was administered 4 × 109 live culture of Bacillus subtilis 3 times a day for 14 days before surgery and 7 days after. In order to eliminate the inflammatory process as a local therapy on the location of HA before surgery and in the postoperative period, patients were prescribed the drug "Ginodek", which is a gel based on high molecular weight hyaluronic acid in the form of 1,5% sodium hyaluronate, antiseptic decamethoxine 20 % and lactate buffer pH 3,8 to 4,5. The pH level of the drug coincides with the indicators of healthy skin, which allows its use topically on the skin of the perineum. Lactate, which is part of the drug, stimulates the growth of connective tissue and collagen production, which is important in wound healing. Decamethoxine has a purely local effect, as it is not absorbed by mucous membranes, skin and wound surface, which eliminates the systemic effect on homeostasis. Its antimicrobial, fungicidal and antiviral effects are realized due to the effect on the phospholipids of the membrane of the pathological agent, which leads to a violation of their permeability and death. Decamethoxine has activity against microorganisms that are resistant to antibiotics. Abstract Sodium hyaluronate enhances angiogenesis, reduces cytokine levels, promotes wound healing and protects blood vessels from damage. In the postoperative period, sodium hyaluronate reproduces a protective film on the skin, which protects the postoperative wound from infection [19]. The drug was administered in the preoperative period at 2.0 ml per affected area 2 times a day for 10 days and 10 days after surgery. In women, according to the indications, in order to rehabilitate the vagina, the drug was also prescribed vaginally 5 ml 2 times a day. Given the data we received during the examination of patients on the violation of microcirculation in the anogenital area in HA, one of the areas of treatment was the impact on 899 the normalization of blood circulation in the anogenital area. For this purpose, we proposed the drug «Tivortin aspartate» (L-arginine). Arginine is a nitrogen donor for the enzyme NO synthase, which is required for the synthesis of nitric oxide by endothelial cells, thereby having the effect of pronounced vasodilation by inhibiting the synthesis of endothelin-1. The latter is a powerful vasoconstrictor. Arginine reduces oxidative stress by inhibiting the synthesis of asymmetric dimethylarginine. Stimulates the activity of the thymus gland, affecting the synthesis of T lymphocytes. Arginine has antihypoxic, antioxidant, immunomodulatory, membrane stabilizing, cytoprotective and detoxifying effects. The drug is able to affect the synthesis of a number of hormones and biologically active substances. According to the treatment design, patients were divided into 2 clinical groups. The first group included 70 patients, among whom patients with multiple lesions of the perianal region were 47 (67,14 %), single – 21 (30,0 %), Bushke-Levenstein warts – 2 (2,86 %). All patients of the first group, except for antiviral and topical therapy, were given L- arginine aspartate 2 weeks before surgery and 10 days in the postoperative period. The drug was administered in a single dose of 5,0 ml, corresponding to 1,0 g of L-arginine, orally, 4 times a day with meals. The second clinical group included patients with multiple lesions of the perianal region – 19 (54,29 %), single – 15 (42,86 %), Bushke-Levenstein warts – 1 (2,86 %), who did not receive L- arginine. Otherwise, the treatment regimen did not differ. The obtained results and their discussion. Abstract The analysis of the somatic status of patients with AW of the anogenital region revealed a high level of chronic diseases, which were observed in 96 (91,43 %). At the same time, 24 (22,86 %) patients presented themselves as somatically healthy, did not seek medical help, which contributed to the chronicity of the pathology. The high frequency of pathology of the digestive system, which was 81 (77,14 %), is noteworthy. On average, one patient in the main group had 2,16 cases of pathology of the digestive system and 3,41 cases of general somatic pathology. In contrast to the main group, the control level of somatic pathology was significantly lower. On average, in the control group per subject there were 0,9 cases of pathology of the digestive system and 1,2 of general somatic pathology, which is lower than in the main, respectively, 2,4 and 2,8 times (p <0.05). The presence of a high level of chronic somatic and infectious pathology in patients with AW area gives reason to think about the presence of chronic immune disorders which are a risk factor for AW in infected HPV and lead to the manifestation of the pathological process. When examining the immune status of 100 % of patients with AW anogenital area was observed significantly lower compared to the control group, the total number of lymphocytes, 900 equal to (1,63 ± 0,07) × 109 /L at (2,15 ± 0,1) × 109 /L (p < 0,05), which indicates the homogeneity of the group on this basis and can be used as a prognostic factor. The relative number of lymphocytes corresponded to the lower limit of the reference values – (28,75 ± 1,75) %, while in the control group it was 1,3 times higher and equal to (35,95 ± 1,61) % (p < 0,05). The presence of absolute and relative lymphopenia can be explained from the standpoint of the influence of long-term viremia on immune homeostasis. Immunological examination in patients of the main group in comparison with the control revealed a proportional decrease in the absolute number of T-lymocytes (CD3 +) and their populations. The number of T-lymocytes (CD3 +) was 1,6 times lower and was equal to (0,70 ± 0,02) × 109 /L against (1,13 ± 0,07) × 109 /L (p < 0,05). Abstract The absolute level of (CD4 +) (T-helpers / inducers) compared with the control group was reduced by 1,6 times – (0,42 ± 0,01) × 109 /L in the control (0,66 ± 0,05) × 109 /L (p < 0,05). The level of CD8 + (T- suppressors / killers) also differed and was 1,5 times lower, respectively – (0.28 ± 0,01) × 109 /L at (0,43 ± 0,03) × 109 /L (p <0,05) in the control. The relative number of T-lymphocyte populations also differed significantly. The level of CD3 + in the main group was equal to (41,64 ± 0,41) %; in the control – (49,45 ± 0,8) % (p < 0,05). CD4 + (25,19 ± 0,45) % and (30,15 ± 0,64) % (p < 0,05), respectively. According to CD8 + in the main (16,48 ± 0,36) % and in the control (19,40 ± 0,52) (p < 0,05). All subjects showed an imbalance in the subpopulation of T lymphocytes with a proportional decrease of 1,2 times. At the same time, the level of CD3 + was reduced relative to the reference values in 100 % of patients, which indicates statistical homogeneity of the population. According to CD4 + indicators, there were 33 (36,7 %) reductions. The level of CD8 + was reduced by 29 (32,2 %). Immunoregulatory index did not differ significantly and was equal in the main group (1,64 ± 0,06) %, in the control – (1,55 ± 0,08) % (p > 0,05), but in the main group of its increase relative to the reference values were observed in 18 (20,0 %), while in the control only in one case (5,0 % ) due to its reduction. At the same time, the level of B-lymphocytes (CD 22) was changed against the background of T-lymphopenia. An increase in the total number of B-lymphocytes relative to the reference values was observed in 81 (90,0 %) patients of the main group and only in 3 (15,0 %) (p < 0,05) in the control with values, respectively (0,52 ± 0,02) × 109 /L and (0,45 ± 0,03) × 109 /L (p < 0,05). Abstract The relative composition was also changed in the direction of increase: (30,41 ± 0,52) % at (20,4 ± 0,38) % in the control (p < 0,05), indicating the presence of chronic inflammation and / or viral inflammation with depletion of the T-link of immunity 901 In accordance with the increase in B-lymphocytes, we observed changes in the link of humoral immunity. The indicators of Ig A and Ig G showed a significant increase. The level of Ig A was equal to (1,84 ± 0,03) g/L in the group of patients and (1,36 ± 0,05) g /L in the control group (p < 0,05). Ig G, respectively (13,96 ± 0,20) g /L and (11,6 ± 0,35) g /L (p < 0,05). The level of Ig M in both groups probably did not differ, but in the main group tended to decrease and was equal to (1,12 ± 0,03) g /L and (1,29 ± 0,17) g /L, respectively (p > 0,05). In the study of the cytokine profile in patients of the main group we found a significant increase in the level of pro-inflammatory interleukin IL-8, pro- and anti-inflammatory IL-4 and tumor necrosis factor (TNFa) The level of IL-4 was 2,4 times higher than in the control respectively (4,14 ± 0,24) pg/ml and (1,73 ± 0,13) pg/ml (p < 0,05). Comparison of IL-4 levels with the level of CD 22, IgG, IgA confirms its role in the stimulation of the humoral immune system, as a compensatory response to the depletion of the T-link in a chronic inflammatory process [5]. IL-8 levels were increased 1,9-fold, respectively (29,82 ± 3,15) pg/ml and (15,18 ± 1,82) pg/ml (p < 0,05). Increase in TNFa – 3,0 times – (7.64 ± 0.93) pg/ml against (2,55 ± 0,22) pg/ml (p < 0,05). The level of IL-6 in patients with AW was 2,7 times higher – (3,77 ± 0,45) pg/ml, while in the control – (1,62 ± 0,15) pg/ml (p < 0,05). Analyzing the cytokine profile data, we found the dependence of IL-6, IL-4 and IL-8 levels on the prevalence of AW and the presence of concomitant perifocal inflammation. Thus, the highest values were in patients with giant Bushke-Levenstein warts, which were accompanied by necrosis, secondary inflammation, severe tissue infiltration. While in patients with single AW cytokine status did not differ significantly from the values in the control group. Abstract Surveys showed that in patients with AW, the state of microcirculation was significantly different from that in the control group. The state of microcirculation in the control group was variable. The average microcirculation (M) was equal to (6,57 ± 0,10) perfusion units (p.u) with a high standard deviation (σ) – (1,95 ± 0,06) p.u and a coefficient of variation (Kv) – (29,13 ± 0,93) %, which indicates high modulation of blood circulation. Data on the state of blood circulation in the main group differed significantly from the control and depended on the nature of the spread of the pathological process. Circulation in single AW in comparison with the control group was characterized by a decrease in all three indicators: M – (4,60 ± 0,06) p.u (p < 0,05), σ – (0,56 ± 0,03) p.u (p < 0,05) and KV – (12,23 902 ± 0.58) % (p > 0,05). This variant of microcirculation is most consistent with the spastic form and, in our opinion, may be one of the factors in the implementation and spread of AW. At the same time in the group of patients with multiple AW there was a significant increase in M with average values of M – (12,29 ± 0,32) p.u (p < 0,05), which was characterized by monotony, σ – (0,66 ± 0,03) p.u (p < 0,05) with a low KV – (5,52 ± 0,22) % (p < 0,05). This type of blood flow in patients with multiple AW corresponds to the hyperemic type of microcirculation that accompanies the inflammatory process, and is characterized by increased blood flow with slowed outflow due to decreased venous circulation. The most pronounced changes in M were observed in patients with giant Bushke- Levenstein warts, in which in different areas there were changes in blood flow from increased hyperemic with average values of M – (18,92 ± 0,55) p.u (p < 0,05), monotonicity σ – (0,55 ± 0,21) p.u (p < 0,05), KV – (2,95 ± 1,17) % (p < 0,05); to a stagnant form at a combination of Bushke-Levenstein's condyloma with a necrosis and a phlegmon of area of a perineum: M – (19,81 ± 0,13) p.u (p < 0,05), monotonicity σ – 0,40 p.u., KV – 2,0 %. Abstract In these patients, we observed maximal values of interleukin levels, which ranged from IL-4 to (6,74 – 8,64) pg/ml; IL-8 – (104,0 – 140,7) pg/ml; IL-6 – (12,90 – 26,91) pg/ml; TNFα – (23,57 – 27,1) pg/ml. When comparing the course of the postoperative period, we observed a slower elimination of symptoms of post-traumatic reaction in patients of the second clinical group. The intensity of pain in the first group averaged (4,74 ± 0,24) points and ranged from weak and insignificant 1 - 4 points, which accompanied 23 (32,86 %) patients, moderate – 5 - 6 points in 28 (40,0 %) to strong – 7 - 8 points – 18 (25,71 %) and intolerable – 1 (1,43 %). In the second group, the average values of pain intensity were higher – (5,54 ± 0,29) points (p1, p2 > 0,05) due to an increase in cases of moderate and severe pain, which was determined with the following frequency: mild and minor – 6 (17,14 %), moderate – 17 (48,47 %), strong – 11 (31,43 %), intolerable – 1 (2,86 %). In both groups, excruciating pain accompanied the postoperative period in patients with giant Bushke-Levenstein warts, which required the appointment of narcotic analgesics on the first day. In the postoperative period in the first group the pain persisted for up to 4 days – (2,93 ± 0,09) days, in the second - up to 5-8 – (5,88 ± 0,17) days (р < 0,05). Post-traumatic perifocal reaction was observed in both groups. But these symptoms were eliminated in the first group for 3-5 days – (3,94 ± 0,10), while in the second for 6-9 903 days – (7,01 ± 0,18), (p < 0, 05), which significantly increased the length of stay in the hospital. In total, postoperative wound healing in 102 patients occurred according to the biological type of "healing under the scab". In 2 patients with giant Bushke-Levenstein warts, healing was secondary, due to the large volume of excision in both cases and the presence of phlegmon of the anogenital region in the patient of the first group. Complete epithelialization of the postoperative wound with discharge of the scab in patients of the first group occurred on average after 12 - 14 days – (12,97 ± 0,11) days in the second after - 14 - 18 – (15,62 ± 0,26) (р < 0,05). Abstract The term of wound healing in patients with giant Bushke-Levenstein warts was continued in the first group in a patient without purulent-septic complications and suturing for 14 days, and in a patient with complicated perineal phlegmon process up to 4 months. In the second group, the process of complete epithelialization occurred after 24 days. We monitored the dynamics of the interleukin profile on the 10th day of the postoperative period, namely in the phase of proliferation of postoperative wound healing. During treatment, we observed a decrease in cytokine levels, which was more pronounced in the first group. The average values of TNFa levels in patients of the first group on the 10th day of the postoperative period decreased in and were equal to (1,56 ± 0,14) pg/ml (p1 < 0,05). Clinically, this was manifested by an acute recurrence of the disease in men and women with multiple HA 12 and 14 days after surgical destruction and was accompanied by multiple formations. The level of IL-4 in the first group on the 10th day of the postoperative period was equal to (1,73 ± 0,07) pg/ml (p1 < 0,05) There was also a decrease in the level of IL-6 and IL-8, respectively (1,35 ± 0,22) pg/ml (p1 <0,05) and (8,34 ± 0,54) pg/ml (p1 <0.05). In the second group on the 10th day of the postoperative period, the dynamics of TNFa was more appropriate – (2,17 ± 0,12) pg/ml (p2 < 0,05) (p1, p2 < 0,05). There was a positive dynamics in the level of interleukins. IL-4 with values (2,0 ± 0,08) pg/ml (p2 < 0,05) (p1, p2 > 0,05); IL-6 – (1,60 ± 0,11) pg/ml (p2 < 0,05) (p1, p2 > 0,05); IL-8 – (12,47 ± 0,53) pg/ml (p2 < 0,05) (p1, p2 <0,05). In the second group, recurrence of the disease was observed in 3 (8.57%) after 1-3 months: in one man and woman with multiple genital warts and women with single. On the background of therapy in patients of the first group with multiple AW (n = 47) on the 10th day of the postoperative period on the background of complex therapy with L- 904 arginine, we observed positive changes in the mean microcirculation, characterized by increased variability of M - flaxmotions (7,34 ± 1,02) p.u, relative to the initial values (p1 < 0,05). Abstract At the same time, there was a significant increase in σ – (1.37 ± 0.03) p.u (p1 < 0,05) and Kv, which was equal to (19,08 ± 0,58) %, indicating the restoration of microcipculation. At single AW at all patients of the first group (n = 21) the shift of indicators of microcirculation towards normalization was also noted: M – (6,21 ± 0,07) p.u (p1 < 0,05); σ – (1,73 ± 0,04) p.u. (p1 < 0,05); Kv – (27,91 ± 0,71) % (p1 < 0,05). In the second group in patients with multiple AW (n = 19) the average microcirculation was equal to M – (8,27 ± 0,42) p.u (p2 < 0.05) (p1, p2 > 0,05); σ – (1,37 ± 0,05) p.u (p2 < 0,05) (p1, p2 > 0,05); Kv – (17,82 ± 1,11) % (p2 < 0.05) (p1, p2 > 0,05). In patients of the second group with single AW (n = 15) there were also significant positive changes: M – (5,60 ± 0,06) p.u (p2 < 0,05) (p1, p2 < 0,05); σ - (1,19 ± 0,05) p.u (p2 < 0.05) (p1, p2 < 0.05); Kv – (21,27 ± 0,85) % (p2 < 0,05) (p1, p2 < 0,05). Among patients of both groups with giant Bushke-Levenstein warts more pronounced positive dynamics occurred in the first group (one case in each), respectively, M – (10,99 ± 0,4) p.u (p1 < 0,05) and (11,02 ± 0,27) p.u (p2 < 0,05) (p1, p2 > 0,05); with a slight increase in σ and Kv, more pronounced in the first group. Conclusions. In patients with AW, there are shifts in the links of cellular and humoral immunity, manifested by depletion of cellular immunity – general lymphopenia, T- lymphopenia with a decrease in their absolute and relative numbers in all subpopulations, increased levels of B-lymphocytes with significant and Ig A. The proportional decrease in absolute and relative indicators of the lymphocyte pool with a significant increase in the level of B-lymphocytes and immunoglobulins in patients of the main group gives reason to consider it a consequence of the impact of HPV on this link. Significant increase in the level of cytokines IL-4, IL-6, IL-8 and TNFa in patients with AW reflects the prevalence of AW and corresponds to the nature of the perifocal inflammatory process, which should be used as a factor in predicting the postoperative period and treatment effectiveness. Abstract Changes in microcirculation in patients with AW of the anogenital region depend on the spread of AW and concomitant factors, such as perifocal inflammation due to bacterial contamination. Changes in microcirculation are realized in 3 variants: spastic - in patients with single AW, hyperemic - in patients with multiple AW, stagnant, which acquired maximum disorders in patients with giant Bushke-Levenstein warts. 905 The obtained results showed a significantly higher efficiency of complex treatment of acute genital warts of the anogenital region in the group of patients receiving a nitrogen donor L - arginine. In the group of patients receiving L-arginine (n = 70), there were 2 (2,86 %) relapses. In the control (n = 35) group – 3 (8,57 %) (p < 0,05). The use of nitrogen donor (L-arginine) in complex therapy allows to eliminate subjective symptoms, signs of secondary bacterial contamination in the preoperative period, postoperative pain, post-traumatic stress and to provide a significantly higher rate of epithelization, which optimizes the patient's stay. prognosis for recurrence of AW. References 1. Al-Awadhi, R, Al-Mutairi, N, Albatineh, A.N. Wassim Chehaden (2019). Association of HPV genotipes with external anogenital warts: across sectional study. BMC Infect Dis 19, 375. doi.org/10.1186/s12879-019-4005-4. 2. Aşkın Özge (2017). Anogenital HPV, Fundamentals of Sexually Transmitted Infections. Internet resours: IntechOpen. doi: 10.5772/intechopen.70017. 2. Aşkın Özge (2017). Anogenital HPV, Fundamentals of Sexually Transmitted Infections. 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https://openalex.org/W3122044517	https://europepmc.org/articles/pmc6164555?pdf=render	English	null	Targeting Amyloid Aggregation: An Overview of Strategies and Mechanisms	null	2,018	cc-by	17,266	Received: 7 August 2018; Accepted: 5 September 2018; Published: 9 September 2018 Abstract: Amyloids result from the aggregation of a set of diverse proteins, due to either speciﬁc mutations or promoting intra- or extra-cellular conditions. Structurally, they are rich in intermolecular β-sheets and are the causative agents of several diseases, both neurodegenerative and systemic. It is believed that the most toxic species are small aggregates, referred to as oligomers, rather than the ﬁnal ﬁbrillar assemblies. Their mechanisms of toxicity are mostly mediated by aberrant interactions with the cell membranes, with resulting derangement of membrane-related functions. Much effort is being exerted in the search for natural antiamyloid agents, and/or in the development of synthetic molecules. Actually, it is well documented that the prevention of amyloid aggregation results in several cytoprotective effects. Here, we portray the state of the art in the ﬁeld. Several natural compounds are effective antiamyloid agents, notably tetracyclines and polyphenols. They are generally non-speciﬁc, as documented by their partially overlapping mechanisms and the capability to interfere with the aggregation of several unrelated proteins. Among rationally designed molecules, we mention the prominent examples of β-breakers peptides, whole antibodies and fragments thereof, and the special case of drugs with contrasting transthyretin aggregation. In this framework, we stress the pivotal role of the computational approaches. When combined with biophysical methods, in several cases they have helped clarify in detail the protein/drug modes of interaction, which makes it plausible that more effective drugs will be developed in the future. Keywords: amyloid diseases; biocomputing; drug design; natural antiamyloids Targeting Amyloid Aggregation: An Overview of Strategies and Mechanisms Soﬁa Giorgetti 1, Claudio Greco 2, Paolo Tortora 3,4,* and Francesco Antonio Aprile 5,* 1 Department of Molecular Medicine, Institute of Biochemistry, University of Pavia, Via Taramelli 3b, 27100 Pavia, Italy; soﬁa.giorgetti@unipv.it 2 Department of Earth and Environmental Sciences, University of Milano-Bicocca, Piazza della Scienza 1 20126 Milano, Italy; claudio.greco@unimib.it y g 3 Department of Biotechnologies and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy 3 Department of Biotechnologies and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy 4 Milan Center for Neuroscience (Neuro-MI), 20126 Milano, Italy 5 Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK * Correspondence: paolo.tortora@unimib.it (P.T.); faa25@cam.ac.uk (F.A.A.); Tel.: +39-02-6448-3401 (P.T.); +44-1223-336-366 (F.A.A.) International Journal of Molecular Sciences International Journal of Molecular Sciences 1. Introduction Proteins generally require speciﬁc three-dimensional conformations in order to be soluble and function correctly in the body. Under stress conditions, normally soluble proteins can undergo structural changes and self-assembly that lead to their aggregation into insoluble deposits, referred as amyloids [1,2]. Amyloids from different proteins share several structural properties: they all have a ﬁbrillar morphology and cross-β structure, whereby intermolecular main-chain hydrogen bonding acts as one major stabilising interaction [1,3]. Frequently, they also have repetitive hydrophobic or polar Int. J. Mol. Sci. 2018, 19, 2677; doi:10.3390/ijms19092677 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 2677 2 of 27 interactions along the ﬁbril axis [3]. They are highly rigid [4], resistant to thermal [5] and chemical denaturation and degradation [6]. Recent technological advances in structural biology, which include solid-state nuclear magnetic resonance (SSNMR) [7], and cryo electron microscopy (cryo-EM) [8], allowed scientists to determine the structure of amyloids at the molecular level directly from patient tissues. These studies have shown that amyloid aggregates formed in vivo consist of differently modiﬁed variants of the amyloidogenic protein and are associated and often co-aggregated with components of the protein homeostasis system, such as molecular chaperones [9]. The presence of amyloids is usually related to pathological conditions generally called amyloidoses [1]. Amyloidoses can be either localized or systemic, according to whether or not the amyloidogenic protein aggregates are in the site of synthesis, respectively, and they can have characteristic molecular and clinical hallmarks, depending on the site of deposition [10]. Neurodegenerative diseases including Alzheimer’s (AD) and Parkinson’s diseases (PD) and corea of Hungtington (CH) represent a highly prevalent class of fatal localized amyloidoses in which amyloid deposits form in the nervous system where they induce the death of speciﬁc neuronal cell types [3]. In systemic amyloidoses, such as immunoglobulin light chain (AL), transthyretin (TTR), and dialysis-related amyloidoses (DRA), several organs are affected as the amyloidogenic protein is distributed in different sites of the body while it travels from the site of synthesis [10]. 1. Introduction The formation of amyloids depends on extremely complicated aggregation processes, in which various aggregation intermediates form through a combination of simultaneous microscopic events [11], namely: (1) primary nucleation, in which initial small soluble aggregates form from monomers interacting in solution; (2) elongation, in which existing ﬁbrils increase in length by monomer addition; (3) secondary nucleation processes, in which the surface of existing aggregates catalyses the formation of new small soluble aggregates and (4) fragmentation, in which existing ﬁbrils break apart, increasing the total number of ﬁbrils [12]. The contributions of each of these microscopic events to the lag and growth phases are highly protein speciﬁc. Small soluble aggregates formed during the amyloid aggregation are generally called oligomers. These protein species are extremely heterogeneous and can rapidly interconvert into protoﬁbrils. They are extremely toxic and believed to play a major role in cell and tissue toxicity, particularly in neurodegenerative diseases [13–16]. The mechanisms of toxicity of amyloid oligomers are still under debate. In vivo and in vitro studies have demonstrated that high levels of amyloid oligomers are able to over-stimulate glutamatergic synaptic transmission and cause synapse loss [17–22]. It has also been reported that oligomers are able to interact with the cell membrane [16,23] and are associated with oxidative stress [14,24], altered calcium homeostasis (which is the most sensitive to alterations of membrane permeability, due to its huge concentration gradient across the plasma membrane, i.e., about four orders of magnitude) [25], mithocondrial dysfunction [26], and inﬂammation [27]. In the case of systemic amyloidoses, the mechanisms of toxicity are probably different, and both insoluble ﬁbrils and soluble oligomers are important for cytotoxicity [28]. For example, it has been reported that a further pathogenic effect besides that played by oligomers results from the massive extracellular accumulation of amyloid ﬁbrils, which cause mechanical stress, and ultimately organ function impairment and failure [10]. In neurodegenerative diseases, the amyloidogenic proteins are often intrinsically disordered, which means that no misfolding event needs to occur to initiate their aggregation. Instead, the transition to amyloid is generally directly triggered by mutations, post-translational modiﬁcations (such as proteolytic cleavages or chemical modiﬁcation of the protein side chains or backbone), and interactions with the environment, such as membranes and lipids [29,30]. 1. Introduction Such a discovery unleashed a ﬂurry of research aimed at designing new biocompatible models of in vitro ﬁbrillogenesis for this and other proteins such as TTR [34], which are especially suitable for understanding the in vivo mechanism of amyloidogenesis and offering a reliable tool for drug discovery. β p g y q the protein acquires a strong propensity to misfold and aggregate. Normally, the intrinsic propensity to misfold is inhibited by the stabilizing interaction with the heavy chain within the MHC I and this observation offers the natural demonstration that amyloidogenesis can be prevented by stabilization through protein/protein or protein/ligand interactions. There is, so far, only one reported mutation associated to β2-m amyloidosis occurring in the absence of haemodialysis and low concentration of circulating β2-m. The study of the mutation Asp76Asn has disclosed a new scenario because it led to the discovery that the partial unfolding and amyloid transition can be obtained by simply playing through the biomechanical forces generated by the turbulent fluid flow of a physiologic fluid at the interface with hydrophobic patches [33]. Such a discovery unleashed a flurry of research aimed at designing new biocompatible models of in vitro fibrillogenesis for this and other proteins such as TTR [34], which are especially suitable for understanding the in vivo mechanism of amyloidogenesis and offering a reliable tool for drug discovery. The present review will focus on and discuss effects and therapeutic efficacy of drugs and nutraceuticals currently in use or under investigation, which are endowed with a well-documented The present review will focus on and discuss effects and therapeutic efﬁcacy of drugs and nutraceuticals currently in use or under investigation, which are endowed with a well-documented capability of inhibiting the appearance of toxic protein aggregates. In doing so, we mainly aim at highlighting the methodological aspects related to the mechanisms of action of such compounds and to the development of new ones, rather than providing a comprehensive survey of this topic, provided this will be ever possible. In particular, their mode of interaction with the proteins committed to amyloidogenesis will be analysed. It should be stressed, however, that many such compounds also act by mitigating some of the aforementioned toxic effects at the cellular level. In any case, evidence of beneﬁcial effects precisely fulﬁlled at the level of protein aggregation will be presented and discussed, when available. 1. Introduction Most of the theoretical body on the mechanism of amyloidogenesis and amyloid toxicity for systemic amyloidoses derives from experimental studies carried out on three types of proteins: LC, TTR and β2-microglobulin (β2-m) [31]. A generic step for the amyloid transition of these globular proteins and the acquisition of cytotoxic properties is the partial unfolding of the native state. Also in this case, Int. J. Mol. Sci. 2018, 19, 2677 I t J M l S i 2018 19 3 of 27 3 f 26 mutations and selective proteolytic cleavages are certainly two major determinants in facilitating the amyloid transition. Studies on β2-m were particularly informative because the clinical counterpart of the experimental and theoretical side is relatively simple [32], at least simpler than for TTR and immunoglobulin. There are only two types of amyloidoses caused by β2-m, one acquired and one genetically transmitted, and both extensively characterized. counterpart of the experimental and theoretical side is relatively simple [32], at least simpler than for TTR and immunoglobulin. There are only two types of amyloidoses caused by β2-m, one acquired and one genetically transmitted, and both extensively characterized. Dialysis-related amyloidosis is the acquired form of the disease and is caused by a substantial increase in monomeric β2-m plasma concentration resulting from haemodialysis. As a consequence, g y y Dialysis-related amyloidosis is the acquired form of the disease and is caused by a substantial increase in monomeric β2-m plasma concentration resulting from haemodialysis. As a consequence, the protein acquires a strong propensity to misfold and aggregate. Normally, the intrinsic propensity to misfold is inhibited by the stabilizing interaction with the heavy chain within the MHC I and this observation offers the natural demonstration that amyloidogenesis can be prevented by stabilization through protein/protein or protein/ligand interactions. There is, so far, only one reported mutation associated to β2-m amyloidosis occurring in the absence of haemodialysis and low concentration of circulating β2-m. The study of the mutation Asp76Asn has disclosed a new scenario because it led to the discovery that the partial unfolding and amyloid transition can be obtained by simply playing through the biomechanical forces generated by the turbulent ﬂuid ﬂow of a physiologic ﬂuid at the interface with hydrophobic patches [33]. 1. Introduction In Figure 1, the general features of the amyloidogenic pathway are shown, also highlighting the step(s) where these compounds interfere with the process, at least in most cases. In Table 1, a wide compilation thereof is provided, whereas the formulas of those compounds, which will be discussed in the present review are presented in Figure 2. capability of inhibiting the appearance of toxic protein aggregates. In doing so, we mainly aim at highlighting the methodological aspects related to the mechanisms of action of such compounds and to the development of new ones, rather than providing a comprehensive survey of this topic, provided this will be ever possible. In particular, their mode of interaction with the proteins committed to amyloidogenesis will be analysed. It should be stressed, however, that many such compounds also act by mitigating some of the aforementioned toxic effects at the cellular level. In any case, evidence of beneficial effects precisely fulfilled at the level of protein aggregation will be presented and discussed, when available. In Figure 1, the general features of the amyloidogenic pathway are shown, also highlighting the step(s) where these compounds interfere with the process, at least in most cases. In Table 1, a wide compilation thereof is provided, whereas the formulas of those compounds, which will be discussed in the present review are presented in Figure 2. Finally, in the last chapter we will highlight how in vitro and in silico approaches have contributed to the present knowledge and how they have complemented each other. Figure 1. Schematic representation showing the intermediates of a generic amyloid aggregation pathway (monomers, oligomers, protofibrils and fibrils). The scheme includes a membrane as well, which in some cases can play a role in the process, such as for α-syn. In the figure, the main classes of anti-aggregation molecules discussed in this review are connected to the aggregated species to which they have been reported to preferentially bind. Figure 1. Schematic representation showing the intermediates of a generic amyloid aggregation pathway (monomers, oligomers, protoﬁbrils and ﬁbrils). The scheme includes a membrane as well, which in some cases can play a role in the process, such as for α-syn. In the ﬁgure, the main classes of anti-aggregation molecules discussed in this review are connected to the aggregated species to which they have been reported to preferentially bind. Figure 1. 1. Introduction Schematic representation showing the intermediates of a generic amyloid aggregation pathway (monomers, oligomers, protofibrils and fibrils). The scheme includes a membrane as well, which in some cases can play a role in the process, such as for α-syn. In the figure, the main classes of anti-aggregation molecules discussed in this review are connected to the aggregated species to which they have been reported to preferentially bind. Figure 1. Schematic representation showing the intermediates of a generic amyloid aggregation pathway (monomers, oligomers, protoﬁbrils and ﬁbrils). The scheme includes a membrane as well, which in some cases can play a role in the process, such as for α-syn. In the ﬁgure, the main classes of anti-aggregation molecules discussed in this review are connected to the aggregated species to which they have been reported to preferentially bind. 4 of 27 Int. J. Mol. Sci. 2018, 19, 2677 Table 1. A compilation of anti-aggregation compounds against amyloid diseases. Table 1. A compilation of anti-aggregation compounds against amyloid diseases. 1. Introduction Molecule Class Target Protein Disease Key References Squalamine * Sterol α-syn PD [35] Trodusquemine Sterol α-syn PD [36] Tetracycline Tetracyclines Prp/Aβ APrP/AD [37] Doxycycline Tetracyclines Aβ/PrP/β2-m/TTR/LC AD/Aβ2-m/ATTR/AL [37–45] 4′Iodo-4′-doxorubicin Anthracyclines AL/SAA/TTR/Aβ/PrP AD/AL/AA/ATTR/Aβ2-m [46,47] Acid fuchsin Triarylmethane dye IAPP AD/diabetes [48] Fast Green FCF Triarylmethane dye IAPP AD/diabetes [49] Crystal violet Triarylmethine dye tau AD [50] N744 Cyanine dye tau AD [51] Congo red Azo dye Aβ/casein/PrP/α-syn AD/systemic amyloidosis/ prion disease/PD [52,53] Resveratrol Polyphenol Aβ/IAPP AD/diabetes [54] Curcumin Polyphenol Aβ/tau/α-syn /htt/PrP AD/PD/CH [54] EGCG Polyphenol Aβ/α-syn/htt/TTR/IAPP/ PAP248–286/HEWL/k-casein and calcitonin/polyQ proteins AD/PD/CH/HIV infectivity [54] Quercetin and myricetin Polyphenol Aβ/α-syn/insulin/IAPP AD/PD/diabetes [54] Olive oil phenols Polyphenol Aβ/IAPP AD/diabetes [54] Oleuropein Polyphenol Baicalein (quinone) Polyphenol tau AD [55] Tafamidis (Vyndaqel) Benzoxazole TTR ATTR [56] Tolcapone Benzophenone TTR ATTR [57] Mds84 Palindromic ligand TTR ATTR [57] Oleocanthal Aldehyde tau AD [55] Cinnamaldehyde Aldehyde tau AD [55] Asperbenzaldehyde Aldehyde tau AD [55] β-Breakers Peptide Aβ AD [58] β-Breakers Peptide IAPP AD/diabetes [59] β-Breakers Peptide IAPP AD/diabetes [60] β-Breakers Peptide Aβ/IAPP AD/diabetes [61] (Bi)Cyclic peptides Peptide Aβ AD [62] Nanobodies Single domain antibodies α-syn/Aβ/lysozyme/β2-m AD/PD/systemic [63–66] Rationally designed antibodies Single domain antibodies Aβ/α-syn/IAPP AD/PD/diabetes [67–69] Aducanumab Monoclonal antibody Aβ AD [70] mAb158 * (BAN2401) Monoclonal antibody Aβ AD [71] Crenezumab Monoclonal antibody Aβ AD [72] Gantenerumab Monoclonal antibody Aβ AD [73] Solanezumab ** Monoclonal antibody Aβ AD [74] Tanshinones Diterpene Aβ AD [75] Dopamine and L-dopa Neurotransmitter Aβ/α-syn/IAPP AD/PD/diabetes [76,77] Methylene Blue Thiazine dye tau/PrP/ Aβ AD [55,78,79] Notes: * α-Syn aggregation is induced by its interaction with biological membranes. Squalamine inhibits the aggregation of α-syn by displacing it from the membranes, Covalent inhibitors, * Murine version of BAN2401, **** not taken further. Information regarding the progress of clinical/preclinical trials of the compounds presented in the table is available at the following link: https://clinicaltrials.gov. Abbreviation: α-syn (α-synuclein); PrP (prion protein); Aβ (amyloid beta); APrP (Prp amyloidosis); β2-m (β2-microglobulin); Aβ2-m (β2-microglobulin amyloidosis); TTR (transthyretin); LC (Immunoglobulin light chain); ATTR (transthyretin amyloidosis); AL (immunoglobulin light chain amyloidosis); SAA (serum amyloid A); AA (serum amyloid A amyloidosis); IAPP (amylin); htt (huntingtin); CH (corea of Hungtington); HEWL (egg-white lysozyme); polyQ (polyglutamine); PAP248–286 (prostatic acidic phosphatase fragment); PD (Parkinson’s disease); AD (Alzheimer’s disease). Notes: * α-Syn aggregation is induced by its interaction with biological membranes. 1. Introduction Squalamine inhibits the aggregation of α-syn by displacing it from the membranes, Covalent inhibitors, * Murine version of BAN2401, ** not taken further. Information regarding the progress of clinical/preclinical trials of the compounds presented in the table is available at the following link: https://clinicaltrials.gov. Abbreviation: α-syn (α-synuclein); PrP (prion protein); Aβ (amyloid beta); APrP (Prp amyloidosis); β2-m (β2-microglobulin); Aβ2-m (β2-microglobulin amyloidosis); TTR (transthyretin); LC (Immunoglobulin light chain); ATTR (transthyretin amyloidosis); AL (immunoglobulin light chain amyloidosis); SAA (serum amyloid A); AA (serum amyloid A amyloidosis); IAPP (amylin); htt (huntingtin); CH (corea of Hungtington); HEWL (egg-white lysozyme); polyQ (polyglutamine); PAP248–286 (prostatic acidic phosphatase fragment); PD (Parkinson’s disease); AD (Alzheimer’s disease). aggregation of α-syn by displacing it from the membranes, Covalent inhibitors, * Murine version of BAN2401, ** not taken further. Information regarding the progress of clinical/preclinical trials of the compounds presented in the table is available at the following link: https://clinicaltrials.gov. Abbreviation: α-syn (α-synuclein); PrP (prion protein); Aβ (amyloid beta); APrP (Prp amyloidosis); β2-m (β2-microglobulin); Aβ2-m (β2-microglobulin amyloidosis); TTR (transthyretin); LC (Immunoglobulin light chain); ATTR (transthyretin amyloidosis); AL (immunoglobulin light chain amyloidosis); SAA (serum amyloid A); AA (serum amyloid A amyloidosis); IAPP (amylin); htt (huntingtin); CH (corea of Hungtington); HEWL (egg-white lysozyme); polyQ (polyglutamine); PAP248–286 (prostatic acidic phosphatase fragment); PD (Parkinson’s disease); AD (Alzheimer’s disease). Int. J. Mol. Sci. 2018, 19, 2677 5 of 27 Figure 2. Chemical structures of the antiamyloid compounds discussed in the present review. Figure 2. Chemical structures of the antiamyloid compounds discussed in the present review. Figure 2. Chemical structures of the antiamyloid compounds discussed in the present review. Figure 2. Chemical structures of the antiamyloid compounds discussed in the present review. Table 1. A compilation of anti-aggregation compounds against amyloid diseases. Molecule Class Target Protein Disease Key References Finally, in the last chapter we will highlight how in vitro and in silico approaches have contributed to the present knowledge and how they have complemented each other. q 2.1. Probes and Diagnostic Molecules Some of these molecules (Table 1) have also been proved to affect amyloid aggregation to different extents and with different mechanisms. Congo red (CR) is probably the most famous case. This molecule is used for determining the amyloid nature of protein aggregates from biological samples. In particular, CR shows green birefringence under polarized light in the presence of amyloid aggregates. Recent studies have shown that CR is able to inhibit the aggregation of a series of proteins, including amyloid beta (Aβ), casein, the prion protein (PrP), α-syn (α-synuclein) [52,53]. In particular, CR is able to accelerate these aggregation processes, thus reducing the life-time of toxic oligomeric species [53]. Another relevant compound in this category is crystal violet, which can be used for the detection of amyloid aggregates in histologic preparations for light microscopy. This molecule has been reported to an effective inhibitor of tau aggregation [50]. Structurally similar molecules such as acid fuchsin and fast green FCF have been reported to have the same effect [55]. It is also worth mentioning methylene blue, a phenothiazine used as a treatment for haemoglobin conditions and as stain for cells and tissues in endoscopic procedures. While not speciﬁcally designed to be a ﬁbril probe, this molecule has anti-amyloid effects. In particular, it has been shown that methylene blue can inhibit the oligomerization of amyloidogenic proteins with different mechanisms. More speciﬁcally, in the case of PrP, methylene blue inhibits the formation of oligomers by affecting its ﬁbrillization [78]. In the case of Aβ, methylene blue increases ﬁbrillization of the peptide, depleting monomers available for oligomerization [79]. q 2.1. Probes and Diagnostic Molecules Tetracycline Tetracyclines Prp/Aβ APrP/AD [37] Doxycycline Tetracyclines Aβ/PrP/β2-m/TTR/LC AD/Aβ2-m/ATTR/AL [37] [38] [37] [39] [40–45] 4′Iodo-4′-doxorubicin Anthracyclines AL/SAA/TTR/Aβ/PrP AD/AL/AA/ATTR/Aβ2-m [46] [47] Acid fuchsin Triarylmethane dye IAPP AD/diabetes [48] Fast Green FCF Triarylmethane dye IAPP AD/diabetes [49] Crystal violet Triarylmethine dye tau AD [50] N744 Cyanine dye tau AD [51] Since the origins of the amyloid ﬁeld, several compounds have been developed for research purposes in order to characterise the mechanisms of formation and structure of amyloids. In particular, many structural analyses are currently based on the use of molecular probes that change their spectroscopic properties upon binding to the amyloid ﬁbrils. This is the case of thioﬂavin-T (ThT), a ﬂuorescent molecule that is now routinely used for monitoring the time evolution of the amyloid aggregates in vitro [80] or the compounds 1-anilinonaphthalene-8-sulfonic acid (ANS) and 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bisANS), which are employed in a similar way to ThT. However, as they recognise solvent-exposed hydrophobic patches of proteins, they are also used for characterising early-stage oligomerization and initial structural rearrangements of amyloidogenic proteins [81] Congo red Azo dye Aβ/casein/PrP/α-syn AD/systemic amyloidosis/prion disease/PD [52,53] Resveratrol Polyphenol Aβ/IAPP AD/diabetes [54] Curcumin Polyphenol Aβ/tau/α-syn /htt/PrP AD/PD/CH [54] Furthermore, an increasing research effort has now focused on improving the afﬁnity of these molecules in order to develop high sensitivity methods for the study of amyloids. For example, it has been show that ThT dimers can have a 70-fold higher afﬁnity for amyloid ﬁbrils than the original molecule while maintaining its ﬂuorescent properties and binding selectivity [82]. The development of molecules of this type will facilitate in vitro studies of amyloids at nanomolar concentration, which represents a more relevant condition for characterizing their mechanisms of toxicity. Int. J. Mol. Sci. 2018, 19, 2677 6 of 27 6 of 27 There are several other molecules, such as Thioﬂavin S or [50-(p-Hydroxyphenyl)-2,20-bithienyl-5-yl]-methylidene}-propanedinitrile (NIAD-4) [83], which have been successfully employed for diagnostic and research applications ex vivo or on tissues thanks to their selective binding to amyloid aggregates. Some of these molecules (Table 1) have also been proved to affect amyloid aggregation to different extents and with different mechanisms. molecules, or There are several other molecules, such as Thioﬂavin S or [50-(p-Hydroxyphenyl)-2,20-bithienyl-5-yl]-methylidene}-propanedinitrile (NIAD-4) [83], which have been successfully employed for diagnostic and research applications ex vivo or on tissues thanks to their selective binding to amyloid aggregates. 2.2. Anthracyclines and Tetracyclines In 1995, staring at the clinical observation that the anthracycline 4′-iodo-4′-deoxy-doxorubicin (IDOX) was able to induce amyloid resorption in patients with AL amyloidosis, Merlini et al. showed the capability of this drug to interact with several types of amyloid ﬁbrils and to inhibit the amyloid conversion of native proteins [46,47]. However, due to its intrinsic cardiotoxicity, the clinical exploitation of the drug was discontinued. The search for structural analogues of IDOX resulted in the identiﬁcation of tetracyclines as good candidates for mimicking the IDOX activity despite the lack of signiﬁcant cardiotoxicity. A conﬁrmation of the hypothesized anti-amyloid efﬁcacy of tetracyclines came from experiments on inhibition of PrP infectivity in animal models [84]. The generic effect of tetracyclines in interfering with amyloid formation inspired further investigation on the mechanism of interaction with amyloid structure and consequent blocking of amyloid growth. Through a molecular mechanic approach, Cosentino et al highlighted the crucial role of the hydrophobic core given by aromatic rings in the generic interaction with amyloid [85]. This study provided insight into how different polar substituents could determine the speciﬁcity of the interaction between various analogues of tetracyclines with different types of ﬁbrils. The fact that the afﬁnity for tetracyclines differs from ﬁbril to ﬁbril is most likely based on the structural heterogeneity and polymorphisms of ﬁbrils now clearly emerging from their structure solved at the atomic level by solid state NMR and cryo-EM [86]. Thus, the overall picture of the drug’s mode of action that emerges from available data is multifaceted. Apparently, tetracyclines not only bind mature ﬁbrils, but can also interact with soluble precursors of insoluble amyloid ﬁbrils: monomers and oligomers. In the case of the Aβ peptide, tetracyclines bind oligomers, but not the monomer [38]; in the case of the globular protein β2-m the binding not only involves oligomers but also the monomer through a binding site highly inﬂuenced by the physical-chemical properties of the environment [39]; furthermore, in the case of ataxin-3 (ATX3), tetracycline only binds oligomers via functional groups, mostly hydrophobic, located on one edge of the molecule, probably shielding to some extent the aggregate from the Int. J. Mol. Sci. 2018, 19, 2677 Int. J. Mol. Sci. 2018, 19, 2677 7 of 27 medium [87,88]. 2.2. Anthracyclines and Tetracyclines It is worth noting that also ﬁbrils, upon binding to tetracyclines, deeply rearrange their structure resulting in the formation of disordered insoluble material lacking the typical features of amyloid ﬁbrils [40]. Regardless of the molecular target and mechanism of binding, the capacity of tetracyclines to inhibit the intrinsic toxicity of these soluble conformers is apparently due to the drugs’ capability to structurally rearrange the toxic oligomers [41], thus converting them into inactive molecules. The best investigated type of tetracyclines is doxycycline (DOX), not only because is one of the most effective conformers on several type of ﬁbrils in vitro, but also because it has been used in vivo for many years as a wide-spectrum antibiotic with no appreciable adverse effects. Its use in amyloidosis just represents the repurposing of an old drug on a new target. Based on the anti-amyloid properties demonstrated in vitro, the clinical efﬁcacy of DOX is now under investigation in at least three types of systemic amyloidoses (http://clinicaltrials.gov). In TTR-related amyloidosis a phase-3 clinical trial is in the stage of patients recruitment. In this study, DOX is used in combination with Tauroursodeoxycholic acid and the trial is designed on the basis of the results of a previous phase-2 study, showing the efﬁcacy of this treatment in stabilizing the disease [42]. DOX was used in an exploratory off-label study on three patients affected by a severe form of dialysis-related amyloidosis (DRA) and although the amyloid mass was not apparently reduced, the patients experienced a very signiﬁcant reduction of the ostheoarticular pain, as well as a remarkable improvement of the active and passive movements [43]. Although the mechanism in vivo is not clariﬁed, the beneﬁts of this treatment were recently conﬁrmed by Piccoli et al., who recommend the DOX treatment as antalgic therapy for this kind of patients [44]. Although clinical trials for the validation of the treatment of this amyloidosis are not currently ongoing, DOX has received the designation of orphan drug by the European Medicines Agency for the treatment of DRA and hopefully a trial will be designed soon because there is no treatment for this very debilitating disease. Several clinical trials are now active or in the pipeline in AL amyloidosis caused by the ﬁbrillar deposition of immunoglobulin light chains. 2.2. Anthracyclines and Tetracyclines In these trials, the purpose is to evaluate potential beneﬁts on the disease outcome by the addition of DOX to standard chemotherapy used in these patients. These studies were strongly encouraged by the data reported by Wechalekar et al. [45], showing that addition of DOX to standard chemotherapy signiﬁcantly reduced the mortality in patients in advanced state of the disease. 2.4. Peptides and Engineered Antibodies One very demanding goal when designing anti-aggregation compounds is the development of highly speciﬁc molecules [92]. For this purpose, scientists have then looked at molecular biology and protein engineering as a solution in order to generate peptides and proteins for therapeutic applications. In particular, small peptides, generally referred to as β-sheet breakers or simply β-breakers, have been reported to affect the formation and stability of amyloid aggregates [93]. β-breakers are soluble short sequence portions of amyloidogenic proteins. As protein aggregation is a self-assembly process, β-breakers interact with the same sequences within amyloidogenic proteins blocking their aggregation or promoting the disaggregation of existing ﬁbrils [93]. They have been shown to be effective in vitro in the case of Aβ [94]. Nevertheless, they are poorly stable, in vivo, being prone to proteolytic degradation and having a relatively short half-life [95,96]. To overcome these limitations, scientists are trying several chemical modiﬁcations including N-methylation, the incorporation of unnatural amino acids, and cyclization [97]. Small engineered protein domains can act as potent inhibitors of amyloid aggregation as well. This is the case, for example, for some antibody mimetics, in particular some afﬁbodies, which mimic the high afﬁnity binding of antibodies, while being structurally distinct. Among them, β-wrapins have been reported to stabilize amyloidogenic proteins in β-hairpin conformations, thus preventing self-assembly or promoting the disaggregation of preformed oligomers [98]. These molecules have been proven to be effective in inhibiting the aggregation of Aβ, α-syn, and IAPP [98–100]. Antibodies and antibody fragments also have anti-aggregation properties. In particular, monoclonal antibodies probably represent at the moment the class of protein therapeutics with the most positive recent result from clinical trials. For example, the antibodies Aducanumab and BAN2401 [70,71] have successfully passed phase 2 clinical trials in the context of passive immunotherapy protocols against Alzheimer’s and Parkinson’s diseases, with Aducanumab currently giving positive results in phase 3 clinical trials where it shows dose-dependent clearance of amyloid deposits and slows down cognitive decline. Also, antibody fragments have been proved to be effective anti-aggregation molecules. For example, camel single domain antibodies, generally referred as nanobodies, are extremely effective inhibitors of the aggregation of several amyloidogenic proteins, including lysozyme [63], α-synuclein (α-syn) [64], Aβ [65], tau [101], and β2-m [66]. They have also been proved to be effective diagnostic tools for distinguishing amyloid ﬁbrils at different maturation stages [102]. 2.3. Sterols Sterols are a class of steroids, which are naturally produced by several organisms, including plants and bacteria. In particular, a broad-spectrum of them has been isolated from the dogﬁsh shark Squalus acanthias, initially for their antibiotic properties against both Gram-negative and Gram-positive bacteria, and fungicidal anti-protozoa activity [89]. Among these molecules, the compound squalamine has been proved to be effective against cancer [90] and, very recently, against PD. In particular, squalamine is able to inhibit the aggregation of the protein α-syn [35], whose deposition into Lewy bodies in a hallmark of PD [91]. In this regard, the mechanism of action of squalamine is an example of indirect effect of a molecule on the aggregation of an amyloidogenic protein. α-syn is known for being very soluble at normal pH, even at very high (mM) concentrations. In order to aggregate, α-syn requires the presence of hydrophobic surfaces, such as lipid membranes, where α-syn monomers are attracted to and nucleate [30]. Squalamine has been proved to inhibit α-syn aggregation by displacing monomers from the membranes [35]. Recently, a squalamine derivative, called trodusquemine, has been shown to affect the aggregation of α-syn as well [36]. In addition to displacing α-syn monomers similar to the mechanism fulﬁlled by squalamine, trodusquemine directly interacts with α-syn to inhibit the secondary nucleation of aggregation [36]. Int. J. Mol. Sci. 2018, 19, 2677 8 of 27 8 of 27 2.4. Peptides and Engineered Antibodies The so-called grafted amyloid-motif antibodies (or gammabodies) represent a valuable alternative class of anti-aggregation antibodies. Gammabodies are single domain human antibodies, where the complementarity-determining regions are replaced by aggregation-prone sequences from amyloidogenic proteins [67]. They then act as β-breakers with the advantage of being more soluble thanks to the stabilizing effect provided by the presence of the single-domain antibody scaffold. In addition, recent advances have disclosed new possibilities for the rational development of anti-aggregation antibody molecules [68]. In this regard, single domain antibodies have been rationally designed to speciﬁcally inhibit the aggregation of α-syn, amylin (the causative agent of islet amyloid in type-2 diabetes, IAPP) and Aβ [68,69]. Rational design has also been applied to other classes of proteins, such as molecular chaperones. They are very well known for being naturally occurring effective inhibitors of protein aggregation [9,103,104], but also for being highly non-speciﬁc, as they interact with any solvent-exposed protein hydrophobic patch. In order to increase their speciﬁcity towards amyloidogenic proteins, scientists have designed chaperone variants carrying peptides, which selectively interact with a given protein when found in aggregated conformation [105,106]. Int. J. Mol. Sci. 2018, 19, 2677 9 of 27 9 of 27 2.5.1. (−)-Epigallocatechin-gallate (EGCG) and Related Compounds 2.5.1. (−)-Epigallocatechin-gallate (EGCG) and Related Compounds 2.5.1. (−)-Epigallocatechin-gallate (EGCG) and Related Compounds EGCG is the major catechin found in the leaves of green tea. They also contain a variety of related, structurally simpler molecules, in particular (−)-epigallocatechin (EGC) and gallic acid (GA), whose effects are qualitatively similar to those exerted by EGCG [107,108]. Current literature shows ECGC’s capability to prevent the formation of aggregates from several potentially amyloidogenic proteins or peptides, including Aβ, α-syn [109], IAPP [110], AL [111], polyglutamine (polyQ)-containing proteins, including huntingtin (htt) [112] and ATX3 [87,113]. Although the precise mechanisms by which EGCG fulﬁls its action differ in details depending on the different target proteins, the trait most often observed is the compound’s capability to redirect the aggregation towards off-pathway, non-toxic, β-sheet-poor aggregates, and/or remodeling the aggregates after their formation (as, for instance, in the case of htt and IAPP), rather than just retarding amyloid aggregation [112,114]. It also can interact with both monomeric protein and oligomeric aggregates. Concerning the non-covalent interactions underlying the antiamyoid action of EGCG and related compounds, plenty of work highlights a complex pattern. In particular, hydrogen bonding with both protein backbone and hydrophilic side chains has been identiﬁed, as well as hydrophobic interactions, including those with aromatic residues [115]. This pattern points to a non-speciﬁc binding, as clearly supported by the large repertoire of proteins, both folded and disordered, EGCG can interact with. Remarkably, EGCG was also proved to covalently bind to lysines of target proteins via Schiff base formation, which might be one factor allowing irreversible protein remodelling into non-toxic aggregates [116]. Still with regard to covalent modiﬁcation EGCG can undergo, it has long been known that this molecule is subject to auto-oxidation, an issue obviously related to its bioavailability [117]. However, it has been recently reported that the oxidation products, i.e., quinone or quinonoid substances, are even more effective in preventing amyloid aggregation, quite likely by covalently binding to target protein [118]. Some literature is also available regarding the antiamyloid effects of smaller polyphenols structurally related to EGCG, in particular EGC and GA. In general, they also were proved to be effective antiamyloid agents, although to a somewhat lesser extent [113,119]. 2.5. Polyphenols Polyphenols are a class of compounds whose structure is characterized by the presence of several phenol units. They include a wealth of structurally diverse molecules, although they also share in part the mechanisms of action. Besides their capability to prevent or retard amyloid aggregation, several additional effects have been assigned to them, which are beneﬁcial for human health. Most notably, they are endowed with antioxidant and anticancer properties, the latter being mediated by inhibition of antiangiogenesis. The main types of polyphenols are discussed below. 2.5.4. Oleuropein Oleuropein, the main phenolic compound of olive oil [139], is endowed with several beneﬁcial effects on human health, the most prominent being anti-tumor [139], anti-inﬂammatory [140] and antioxidative activities [141], besides its capability to prevent the formation of toxic amyloid aggregates. This compound and its aglycone form have been proved to exert anti-amyloidogenic effects on Aβ [142], α-syn [143], β2-m [144], TTR [145], IAPP [146], and tau protein [147]. Similar to other polyphenols, the described modes of action are somewhat different depending on the protein assayed, but also share the basic features. Most often, the aggregation is redirected towards non-toxic, off-pathways. Furthermore, oleuropein displays the remarkable capacity of hindering protein binding to the plasma membrane, a key event in inducing cytotoxicity, as shown in the case of α-syn, IAPP and TTR. Interestingly, the decreased toxicity of the aggregates generated by this latter protein is likely related to the poor interaction between the resulting TTR/oleuropein aglycone complexes and monosialotetrahexosylganglioside 1 (GM1) found in the lipid rafts domains of the plasma membrane [145]. Thus, although no data are available as regards oleuropein’s functional groups involved in protein interaction, it is well established that its action on the amyloidogenic pathway results in signiﬁcant cytoprotective effects. 2.5.3. Curcumin Curcumin ((1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is abundantly found in the rhizomatous plant turmeric that grows in southeast Asia. It is a major component of Indian curries [127,128]. This molecule has been proved to interfere with the amyloid aggregation of Aβ [129–131], α-syn [132], tau [133,134] and PrP, although in this latter case there is no evidence that this interaction prevents of the appearance of toxic aggregates [135]. Overall, curcumin has been shown to prevent oligomerization, as in the case of Aβ and tau [134,136], redirect the aggregation towards nontoxic oligomers (Aβ and α-syn) [130,132] and even disaggregate preformed ﬁbrils (Aβ, tau and α-syn) [132,134,136]. Stacking interactions between aromatic rings of the compound and aromatic residues have been implicated in the aforementioned effects, as well as hydroxy groups on the aromatic rings [137,138]. Plenty of evidence conﬁrms that, irrespective of other well-known cytoprotective effects exerted by the molecule at the cellular level, curcumin also mitigates the neurotoxicity by directly interacting with the amyloidogenic proteins, thus preventing or reducing the appearance of the cytotoxic oligomeric forms [129–131,135]. Interestingly, much effort is being put into developing more potent and water-soluble curcumin analogues, solubility being a major constraint thwarting its therapeutic efﬁcacy [129,132,133]. 2.5.2. Resveratrol Natural sources rich in resveratrol (3,5,4′-trihydroxy-trans-stilbene, RES), are grapes, berries, red wine and other plants [120]. Several protective effects have been assigned to this compound, including antioxidant, antiinﬂammatory, anticarcinogenic properties, as well as a neuroprotective action in models of neurodegenerative diseases [121,122]. RES has been shown to substantially affect the amyloidogenic pathway of Aβ and IAPP. In particular, the effects on Aβ amyloid aggregation are the best characterized. This drug does not prevent oligomer formation, but retards ﬁbril formation and even disaggregates preformed ﬁbrils. Concomitantly, it mitigates Aβ-induced toxicity, suggestive of signiﬁcant structural modiﬁcations in the oligomeric species [123]. A recent study provides structural details on the effects of RES on the mode of Aβ oligomerization [124]. In particular, it was shown that Aβ42 forms disc-shaped low molecular weight and high molecular weight oligomers (1.5–2 and Int. J. Mol. Sci. 2018, 19, 2677 10 of 27 10 of 27 3–5 nm high, respectively), the latter resulting from the stacking of the former, and that RES prevents the stacking process, which also suggests that the largest aggregates are the most toxic ones. The capability of this polyphenol of inhibiting the aggregation of the membrane-associated IAPP has been observed even in the presence of aggregation-fostering negatively charged lipid interfaces [125]. Based on NMR data and molecular simulations, it has been suggested that this molecule prevents ring stacking intermolecular interactions between the residues His18 and Tyr37 from adjacent polypeptide chains, quite likely via its aromatic rings [126]. 2.6. Compounds Retarding Transthyretin Aggregation In the context of the present review, a special mention deserves the class of rationally designed compounds capable of retarding TTR aggregation. This protein can cause familial forms of systemic amyloidosis in the presence of gene mutations, but also the wild type can form in the elderly amyloid deposits, mostly localized in the heart. Int. J. Mol. Sci. 2018, 19, 2677 11 of 27 TTR is a homotetrameric plasma protein presenting a well deﬁned binding site for a maximum of two tyroxines that bind the protein through a negatively cooperative manner resulting in two different afﬁnity constants in the nanomolar and micromolar range. TTR is a homotetrameric plasma protein presenting a well deﬁned binding site for a maximum of two tyroxines that bind the protein through a negatively cooperative manner resulting in two different afﬁnity constants in the nanomolar and micromolar range. In 1992, the pionieering work of Jeff Kelly [148] revealed that aggregation of TTR requires the disassembly of the tetramer into monomer and dimers and that the native tetramer is otherwise protected from the aggregation. The demonstration that tetramer disassembly, achievable in vitro under denaturing conditions, can be inhibited when the binding pockets are occupied by the natural ligand (thyroxine) or analogues have triggered an extraordinary pharmaceutical action in order to make drugable analogues of thyroxine suitable for stabilizing the TTR tetramer in vivo. In particular, the screening of a library of substituted benzoxazoles led to the identiﬁcation of tafamidis meglumine as an effective TTR stabilizer [56]. This compound is now tested in clinical trials and the early results suggest that even though a mild beneﬁt from the treatment is achievable, there is space for improving the efﬁcacy of this approach because a discrepancy still exists between the non physiologic in vitro model of aggregation used, so far, in drug discovery and the pathologic process occurring in vivo. g Bellotti’s group has recently discovered that disassembly of TTR and ﬁbrillogenesis can be achieved under physiologic conditions by mechano-enzymatic mechanism consisting of a proteolytic cleavage permitted by the perturbation of the folded state in the presence of physiologic biomechanical forces [149,150]. Fibrils formed through this procedure display chemical and structural properties extremely similar to those extracted from natural deposits and highlight the role of a truncated form of TTR [149] in priming the amyloidogenesis. 2.6. Compounds Retarding Transthyretin Aggregation TTR ligands, including tafamidis, can protect TTR from the mechanoenzymatic mechanism of amyloidogenesis; however, the efﬁcacy highly depends on the capacity of ligands to occupy both binding sites and this task is difﬁcult to achieve in vivo due to the negative cooperativity and the low afﬁnity of most of ligands for the second site. A good candidate for a better inhibition of the mechanoenzymatic mechanism might be Tolcapone and analogues for their property of high afﬁnity for both binding sites and a lack of negative cooperativity. Very promising drug candidates are bivalent compounds that not only simultaneously occupy the two binding sites, but also occupy the inner channel of TTR [57]. 2.7. Nanoparticles Besides small molecule compounds and protein therapeutics, in recent years an increasing number of studies have focused on nanoparticles as potential inhibitors of amyloid aggregation. Nanoparticles are intriguing because they are able to cross the blood brain barrier at low concentrations, and show a certain degree of speciﬁcity towards amyloid deposits depending on their composition. In particular, while gold nanoparticles have been proved to be effective anti-aggregation molecules for insulin [151] and Aβ [152,153], polytrehalose nanoparticles have been shown to inhibit the aggregation of poly-Q proteins [154], and silver and iron oxide nanoparticles are instead capable of interfering with the aggregation of amylin [155]. Thus, despite concerns about their toxicity in some cases, nanoparticles represent a growing ﬁeld, which could lead to novel anti-amyloid therapeutic approaches. 4. The Contribution of the Computational Approaches The high structural ﬂexibility of many polypeptides involved in amyloid plaques formation is such that the application of molecular docking techniques to the study of ligand-target binding has generally proved to be a challenging task. In fact, docking strategies based on classical, molecular mechanics force ﬁelds usually require that the overall structure of the receptor be little inﬂuenced by the interaction with inhibitors. When this is actually the case, as for example in the functional interaction between EGCG or related lower-weight compounds (EG, EGC) and the Josephin domain—which triggers the amyloid aggregation in expanded ATX3 variants [165–167]—well-established docking approaches are able to provide detailed information on the structural basis of the action of inhibitors [113]. However, simulations of thermodynamic ensembles by molecular dynamics (MD)-based modeling of Aβ42 dimers, either in the presence or the absence of EGCG, showed that Aβ-EGCG interactions lead to a signiﬁcant reduction in the β-content of speciﬁc regions of the peptide [168]. Similar secondary structure destabilization, accompanied by a concomitant increase in α-helix content, is common ﬁnding in MD studies of Aβ-inhibitor interaction [169,170]. Nonetheless, the structural information provided by MD simulations turned out to be a valuable starting point for extensive docking efforts, which led to the identiﬁcation of key residues for the interaction with several inhibitors. Exemplary cases are the curcumin and RES interactions with Aβ peptides, which turned out to be mainly—but not exclusively—mediated by a speciﬁc stretch of peptide backbone (F19-E22) and by the side chains of two phenylalanine residues (F19 and F20) [171]. Notably, quinone derivatives (e.g., 1,4-naphthoquinon-2-yl-L-tryptophan) exert a similar mechanism of action [170]. Moreover, similar to the effects they exert on lysozyme [118], quinones are possibly able to form covalent bonds with lysine residues of Aβ peptide, which would contribute to disfavour peptides aggregation [172]. Noteworthy, in this respect, is that quinone intermediates can be formed also upon in vivo oxidation of polyphenols containing catechol residues, which may in part explain the superior inhibitory activity of some catechol-containing ﬂavonoids [172]. Computational investigation focusing on molecules such as myricetin, quercetin and baicalein, which contain either catechol groups or adjacent dihydroxy substituents, evidenced other elements that favour inhibition. In fact, molecular docking investigations of such compounds on a tetrameric assembly of Aβ16-21—a relatively rigid scaffold of biochemical signiﬁcance—highlighted the capability of the ﬂavonoids under investigation of forming both polar and non-polar interactions with Lys, Phe, and Leu residues. 3. Lipid-Modulated Amyloid Aggregation and Antiamyloid Drugs As mentioned in the introduction, it is well known that the toxicity of amyloid oligomers is largely mediated by their capability of interacting with and perturbing biological membranes [16,23]. However, further data, mostly acquired in recent times, conversely show that such interactions may also affect in several ways the mode of aggregation, thus enhancing, at least in some cases, the appearance of toxic species. Obviously, the underlying mechanisms are diverse, depending on both the protein and the membrane component involved. The interaction of speciﬁc residues of a protein with hydrophobic or charged groups in the membrane may result in unfolding and generate aggregation-prone conformations [156]. In this respect, the lipid composition plays a key role in modulating the process [157]. In particular, the interaction with negatively charged Int. J. Mol. Sci. 2018, 19, 2677 12 of 27 groups, such as those of anionic phospholipids, may trigger protein misfolding [158]. Furthermore, monosialotetrahexosylganglioside 1 (GM1), an abundant ganglioside that is a major component of lipid rafts, is strongly involved in favouring protein aggregation and cytotoxicity, quite likely mediated by interactions with the negatively charged sialic acid residue [159,160]. As far as cholesterol is concerned, there are contrasting reports regarding its effects on amyloid aggregation, in that it can apparently either promote or inhibit the process. Indeed, a complex picture emerges from the available data [161–163]. Despite the diversity of mechanisms by which membrane components stimulate amyloid aggregation, antiamyloid agents may also prevent protein/membrane interactions besides displaying classical inhibitory mechanisms of amyloid aggregation as such, which is relevant to the present review. In addition to the case of squalamine (discussed in Section 2.3.), another proof supporting this possibility was provided by experiments, whereby the interaction of α-syn with plasma membrane models was investigated in the presence or the absence of EGCG. Actually, EGCG rescued the toxicity of oligomers by reducing the ﬂexibility of the C-terminus, which in turn completely prevented membrane permeation or disruption. Nevertheless, the ﬂavonoid did not change the secondary structure or the size of the isolated αSN oligomers, as substantiated by solid state NMR [164]. These observations suggest that, when developing new antiamyloid drugs, their capability of interfering with protein/membrane interactions should be also carefully assessed. 4. The Contribution of the Computational Approaches Most importantly, the docking poses Int. J. Mol. Sci. 2018, 19, 2677 13 of 27 obtained indicated that their 2-phenylchromen-4-one pharmacophore plays a key role by inserting itself into the core of the Aβ16-21 tetramer [107]. Docking studies have been useful to also clarify the antiamyloidogenic activity of tetracyclines. As above mentioned, modelling studies based on molecular mechanics were successful in clarifying some key aspects of the mechanism of this class of inhibitors [85]. However, more recent studies have gone beyond the exploration of the conformational space of tetracyclines for the search of a pharmacophore, and aimed at the explicit modelling of protein-receptor interactions. Most notably, docking calculations on tetracycline and PrP as a receptor demonstrated that the antibiotic can speciﬁcally bind the C-terminal helix 2 of human PrP [173]. This solvent-exposed fragment of PrP is known as a potential site of nucleation toward conversion from the cellular to the pathogenic form of PrP. Such tetracycline-PrP interaction can be particularly critical because it can modulate the local geometric features of the target, which has no deﬁnite preference between α and β structure in the targeted region [173,174]. g g Computational studies offer the perspective to identify novel classes of inhibitors also by means of the application of virtual screening techniques on large libraries of molecular structures. Such approach requires reliable structural determination of the receptor protein as a premise. When the latter is available, ligand-receptor docking calculations making use of virtual libraries containing thousands of small molecules can lead to the identiﬁcation of novel scaffolds for the development of new drugs. This kind of study was actually performed by Jiang and coworkers [175]. They used the experimentally determined structure of the Aβ16-21 segment after complexation with the Orange G dye as the receptor structure for a virtual screening effort on a library containing 18,000 small molecules. This allowed them to test the ability of the latter to bind the receptor efﬁciently, which led to the identiﬁcation of a number of promising π-conjugated interactors featuring a mainly ﬂat geometry. Subsequently, such molecules were tested on cell cultures, in order to experimentally evaluate their ability to protect cells from Aβ toxicity. The novel compounds probed in this way were actually capable of reducing the toxicity, but there was no evidence that they led to a reduction in the abundance of protein aggregates. 4. The Contribution of the Computational Approaches These observations are consistent with the hypothesis that most of the toxic effects are sustained by fragments arising from the ﬁbrils, rather than by the ﬁbrils themselves. g g y Finally, it is important to underline that high-throughput computational methods were recently proved capable to efﬁciently screen and design peptide inhibitors against Aβ toxicity and aggregation. By using quantitative structure-activity relationship (QSAR) approaches combined with MD simulations, Wang and coworkers demonstrated that high-throughput-based strategies hold a remarkable potential for the development of peptide inhibitors sharing no sequence relationship with natural peptides [176]. By taking into account six ﬁngerprint factors for controlling self-assembling properties of hexapeptides—i.e., bulky property, hydrophobicity, local ﬂexibility, alpha and turn propensity, electronic properties and compositional characteristics—these authors constructed their QSAR model, training it against experimentally veriﬁed amyloidogenic databases of hexapeptides. The obtained model was used to screen and identify thousands of peptides predicted to be able to self-assemble into amyloid-like aggregates, as molecules with such a property were considered to be possibly good interactors with the Aβ peptide. A selection of the hexapeptides thus identiﬁed was further tested for the actual ability to form aggregates, using MD simulations. Then, the most promising hexapeptides were successfully probed for their inhibition activity against Aβ aggregation using biophysical experiments. Notably, in very broad terms these outcomes might also inﬂuence future developments of computational strategies devoted to the de novo design of anti-amyloid antibody drugs. In fact, among the most promising theoretical approaches in this context, it should be mentioned the one that focuses on the design of speciﬁc structural features of the complementarity-determining regions, the latter being relatively short sequence stretches of the antibody molecules that directly interact with the target peptides. Int. J. Mol. Sci. 2018, 19, 2677 14 of 27 14 of 27 14 of 27 Recently, a complementary high-throughput method based on a quasi-structure-based drug discovery and chemical kinetic [177] has been successfully developed in order to potentiate the anti-aggregation activity of small molecules [178] towards the aggregation of Aβ. We anticipate methods of this kind, which look at the activity rather than the binding of potential inhibitor, to provide further advance of the computational design of anti-aggregation inhibitors. 4. The Contribution of the Computational Approaches The increasing level of sophistication of in silico analysis methods (such as for measuring the amount of β aggregates in a system) [179] and the development of approaches that combine computational and complementary experimental techniques have opened new possibilities in drug discovery and allowed the design of new types of anti-aggregation small molecules and peptides. For example, by combining molecular dynamic simulations and experimental biophysics techniques, various peptides with high afﬁnity for amyloidogenic sequences and β-breaker properties have been identiﬁed and optimized for stability and potency [180,181]. Furthermore, thanks to multidisciplinary approaches, novel hybrid anti-aggregation molecules, which combine potential therapeutic properties from different types of small molecules and peptides have been developed, such as potent inhibitors of amyloid aggregation that consist of β-breaker motifs or compounds fused to amyloid-binding elements. In particular, quinone-tryptophan hybrid molecules [179] and endomorphin analogues conjugated to α-aminoisobutyric acid [182] have been proved to be effective in inhibiting the formation of toxic aggregates of Aβ in vitro and in cellular and ﬂy models of disease. 5. Final Remarks and Perspectives In the present review, we have discussed relevance and mechanism of action of several classes of compounds capable of contrasting amyloid aggregation. As far as low-molecular weight molecules are concerned, they can be classiﬁed into two subgroups: (i) natural compounds and (ii) synthetic molecules, the latter generally developed on the basis of drug design approaches. As regards natural compounds, in the present review we have highlighted that they exert a wealth of beneﬁcial effects (antioxidant, antiangiogenetic, anti-inﬂammatory, etc.); not only their antiamyloidogenic effect is well established, but plenty of evidence also supports the idea that this latter underlies much of the observed cytoprotective effects. The mechanism of action of these compounds appears to be—to a certain extent—unspeciﬁc, as supported by both their capability of inhibiting the aggregation of several unrelated proteins, and by their binding afﬁnities, in the order of micromolar. Outcomes of molecular modelling studies are in line with evidence provided by the in vitro experimentation, which indicates that rigid hydrophobic groups in active polyphenols, tetracyclines/anthracyclines and sterols play a major role in interfering with the amyloid aggregation. Also, these inhibitors generate stable patterns of hydrogen bonds with the target proteins, which are crucial in establishing a signiﬁcant inhibition of the amyloidogenic pathway. In the framework of these achievements, it comes with little surprise that compounds belonging to the cited classes of inhibitors display largely superimposable effects, notwithstanding the signiﬁcant structural differences they hold. In view of their cytoprotective action and their natural origin, many of the anti-amyloid compounds can be regarded as molecules to be used not only in therapy, but also in the context of amyloidosis prevention. Instead, synthetic molecules are meant to be used exclusively in the case of overt amyloidoses. Design and development of such molecules can take advantage of knowledge stemming from both theoretical and experimental investigations on the mode of action of natural compounds. In particular, there is surely room for developing more effective compounds starting from the natural ones used as lead compounds. High-throughput screening studies of compounds libraries (also in the form of virtual libraries) have built on previous knowledge on inhibition mechanisms, thus disclosing new perspectives for the development of novel classes of inhibitors. 5. Final Remarks and Perspectives Interestingly, high-throughput computational methods are expected to become increasingly useful not only in view of the development of low-molecular weight organic molecules as anti-amyloid agents, but also as a support in the efﬁcient 15 of 27 f-grafted veral 3D Int. J. Mol. Sci. 2018, 19, 2677 amyloid sequences). N antibodies developmen screening of peptide libraries for the selection of the most effective compounds against amyloid aggregation (i.e., for instance, peptide-based inhibitors derived from original amyloid sequences). Notably, this might have a signiﬁcant impact also on the area of motif-grafted antibodies development, at least on the long run. Also noteworthy, in this respect, is that several 3D structures of drug/protein complexes are currently available, which quite likely will help design more and more effective molecules (some representative examples are shown in Figure 3). g p p y q y p g and more effective molecules (some representative examples are shown in Figure 3). In conclusion, although specificity represent at the moment a big challenge in the field of drug discovery against amyloidoses, we anticipate that the use multidisciplinary approaches that combine computational and high-throughput experimental methods on molecules of different nature could lead to anti-amyloid compounds of potential therapeutic interest and capable to inhibit in a specific manner the aggregation of amyloidogenic proteins. Figure 3. Cont. Figure 3. Cont. Figure 3. Cont. 16 of 27 16 of 26 Int. J. Mol. Sci. 2018, 19, 2677 Int J Mol Sci 2018 19 x Figure 3. Three representative complexes of anti-amyloyd molecules with their respective targets: (A) TTR binding sites in the presence of tolcapone, with ligand shown as solvent accessible surface. For clarity, H2O oxygen atoms are shown as spheres with 50% of the van der Waals radius (PDB code 4D7B); (B) TTR binding sites in the presence of mds84, with ligand shown as solvent accessible surface as above (PDB code 3IPE); (C) crenezumab Fab in complex with Aβ, with backbones of the interactors coloured in red and green, respectively. Carbon atoms of selected side chains in the Fab are coloured in grey, whereas those belonging to Aβ are green (O, red; N, blue; S, yellow; PDB code: 5VZY). The pictorial representations in panels (A), (B) were taken from Ref. 134 and licensed under a Creative Commons Attribution 4.0. The image in panel (C) was created by means of PyMOL (v. 5. Final Remarks and Perspectives 0.98), using the set of atomic coordinates available in the Protein Data Bank. Figure 3. Three representative complexes of anti-amyloyd molecules with their respective targets: (A) TTR binding sites in the presence of tolcapone, with ligand shown as solvent accessible surface. For clarity, H2O oxygen atoms are shown as spheres with 50% of the van der Waals radius (PDB code 4D7B); (B) TTR binding sites in the presence of mds84, with ligand shown as solvent accessible surface as above (PDB code 3IPE); (C) crenezumab Fab in complex with Aβ, with backbones of the interactors coloured in red and green, respectively. Carbon atoms of selected side chains in the Fab are coloured in grey, whereas those belonging to Aβ are green (O, red; N, blue; S, yellow; PDB code: 5VZY). The pictorial representations in panels (A), (B) were taken from Ref. 134 and licensed under a Creative Commons Attribution 4.0. The image in panel (C) was created by means of PyMOL (v. 0.98), using the set of atomic coordinates available in the Protein Data Bank. Fi 3 Th i l f i l d l l i h h i Figure 3. Three representative complexes of anti-amyloyd molecules with their respective targets: (A) TTR binding sites in the presence of tolcapone, with ligand shown as solvent accessible surface. For clarity, H2O oxygen atoms are shown as spheres with 50% of the van der Waals radius (PDB code 4D7B); (B) TTR binding sites in the presence of mds84, with ligand shown as solvent accessible surface as above (PDB code 3IPE); (C) crenezumab Fab in complex with Aβ, with backbones of the interactors coloured in red and green, respectively. Carbon atoms of selected side chains in the Fab are coloured in grey, whereas those belonging to Aβ are green (O, red; N, blue; S, yellow; PDB code: 5VZY). The pictorial representations in panels (A), (B) were taken from Ref. 134 and licensed under a Creative Commons Attribution 4.0. The image in panel (C) was created by means of PyMOL (v. 0.98), using the set of atomic coordinates available in the Protein Data Bank. Figure 3. Three representative complexes of anti-amyloyd molecules with their respective targets: (A) TTR binding sites in the presence of tolcapone, with ligand shown as solvent accessible surface. AA serum amyloid A amyloidosis Aβ amyloid-beta Conﬂicts of Interest: The authors declare no conﬂict of interest. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations AA serum amyloid A amyloidosis Funding: F.A.A. is supported by a Senior Research Fellowship award from the Alzheimer’s Society, UK (Grant 317, AS-SF-16-003). S.G. is supported by grants from the Cariplo Foundation (2014-0700), the Italian Ministry of Health (Ricerca Finalizzata RF-2013-02355259), Italian Ministry of Education, University and Research (grant: ”Dipartimenti di Eccellenza 2018–2022”). 5. Final Remarks and Perspectives For clarity, H2O oxygen atoms are shown as spheres with 50% of the van der Waals radius (PDB code 4D7B); (B) TTR binding sites in the presence of mds84, with ligand shown as solvent accessible surface as above (PDB code 3IPE); (C) crenezumab Fab in complex with Aβ, with backbones of the interactors coloured in red and green, respectively. Carbon atoms of selected side chains in the Fab are coloured in grey, whereas those belonging to Aβ are green (O, red; N, blue; S, yellow; PDB code: 5VZY). The pictorial representations in panels (A), (B) were taken from Ref. 134 and licensed under a Creative Commons Attribution 4.0. The image in panel (C) was created by means of PyMOL (v. 0.98), using the set of atomic coordinates available in the Protein Data Bank. Author Contributions: S.G. wrote and revised the paper; C.G. wrote and revised the paper; P.T. designed, wrote, revised the paper and performed the final editing; F.A.A. wrote, revised the paper and performed the final editing. Funding: F.A.A. is supported by a Senior Research Fellowship award from the Alzheimer’s Society, UK (Grant 317, AS-SF-16-003). S.G. is supported by grants from the Cariplo Foundation (2014-0700), the Italian Ministry of In conclusion, although speciﬁcity represent at the moment a big challenge in the ﬁeld of drug discovery against amyloidoses, we anticipate that the use multidisciplinary approaches that combine computational and high-throughput experimental methods on molecules of different nature could lead to anti-amyloid compounds of potential therapeutic interest and capable to inhibit in a speciﬁc manner the aggregation of amyloidogenic proteins. Health (Ricerca Finalizzata RF 2013 02355259), Italian Ministry of Education, University and Research (grant: "Dipartimenti di Eccellenza 2018–2022”). Author Contributions: S.G. wrote and revised the paper; C.G. wrote and revised the paper; P.T. designed, wrote, revised the paper and performed the ﬁnal editing; F.A.A. wrote, revised the paper and performed the ﬁnal editing. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations AA l id A l id i Funding: F.A.A. is supported by a Senior Research Fellowship award from the Alzheimer’s Society, UK (Grant 317, AS-SF-16-003). S.G. is supported by grants from the Cariplo Foundation (2014-0700), the Italian Ministry of Health (Ricerca Finalizzata RF-2013-02355259), Italian Ministry of Education, University and Research (grant: ”Dipartimenti di Eccellenza 2018–2022”). AA serum amyloid A amyloidosis Aβ amyloid-beta Conﬂicts of Interest: The authors declare no conﬂict of interest. 17 of 27 Int. 5. Final Remarks and Perspectives J. Mol. Sci. 2018, 19, 2677 Abbreviations Abbreviations AA serum amyloid A amyloidosis Aβ amyloid-beta Aβ2-m β2-microglobulin related amyloidosis AD Alzheimer’s disease AL immunoglobulin light chain amyloidosis APrP Prp amyloidosis α-syn alpha-synuclein ATX3 ataxin-3 ATTR transthyretin amyloidosis β2-m β2-microglobulin CH corea of Hungtington CR Congo red cryo-EM cryo electron microscopy DOX doxycycline DRA dialysis-related amyloidosis EGC (−)-epigallocatechin EGCG (−)-Epigallocatechin-gallate GA gallic acid GM1 Monosialotetrahexosylganglioside 1 HEWL egg-white lysozyme htt huntingtin IAPP amylin IDOX 4′-iodo-4′-deoxy-doxorubicin LC immunoglobulin light chain MD molecular dynamics PD Parkinson’s disease polyQ polyglutamine PrP prion protein RES resveratrol (3,5,4′-trihydroxy-trans-stilbene) QSAR quantitative structure-activity relationship SAA serum amyloid A SSNMR solid-state nuclear magnetic resonance TTR transthyretin References Bucciantini, M.; Giannoni, E.; Chiti, F.; Baroni, F.; Formigli, L.; Zurdo, J.; Taddei, N.; Ramponi, G.; Dobson, C.M.; Stefani, M. 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https://openalex.org/W4389100426	https://dergipark.org.tr/tr/download/article-file/3259899	English	null	Parent Attitudes and Behaviors Affecting Teacher Motivation	Research on education and psychology	2,023	cc-by	10,688	RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) Received: July 16, 2023 Accepted: November 14, 2023 http://dergipark.org.tr/rep Research Article e-ISSN: 2602-3733 Copyright © 2023 December 2023  7(3)  408-429 https://doi.org/10.54535/rep.1326432 Ali Ünal2 Necmettin Erbakan University 1 Ministry of Education, Konya, Türkiye. E-mail: yasingolezlioglu@gmail.com ORCID: 0000-0002-1012-2146 2 Correspondence to: Necmettin Erbakan University, Ahmet Keleşoğlu Faculty of Education, Konya, Türkiye. E- mail: aliunal@erbakan.edu.tr ORCID: 0000-0003-2967-2444 Parent Attitudes and Behaviors Affecting Teacher Motivation Ali Ünal2 Necmettin Erbakan University Yasin Nuri Gölezlioğlu1 Ministry of Education Citation: Gölezlioğlu, Y.N., & Ünal, A. (2023). Parent attitudes and behaviors affecting teacher motivation. Research on Education and Psychology (REP), 7(3), 408-429. 1 Ministry of Education, Konya, Türkiye. E-mail: yasingolezlioglu@gmail.com ORCID: 0000-0002-1012-2146 2 Correspondence to: Necmettin Erbakan University, Ahmet Keleşoğlu Faculty of Education, Konya, Türkiye. E- mail: aliunal@erbakan.edu.tr ORCID: 0000-0003-2967-2444 Citation: Gölezlioğlu, Y.N., & Ünal, A. (2023). Parent attitudes and behaviors affecting teacher motivation. Research on Education and Psychology (REP), 7(3), 408-429. Abstract The aim of the study was to determine the attitudes and behaviors of parents that positively or negatively affect teachers' motivation based on their perceptions. The research was conducted using a case study methodology on 19 teachers working in public secondary schools in Karatay district of Konya, Turkey. The data were collected through semi-structured interviews and analyzed using content analysis. The study group was determined according to the maximum diversity sampling method. As a result of analysis, six parental behaviors that increase or decrease teachers' motivation were identified: recognition and appreciation, involvement and support, communication, trust in teacher expertize valuing holistic development, and support in terms of resources and materials. Teachers observed that their motivation increased when parental attitudes and behaviors aligned with their expectations and decreased when they did not. Based on the results, two recommendations were made: (1) schools should see parents as equal partners, and (2) schools should initiate parent involvement practices that will ensure appropriate behaviour without waiting for parents' appropriate behaviour. Key Words Parent behaviour • Parent involvement • Teacher motivation Key Words Gölezlioğlu, Ünal / Parent attitudes and behaviors affecting teacher motivation Teacher motivation has attracted the attention of researchers in every period. The reason for this interest is that teachers and teacher performance is one of the most important variables in schools for student achievement. One of the most important determinants of teacher performance is teacher motivation. In this study, acting with the same motivation, parent behaviours affecting teacher motivation were determined as the subject of this research. Teacher motivation is a factor that attracts individuals to the teaching profession, makes them endure difficult training to become a teacher, and then determines how long they will stay in the profession and how much they will concentrate on their profession. This factor includes attraction, persistence and effort in relation to the teaching profession (Sinclair 2008). Research shows that teachers' motivation influences their approach to professional challenges, frustrations and rewards, their professional plans, their behaviors and, consequently, student outcomes (Abazaoğlu & Aztekin, 2016; Richardson & Watt, 2010). Dörnyei and Ushioda (2011) draw attention to the existence of frustrated, dissatisfied, or simply bored teachers at all levels of education. Accordingly, this situation, which negatively affects perseverance and effort, is a motivational crisis, and the causes of this crisis are stress, restricted autonomy, stress, ınsufficient self-efficacy, lack of intellectual challenge, ınadequate career structure. The source of stress, which is one of the factors that cause teachers' demotivation, according to some studies (e.g. Müller et al., 2009; Skaalvik & Skaalvik, 2009), is teachers' relationships with students' parents. According to these studies, the stress experienced by teachers who have difficulties in building a relationship with their parents causes their motivation to decrease. The key point to remember about demotivation is that it does not eliminate all the positive effects that underlie motivational behavior. Other motivational factors can still provide motivation despite the presence of demotivation (Dörnyei & Ushioda, 2011). In other words, the stress experienced by the teacher in his/her relationship with the parents may negatively affect the teacher's motivation, but it may not completely eliminate it. For example, a teacher who loves his/her profession very much can maintain his/her motivation by coping with the stress caused by the parents. Studies in Türkiye also show that teachers' relationships with parents affect their motivation positively or negatively. Key Words For example, participatory (Aydemir, 2008), interested, satisfied, collaborative, competent, and educated (Ada et al., 2013), supportive (Aydemir, 2008; Karabağ-Köse et al., 2018), and respectful (Kızıltepe, 2011) parents increase teachers' motivation. On the other hand, parents' indifference and insensitivity (Ada et al., 2013; Karabağ- Köse et al., 2018), negative attitudes (Ada et al., 2013), and confrontational behavior (Kızıltepe, 2011; Özge-Sağbaş & Özkan, 2022) cause teachers' motivation to decrease. When the researches are examined, it is observed that parent behaviours that affect or decrease teacher motivation in teacher-parent relationships are divided into various categories: interested-uninterested, sensitive-insensitive, cooperative-uncooperative, sufficient-insufficient, respectful-disrespectful, supportive-non-supportive-blocking. However, the content of these categories is not sufficiently focussed. For example, the meaning of parents' respectful behavior toward the teacher is unclear. In addition, the findings are not directly related to the relationship between parental behavior and teacher motivation, but as one of the factors affecting teacher motivation or performance in general. However, parents play an important role in teacher motivation. Comprehensive research to understand the attitudes and behaviors of parents that affect teacher motivation can provide valuable information to improve teacher-parent relationships and increase teacher 409 RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) motivation (Anastasiou & Papagianni, 2020). It can also contribute to policies and practices aimed at improving teacher-parent collaboration and the quality of education. Although schools are generally considered responsible for students' academic performance, research shows that a large part of students' academic performance is explained by family characteristics (Carneiro, 2008, Coleman et al., 1966). In other words, students' academic success is based on a supportive home environment (Cabus & Ariës, 2016). However, it is not possible to completely ignore schools and what is done at school. At the very least, teachers and parents can work together to shape children's learning and therefore their upbringing, and development. It can be argued that it is imperative that parents and teachers share responsibility for supporting students' success and for promoting their overall well-being (Smith et al. 2022). Indeed, research shows that when teachers are able to share this responsibility, there are positive improvements in student achievement and behavior (e.g. Ateş, 2021; Fan & Chen, 2001; Kim et al., 2013; Pushor & Amendt 2018). However, the literature reveals that neither parents nor teachers are satisfied with the extent and depth of parental involvement in schools (Christenson, 1995; Gokturk & Dinckal, 2018). Key Words Gokturk and Dinckal (2018) attribute this to the fact that both parties have different understandings of the concept of the parental involvement. It is possible to find the source of the different understandings identified by Gokturk and Dinckal (2018) in Schlechty's (2009) metaphors of the school as a factory, a producer of professional services, a warehouse, a prison, and a learning organization In these metaphors, Schlechty (2009) explained how the role expected of parents changes according to the different designs of the school as follows. In schools designed as factories, students are seen as raw materials and products. Parents are the source of supply and the determinant of product quality. From this perspective, who the parents are, what the parents do, and the nature of the social and cultural trust provided by the family are the main determinants of the performance expected of children. When a school is conceived as a professional service-producing organization, students are seen as customers and parents as guarantors and suspicious allies. In this context, teachers are experts and the parents are expected to ensure that the teachers do what they ask of their children without question. Parents who refuse to play the role expected of them are treated as outsiders and rule breakers. In such cases, the principal is usually expected to protect teachers from interference by uncooperative parents. However, for reasons such as incompetence, mistrust, view of the school, or self-perceived superiority over teachers, parents may refuse to fulfill the role of guarantor unqualifiedly. In warehouse or prison school designs, the school has begun to have warehouse or prison features by incorporating students who do not fit into the factory system. In this design, students play the role of objects or prisoners and parents play the role of strangers. The primary role of the parent is that of sender and receiver, ensuring that the student goes to school and has a place to return to when school is over. Parents are always a dubious ally, not to be trusted. They are viewed with suspicion, treated as outsiders and forced to follow certain rules, limit their interactions, and not go where they are not invited. 410 Gölezlioğlu, Ünal / Parent attitudes and behaviors affecting teacher motivation In the school as a learning organization perspective, students are seen as knowledge workers and parents as partners. Key Words The role of parents as partners is to participate with teachers in the education of their children. This means, in addition to parent teacher association meetings, regular contact with teachers through intensive use of the Internet and other electronic means of communication, and having a say in their child's education together with teachers. Anastasiou and Papagianni's (2020) study revealed that teachers generally have a positive attitude about parental involvement. At the same time, this research shows that teachers' attitudes about parental involvement are negative in cases where parents interfere with their work. Comparing Anastasiou and Papagianni's (2020) findings with Schlechty's (2009) metaphors, teachers' school design is: "school is an institution that produces professional services". They do not accept parental behaviour that contradicts this design in their minds. If there is a gap between the design of the school and parents' perception of their own role in the child's education, conflicts between parents and teachers are inevitable. To manage these conflicts properly, it is important to know what roles the school design assigns to parents and what roles parents assign to the school, and what mutual expectations they have. Research conducted by Ünal (2012a) revealed that there are six types of expectations from parents in school-parent relationships in Turkey: Supporting educational activities: Parents check their children's homework and help them with their homework. Supporting educational activities: Parents check their children's homework and help them with their homework. Participation in social activities: Parents should participate in and financially support school ceremonies and social events. Participation in social activities: Parents should participate in and financially support school ceremonies and social events. Taking part in decision making and following instructions: Parents should make suggestions to the school management and follow the school's policies. Taking part in decision making and following instructions: Parents should make suggestions to the school management and follow the school's policies. Communication: Parents should be in constant communication with the school management and teachers about their children and should take action regarding the student as directed by the teachers. Communication: Parents should be in constant communication with the school management and teachers about their children and should take action regarding the student as directed by the teachers. Financial Contribution: Parents should contribute financially to the school to meet its physical and educational needs. Financial Contribution: Parents should contribute financially to the school to meet its physical and educational needs. RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) in their work. According to educators, parents' focus only on academic success, lack of interest in education, financial concerns, lack of time, communication problems, and lack of self-confidence also negatively affect communication with parents (Ünal, 2012a). Mann and Gilmore (2021) also found in their study that teachers value parent-teacher partnerships less than parents, invest less in the relationship, and participate less. Perhaps because their expectations are not met, educators may take their negative feelings and thoughts further and display rude, abusive, insulting, and scolding behavior toward parents (Ünal, 2012b). Research results show that one of the important variables in teacher-parent interaction is the region where the school is located and the socioeconomic characteristics of the parents, which affect the mutual perception of teacher- parent relationships and the quality of these relationships. For example, teachers in schools located in socio- economically advantaged regions are more satisfied with their relationships with parents, while they believe that parents who do not cooperate with them do not value education (Bellibas & Gumus, 2013). On the other hand, as the socioeconomic characteristics of families improve, parental interaction with schools increases, but parental satisfaction with the schools decreases (Can, 2009). Although the findings clearly show that the socio-economic characteristics of parents affect the teacher-parent relationship, there is a large body of literature on the source of barriers to healthy teacher-parent relationships reported by both parents and teachers (Mann & Gilmore, 2021). Christenson (2004), in his review, explained the parent-based foundations of the problems experienced in teacher-parent relationships under the categories of structural and psychological barriers as follows. Structural barriers: (1) Lack of role models, the lack of information, and the lack of knowledge about resources. (2) Lack of supportive environment and resources (e.g. poverty, limited access to services). (3) Economic, emotional, and time constraints. (3) Economic, emotional, and time constraints. (4) Lack of child care and transport. Psychological barriers: Psychological barriers: (1) Feeling of inadequacy; low self-efficacy. (2) Taking a passive role, leaving education to schools. (3) Ignorance of linguistic and cultural differences, school policies and practices, and the role of parents in education. (3) Ignorance of linguistic and cultural differences, school policies and practices, and the role of parents in education. (4) Scepticism about how they will be treated by the educators. (5) Perceived insensitivity to parents' needs or wishes. (5) Perceived insensitivity to parents' needs or wishes. Key Words Meeting their children's educational needs: Parents should send their children to school by meeting their needs. Meeting their children's educational needs: Parents should send their children to school by meeting their needs. Kıral (2019) also conducted a study in Turkey and found that educators had similar expectations from parents to those identified by Ünal (2012a), but these expectations were not met by most parents. The study by Ünal et al. (2010) also shows that educators' expectations from parents are not met. According to the data obtained by Ünal et al. (2010) in this study, educators see parents as unconscious, indifferent, not knowing what is good for their children, not cooperating enough with the school, and instinctively showing protective behavior toward their children. Another finding of the study is that educators do not see parents as stakeholders in educational activities and they expect parents to be people who accept themselves as experts, do only what they say, and do not intervene 411 Research Design This study determines the attitudes and behaviors of parents that affect teacher motivation through the views of teachers. As the aim was to describe and analyze a limited system in depth, the study used a case study methodology in line with Merriam's (2013) explanation. The use of the case study design provided teachers with the opportunity to understand and explain in detail their experiences, thoughts and feelings about the attitudes and behaviours of parents. RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) Christenson (2004) also identified structural and psychological barriers related to barriers arising from educators and school-family relationships. This means that parents are not the only source of problems in teacher-parent relationships. This study focused only on the attitudes and behaviours of parents that affect teacher motivation, and 412 Gölezlioğlu, Ünal / Parent attitudes and behaviors affecting teacher motivation did not address the systemic and teacher-related issues that affect relationships. The aim of this study was to determine the attitudes and behaviors of parents that, in the view of teachers, have a positive or negative impact on teacher motivation. did not address the systemic and teacher-related issues that affect relationships. The aim of this study was to determine the attitudes and behaviors of parents that, in the view of teachers, have a positive or negative impact on teacher motivation. Data Collection Data were collected using a semi-structured interview technique. First, a literature review was conducted and the relevant research was analyzed to prepare the interview questions. The prepared questions were presented to three different experts who have studies on parental involvement in the field of educational administration. After receiving the experts' opinions, pilot interviews were conducted. After the expert opinions were received, pilot interviews were conducted and the questions were finalised by taking these interviews into consideration.The main question decided to be asked in the semi-structured interview is as follows Do the positive/negative attitudes and behaviours exhibited by student parents have an effect on your professional motivation? During the interview, questions such as "Can you elaborate a little more?" and "Can you explain a little more?" were also asked to allow participants to elaborate their thoughts. The interviews were carried out face - to - face by the first researcher. Participants were contacted by phone or e- mail before the interview. They were informed about the study. Teachers who agreed to be interviewed were also given written information before the interview. They were also asked to do a think piece on the research. The interviews were carried out in the schools where the participating teachers worked. An appointment was made in advance at a time that was convenient for the teachers. Before the interview, the participants were given the voluntary participant consent form. The interviews were recorded with a voice recorder with the permission of the participants. During the interview, notes were taken by the interviewer. The interviewer took note of the participants' gestures and facial expressions and their verbal responses. The shortest interview lasted 12 min and the longest 21 min. The recordings of the interviews were then transcribed. There was no transcription of any information that would be an indication of the identity of the participants. The transcribed versions of the interviews were between 4,300 and 12,000 words in length. Study Group The study group of the research consists of 19 teachers working in official secondary schools and secondary schools for imams and preachers in the center of Karatay district, Konya province, Turkey. The maximum diversity sampling method, one of the purposeful sampling methods, was used to determine the participants. In this context, school type (secondary school [SS] and secondary schools for imams and preachers [SSIP]) and socioeconomic level of the environment where the school is located, gender, branch, educational status, and being a parent of a student were determined as diversity factors. The determination of the diversity factors was based on the studies of Bellibas and Gumus (2013) and Can (2009). Information about participants is presented in Table 1. The real names of the participants were kept confidential, and each was given a code name. Table 1: Personal and professional characteristics of the study group Table 1: Personal and professional characteristics of the study group Participant Name Branch Gender Seniority (years) Graduation Institution Is he/she a parent? The socioeconomic status of parents Göktuğ Informatics Technology Male 0-5 Undergraduate SSIP No Low Necip Religious Culture and Ethics Male 5-10 Undergraduate SSIP Yes Low Cemal English Male 10-15 Master’s SSIP No Low Özge Visul Arts Woman 20+ Undergraduate SSIP Yes Low Nazım Turkish Male 5-10 Undergraduate SS No Low Cahit Technology Design Male 15-20 Undergraduate SSIP Yes Low Lale Turkish Woman 5-10 Undergraduate SSIP No High Leyla Math Woman 10-15 Master’s SS Yes High Edip English Male 15-20 Master’s SS Yes High Orhan Social Studies Male 15-20 PhD. SSIP Yes Medium Nilgün Math Woman 10-15 Undergraduate SSIP No Medium Tezer Social Studies Woman 15-20 Undergraduate SS Yes High 413 RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) Attila Science Male 20+ Master’s SS Yes High Sezai English Male 15-20 Master’s SSIP Yes High Alev Religious Culture and Ethics Woman 15-20 Master’s SS No Medium İlhan Guidance Counselling Male 20+ Master’s SS Yes Medium Enis Physical education and Sport Male 15-20 Undergraduate SSIP No Medium Tomris Math Woman 0-5 Undergraduate SS No Medium Yahya Science Male 10-15 Master’s SS Yes Medium C i Results Based on the interviews with the teachers, parent behaviors affecting teacher motivation are given below under two headings as behaviors that affect teacher motivation positively and behaviors that affect teacher motivation negatively. Data Analysis Data were analyzed using content analysis. For this purpose, the written data were first read several times. Meaningful units were coded while preserving their integrity. The coded data were first categorized by the first researcher. The categories were then reviewed and finalized jointly by two researchers. 414 Gölezlioğlu, Ünal / Parent attitudes and behaviors affecting teacher motivation Merriam's (2013) recommendations were taken into consideration to ensure the validity and reliability of the research. Participant confirmation was obtained during the interview to ensure the validity of the research. During the interview, the participants were also asked confirmation questions such as "Is this what you meant?" and "Is this what I should understand from your words?". To increase internal validity, interviews were continued until data saturation was reached. Purposive sampling was used to increase external validity. Participants were selected from different schools and teachers with different characteristics. In this respect, an attempt was made to make the findings of the research easily testable in similar settings. To increase external validity, an attempt has been made to have a rich and intense narrative presentation of the participants' descriptions. This was done to ensure that readers of the research could better compare the research with other research. To ensure internal reliability, first of all, the way the data would be analyzed was determined in detail before the participants were interviewed. This was done to ensure internal reliability. In addition, during the process of analyzing the data collected during the research, the second author checked the compatibility of the data with the findings and participated in the process as a second researcher and expert experienced in parent involvement. To ensure external reliability, the research was conducted and reported transparently. In addition, throughout the entire analysis and reporting process, the process was completed by making constant comparisons about whether the findings were compatible with previous research. RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) Tomris: "The parent came to school immediately and brought flowers to celebrate the student's success, which made me jubilant. Tomris: "The parent came to school immediately and brought flowers to celebrate the student's success, which made me jubilant. Özge: "The parents' approval of my work motivated me a lot. At the end of the process, reaching even one person and bringing about a certain change in one person is the main source of motivation". Participation and support: Teacher motivation increases when parents are actively involved in their children's learning by attending parent-teacher meetings, helping to solve problems or providing help and resources, setting goals, addressing problems together, and supporting solutions. In practice, this means: Parents show their commitment to their children's education and make teachers feel that they are not alone. For example, parents' help with homework shows that they are invested in their children's education and are willing to contribute to their academic success. This willingness increases teachers' confidence and makes them believe that they will succeed. It creates a positive environment for teachers to continue their work with enthusiasm. Attila: "When a student is given an assignment, the parents control it as much as they can. They call and ask what they can do. In fact, I have seen parents who are very interested in their students even though they do not know much about the school". Yahya: "The mother of a student with orthopedic disabilities asked me for permission to follow my classes with her child. She was a very interested parent. When the mother attended the classes with her child, the student's success increased significantly. Parents value education so much that they are so interested in their children". Tomris: "Sometimes I contact the parents and tell them about the problem their student has. The parent immediately comes to the school and asks me what to do. This shows how much parents care about their students and it makes us very happy to meet such parents". Enis: "What makes us happy is that some parents tell us that if there is a problem they are ready to do everything they can when we ask for a solution". Enis: "What makes us happy is that some parents tell us that if there is a problem they are ready to do everything they can when we ask for a solution". Parent Attitudes and Behaviours that Positively Affect Teacher Motivation Recognition and appreciation: Teachers reported that when parents recognize and express their gratitude or appreciation for the work and effort they put into their children's education, it has a positive impact on their motivation. This includes parents recognizing and thanking teachers for their students' achievements, high test scores, improved behavior increased achievements, or other positive outcomes. The theme of recognition and appreciation generally emphasizes that parents' recognition and appreciation of teachers' works increase teachers' motivation. Leyla: "I really like it when the parents thank me when the student does well in class or gets good grades in exams." Leyla: "I really like it when the parents thank me when the student does well in class or gets good grades in exams." Tezer: "Teacher, what have you done to our child? Our child talks about you all the time during the day, and they say that they get excited when it is time for your class. It is wonderful to hear these words from a parent. Sezai: "When I met his father in the following process, he said that English was his favorite subject and that he tried to do the homework I gave him before his other homework. I worked hard with this student and I was very happy to be rewarded for my efforts". 415 RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) Communication: This theme shows that when parents actively communicate with teachers and provide information about their children, this helps motivate teachers. When parents provide teachers with information about their children's private life, health problems, or daily life, they are better able to respond to the child's individual needs, touch the child's life more, find solutions one on one with the parents, and overcome problems by working together. Edip: "If we establish a positive communication channel with the parents, we can touch the child's life. It does not necessarily have to be educational. It is also at this stage that we start to look at the student's abilities. We are interested in criteria such as character, behavioral development, and moral development. The fact that the parents inform us and that we can take care of the student makes us happy". Attila: "We are an overcrowded school in terms of the number of students. We have over 1500 students. You cannot even learn the names of so many students, let alone their problems. In my opinion, it is a very 416 Gölezlioğlu, Ünal / Parent attitudes and behaviors affecting teacher motivation noble behavior when a parent comes to you and opens up and expresses the problems of his/her child. Although you do not have the opportunity to know the problem personally, the parent informs you and you can overcome the problem by working together. This is a motivating situation for the teacher. I think it is the same for the parents". Trust in teachers' expertise: Teachers stated that parents should recognize their professional expertize and respect their authority in the classroom. Parents avoiding excessive intervention, trusting teachers judgment and allowing them to fulfill their teaching responsibilities contribute to teachers' motivation. The emphasis of the theme is that they expect teachers expertize to be trusted and parents to empower teachers to make decisions without undue interference. In this way, the ability of teachers to fulfill their teaching responsibilities autonomously is a source of teacher motivation. Edip: "Some of our parents have developed an awareness of education. They say that we are experts in the subject, that they will do what we say for the success of their students and that they will always show their support throughout the process". İlhan says: " After the student misbehaves, we call the parents and explain what happened. RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) well-being and recognise efforts to support pupils' holistic development. The theme implies that teachers are more motivated by parents' recognition and appreciation of the wider aspects of a child's development and well-being beyond academic achievement than by a concern solely for exam success. It underlines how important it is for teachers to help children develop intellectually but also to help them develop physically, emotionally, socially, and creatively. Teachers also indicated that they are motivated by parents who prioritize the physical and emotional well- being of their children. This includes parents who encourage healthy habits such as proper nutrition, regular exercise, and adequate rest. Göktuğ: "Some parents focus on subjects such as math and science. When the parents support my work, it opens up a new field of study for the student. When I get the necessary support from my parents, I can work with more enthusiasm". Sezai: "Conscious parents who give up the idea of preparing their children for constant competition are also motivation for us. Cemal: "Once, I had a class counseling session. I had 25 students in my class, and we had a parent meeting. ... One parent raised their hand, and I said, 'Please go ahead.' ... They said that I was constantly involved in cinema and theater and did not allocate time for other things. They said, 'I wish my child was in another class.' ... After that incident, it took me three or four months to recover. I even considered quitting teaching." Enis: "We have parents who forcibly take their children to different places and prevent them from participating in sports activities. We have parents who say, 'Just don't go today' and show no concern. A lack of training can disrupt our work, but unfortunately, parents do not appreciate us as much as we would like." Resources and material support: Teachers have mentioned that providing financial support, such as assisting in acquiring necessary materials, contributing to classroom resources, or helping with equipment repairs, positively impacts the motivation and effective teaching skills of parents. Required materials for the classroom may include textbooks, workbooks, stationery supplies, art materials, science equipment, or technology tools. Cahit: "Due to our subject, we produce tangible products. RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) Sometimes, after the smallest mistake, the parents come to the school and say: 'Teacher, let's do whatever it takes to prevent the problem from growing. When we have this kind of reaction, it is a great boost to our self- confidence. Necip: "For example, last year we got a student from our neighborhood into Science High School for the first time. In this case, the family's positive attitude toward us during the examination process was very effective. The phrases they said to us, like we are willing to do whatever you say, we are willing to sacrifice our lives according to your desires, really affected our attitude toward that student. It made us both run to school and love the profession more". Trust in teachers' expertise also means that when teachers trust parents' professional judgment and take their advice, it increases their motivation and confidence. Trust increases teachers' motivation by making them feel supported and respected in their role. Lale: "Parents must trust me. When I feel that, I feel more comfortable". Lale: "Parents must trust me. When I feel that, I feel more comfortable". Göktuğ: "The main livelihood in this region is animal raising and construction work, which can affect the level of personal care our students receive. When I took the initiative to address this aspect, they expressed their gratitude by saying, 'Teacher, you are doing an excellent work', and it brought me great joy. Some parents cannot go to the city center to provide for their children's needs. But the parents who trust us say, 'Teacher, you understand the child's needs and provide what is necessary, and we will support you. Acting as a member of the student's family and bridging these gaps gives me immense satisfaction. Valuing holistic development: Arts, sports and culture teachers in particular expressed that they are motivated to pursue their work with passion when parents support extra-curricular activities, promote physical and emotional 417 RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) We would like our parents to visit our workshop and support us in obtaining materials." Necip: "We had a parent who graciously volunteered to meet all the material needs of the school. It is a great act of generosity for a parent to contribute to beautifying the school like this." Positive and Respectful Behavior: Teachers are motivated by parents who exhibit positive and respectful behavior both in front of their children and in direct interactions with teachers. Parents who adopt a positive and respectful communication style also contribute to a healthy teacher-parent relationship as well. Nazım: "The knowledge that there will be no abuse, bad words or violence from parents makes me more comfortable in my job." Nazım: "The knowledge that there will be no abuse, bad words or violence from parents makes me more comfortable in my job." 418 Gölezlioğlu, Ünal / Parent attitudes and behaviors affecting teacher motivation Leyla: "Even if we have problems with a parent, the student remains unaware of it. The child continues to show you the respect you deserve. Even if the incident with the parent is negative, I am happy when they display this attitude. I believe the parent also respects me." Attila: "There is a certain boundary here, and I will not cross that boundary, and the parent will encroach on my territory. As long as the parent and teacher respect each other's personal spaces, there will not be any issues." Parent Attitudes and Behaviors that Negatively Impact Teacher Motivation Parent indifference and lack of involvement: The theme indicates that the lack of parental involvement in their children's education and school community negatively impacts teacher motivation. Parents who see their role as minimal or even perceive education as solely the responsibility of the school and teachers can make teachers feel unsupported and undervalued. Examples of parental indifference and lack of involvement include parents not coming to school, not inquiring about their child's progress, not participating in virtual meetings, or not knowing their child's classroom or name. Necip: "There are parents whom I have never seen in the classroom, whose names I do not even know. I invite parents to come to school, but they never show up." Necip: "There are parents whom I have never seen in the classroom, whose names I do not even know. I invite parents to come to school, but they never show up." Nazım: "I rarely encounter parents who come to ask about their child's progress." Leyla: "Parents do not come to school or refuse to participate in virtual meetings. I have come across parents who do not even know their child's classroom or name." Tomris: " What parents generally do is send their children to school and then take no interest in anything else. The developmental period, especially for 7th- and 8th-grade students, requires social support. The biggest negative is that parents ignore this need of the student." Teacher İlhan: "I drop off my child at school, and you educate, nurture, teach, and handle everything. Don't bother with the rest. regarding discipline issues with their children, you receive very little feedback from oblivious parents." Blaming teachers and schools: The theme indicates that some parents tend to shift responsibility and attribute their child's negative behavior solely to external factors such as the school or the teacher, which negatively impacts teacher motivation. These parents, instead of recognizing and addressing their child's behaviour try to blame educators for absolving themselves and their children from any responsibility. Tezer: "Children can make mistakes, but these parents try to blame us every time by ignoring their child's faulty behavior to ease their conscience. They always support the student. While parents try to raise princes and princesses, we face the consequences." Leyla: "I provided extra resource books to my successful students who were financially struggling. I motivated them to solve more tests. I also volunteered to support them outside class hours. Parent Attitudes and Behaviors that Negatively Impact Teacher Motivation The parents filed 419 RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) a complaint with the Ministry of Education, claiming that I was not interested in their children. Since that day, I could not focus on private lessons with students." Cahit: "I noticed that a student did not bring any materials. When I asked why, he said that he was engaged in animal husbandry, that his family worked hard and that he forgot because he was busy. I phoned her mother. The mother said that she would do everything for her child's education and that they would not make him work hard at home. We talked about what they would do and hung up the phone (without any problems). The next day, the child's mother came to the principal and complained about me. The thing she complained about me was that I called her and talked about her child." Lack of trust in teacher's professional expertize: The theme indicates that when parents violate the boundaries of teachers' professional expertise it negatively impacts their motivation. In conversations, teachers mentioned incidents where parents interfered with their work, questioned their competence, and tried to dictate certain aspects of their teaching or classroom management. According to teachers, such boundary violations weaken their professional autonomy and expertize decreasing motivation and morale. Lale: "Although it was not in any of the acquisitions of my lesson, (a parent) sent a message saying "Teacher, you did not teach this subject, it would have been better if you had taught it and I wish you had taught it in this way". He is even trying to decide the method and technique I will use, not to mention the subject I will teach. I was obsessed with this message in my head for two days. It really lowered my motivation. It made me feel inadequate. Anyway, after the parent says "Teacher, you know better, but", I think any word that comes after "but" is going to interfere with your work." Necip: "Once, a parent started telling me how to teach the lesson, how to communicate with the students, how to grade, and so on. I did not flinch and when they finished talking, I asked them what they do for a living. ‘I'm an electrician,’ he said. Parent Attitudes and Behaviors that Negatively Impact Teacher Motivation I said, ‘If I interfered with your work by saying, 'Do this here, do that there,' wouldn't you hit me on the head with the spanner in your hand?’ He laughed and said, ‘I would’. I said, ‘Then don't interfere with my work." Göktuğ: "While inspecting the students' attire for inappropriate dress, I warned a student about their hair. The parent came to school and, in front of everyone, reacted in a loud and inappropriate manner, asking me why I was concerned about their child's hair. This situation damaged my respect for my profession." Unbalanced communication and lack of positive feedback: The theme refers to the imbalance in how parents communicate with teachers and the limited or absent positive feedback from parents, which leads to a decrease in teacher motivation. In discussions, many teachers mentioned that parents often complain or criticize when their children are involved in a negative situation or face a problem. However, when students achieve success or display positive behaviors parents tend not to share or express their appreciation toward teachers. Cemal: "Parents only seem to come to school when something bad happens. They do not inquire about their child's achievements or any school activities. It demotivates us and exhausts us professionally when 420 Gölezlioğlu, Ünal / Parent attitudes and behaviors affecting teacher motivation parents who quickly react to negative incidents do not appreciate the good work we do. This situation pushes us toward burnout syndrome." Yahya: "Some parents can create chaos at school when any incident occurs involving their child. We face situations where they shout, scream, and even insult teachers. Parents do not fully evaluate the incident and directly blame the other party (teachers) and unfortunately, the incident does not only remain in blame. We can be subjected to insults even in front of other teachers and students. Such incidents frequently at the school where I work. We inevitably fear that a parent will enter the classroom and cause a disturbance. There is also the fear that a child will go home and make baseless accusations, leading to the parent coming to school and causing a fight. This situation prevents us from moving freely in the classroom. RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) Leyla: "In my experience, the root cause of the problems I face lies in the parents' illiterature or their lack of knowledge about their child's educational process. It is easier for a parent who is at least somewhat educated or has a diploma to empathize with their child." Enis: "I believe that parents who display negative behavior have not received sufficient education or have never sat in these classrooms." İlhan: "In ignorant parents, there is usually a tendency like this: 'I drop off my child at school, you educate, teach, do everything, look after their appearance, solve their problems. Don't bother me with the rest.' When their child has a problem, you receive very little feedback from oblivious parents." Edip: "We have parents who are incapable of fulfilling their duties as mothers and fathers. When parents become more aware in this regard, it will increase our motivation as well." Parent Attitudes and Behaviors that Negatively Impact Teacher Motivation It affects not only the teacher directly involved in the incident but also other teachers." Nazım: "When there is a problem caused by a student and the parent comes to school, instead of resolving the issue, they escalate it into a bigger problem. There have been instances where a parent pulled a knife on me and other teachers. We have experienced parental violence firsthand. The parent was called to the school because of his child's indiscipline. The incident ended with the parent pulling a knife on the teachers." Disregard for teachers' personal lives: The theme indicates that parents' behaviour of disregarding teachers' personal lives negatively impacts teacher motivation. The statements obtained from discussions emphasize that parents exhibit behavior that crosses these boundaries and disregards teachers' personal well-being, which in turn negatively affects teacher motivation. These behaviors include the constant expectation of accessibility and a lack of respect for teachers' personal lives. Özge: "A parent asked me about their child's exam results through WhatsApp. Even though the parents could access the results through the e-school system, they felt entitled to ask me about it in the middle of the night. I contacted him, wondering if she had access to the system. She said they could access it but wanted me to send the results. Unfortunately, parents try to establish excessive familiarity with teachers through social media. This certainly affects our morale. When a parent behaves like this, the student eventually starts to lose respect for the teacher." Lale: "The parent of a student for whom I am the class advisor messaged me at 10:00-PM asking about homework. I had previously informed my parents that I may not respond to messages sent after a certain hour. When they did not receive a response, they messaged again at midnight. When I still did not respond, they attempted to call me. When I asked why they were calling at this hour, they used disrespectful language, saying that it was my duty to answer. As if that was not enough, the next day they complained about me to the school administration, claiming that I was not helpful to them." The illiterature and understanding of parents: The theme refers to the limited knowledge and awareness of parents regarding their children's education and the general education system. This illiterature and understanding can negatively impact teacher motivation. 421 Discussion, Conclusion & Suggestions Based on the findings from teacher interviews, two conclusions can be drawn regarding parental attitudes and behaviors that affect teacher motivation. First, when parents recognize and appreciate teachers' work, participate in school activities and provide support, engage in effective communication, demonstrate trust in their expertize value holistic development, provide resources and material support, and exhibit respectful behavior it positively impacts teacher motivation. Second, when parents act in a neglectful manner and do not participate in their child's education, blame teachers and schools for educational outcomes, show distrust in teachers' expertise, engage in unbalanced communication, and disregard teachers' personal lives, it negatively affects teacher motivation. The findings align with previous research results that indicate both positive (Ada et al., 2013; Aydemir, 2008; Christenson, 2004; Karabağ-Köse et al., 2018; Kıral, 2019; Kızıltepe, 2011; Mann & Gilmore, 2021; Ünal, 2012a,b) and negative (Ada et al., 2013; Christenson, 2004; Karabağ-Köse et al., 2018; Kızıltepe, 2011; Mann & Gilmore, 2021; Özge-Sağbaş & Özkan, 2022) parental behaviors that impact teacher motivation. Additionally, it can be stated that the results largely support Anastasiou and Papagianni's (2020) finding that teachers support parental involvement but do not want parents to interfere in their work. When analyzing the parental behaviors that affect teacher motivation, based on the findings from teacher interviews, seven parental behaviors can be identified: recognition and appreciation, participation and support, communication, trust in teacher expertize valuing holistic development, and resource and material support. Recognition and Appreciation: Expressing gratitude and appreciation for teachers' efforts positively influence their motivation. Feeling valued and appreciated by parents enhances teachers' sense of accomplishment and encourages them to continue working hard. On the other hand, the lack of recognition or appreciation can decrease teacher motivation, making them feel undervalued and unappreciated. Participation and Support: Active parental involvement and support in their children's education create a positive environment that motivates teachers. Parents who attend meetings, collaborate in problem solving, and provide resources demonstrate their commitment to their children's education, which in turn boosts teacher motivation. 422 Gölezlioğlu, Ünal / Parent attitudes and behaviors affecting teacher motivation Conversely, the lack of participation and support can lead to feelings of being unsupported and overwhelmed, negatively impacting teacher motivation. Communication: Open and informative communication between parents and teachers is crucial for understanding each other's perspectives and working together effectively. Discussion, Conclusion & Suggestions When parents communicate openly and provide information about their child's situation, it allows teachers to meet the individual needs of the student and overcome challenges. Conversely, unbalanced communication that focuses primarily on criticism without providing constructive feedback can create a hostile environment and decrease teacher motivation. Trust in Teacher Expertise: Trusting and respecting teachers' professional expertize is vital for teacher motivation. When parents accept and trust teachers' knowledge and skills, it enables teachers to exercise their professional autonomy and make decisions that benefit students. On the other hand, parents who question or excessively interfere with teachers' competence undermine professional boundaries and hinder teacher motivation. Additionally, when parents reach the point of blaming teachers or schools for their child's problems, it can make teachers feel unappreciated and unjustly targeted, leading to a decrease in motivation. Valuing Holistic Development: Parents who support their child's holistic development and appreciate teachers' efforts in fostering various aspects of growth have a positive impact on teacher motivation. Recognizing the importance of extracurricular activities and promoting students' overall well-being creates a positive partnership between parents and teachers. Conversely, parents who fail to appreciate the significance of holistic development may undermine teachers' efforts in these areas, particularly for teachers of subjects such as art and sports, leading to a decline in motivation. Resource and Material Support: Financial contributions from parents for classroom materials alleviate resource constraints and positively influence teacher motivation. Parents providing the necessary supplies for their children and contributing to the procurement of classroom materials enable teachers to create a conducive learning environment. The recognition of both parental interest and support, along with the availability of an appropriate educational setting, naturally enhances a teacher's enthusiasm. Conversely, the lack of resources and materials can generate concerns about failure due to limited resources, resulting in disappointment and decreased motivation for teachers. Positive and Respectful Behaviour: Parents who exhibit positive and respectful behavior including respecting teachers' personal lives, promote a healthy teacher-parent relationship. This positive relationship creates a supportive and collaborative environment that motivates teachers to perform at their best. Conversely, parents' negative or disrespectful behaviours can strain the relationship and negatively impact teacher motivation. When analyzing parental behaviors that positively or negatively affect teacher motivation, the impact of these behaviors can vary depending on the teacher and the context. Additionally, it should be recognized that these behaviors are not mutually exclusive and can coexist within the same parent. RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) All the parental behaviors that influence teacher motivation indicates that teachers perceive schools as professional institutions that produce professional services. In other words, teachers see themselves as experts and expect parents to be allies who do what is expected of them without questioning. However, in today's school design as a learning organization, parents are expected to be partners. The role of parents in a learning organization entails their active involvement in their children's education, regular communication with teachers, and having a say in their child's education. While the themes of communication, parental participation and support in this study may suggest that teachers' mental model of the school design aligns with a learning organization, teachers' perspectives reveal that the expected communication and participation from parents are seen more as subordinates who simply do as the teacher says. This finding aligns with the study conducted by Ünal et al. (2010), which indicates that educators do not view parents as stakeholders in educational activities, but rather expect them to be passive individuals who accept their expertize and do not intervene in their work. Therefore, it can be inferred that there has been no change in the school and parent design in educators' perspectives over time. This lack of change is natural as there has been no explicit discussion or policy change regarding school design in Turkey during this period. The main contribution of this research to the literature lies precisely in this point. Teachers develop expectations from parents based on their mental model of the school, and when parental attitudes and behaviors align with their expectations, their motivation increases. Conversely, when parental attitudes and behaviors diverge from their expectations, their motivation decreases. In this sense, teachers' motivation may be the result of themselves not perceiving parents as equal partners in line with their mental model of the school. When teachers correctly position the school, themselves, and parents, some motivation problems can be resolved. Christenson (2004) identified "lack of training for educators on how to establish and maintain partnerships with families" as a barrier to the parent involvement. This lack of training also applies to Turkey. In Turkey, teachers do not receive education on how to establish partnerships with families, how to maintain communication and collaboration with them, neither during their pre-service nor in-service training. Discussion, Conclusion & Suggestions For example, a parent may exhibit supportive behavior in certain situations while displaying negative behavior in others. The impact of parental behavior on teacher motivation can vary based on the frequency and intensity of these behaviors 423 RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) It is clear that teacher candidates and teachers need to be trained on how to establish partnerships with families, how to maintain communication and collaboration. Although the findings include the illiterature and understanding of parents as one of the parental behaviors that negatively affect teacher motivation, it is not explicitly mentioned as one of the six effective parental behaviors on teacher motivation. The reason for this is that the lack of parental education and understanding is not a behavior of parents but rather a characteristic. Therefore, it seems more accurate to consider the lack of parental education and understanding as a factor that increases parental behaviors that decrease teacher motivation, rather than labeling it as a parental behavior that negatively affects teacher motivation. Based on the perceptions of teachers whose motivation is affected by the lack of parental education and understanding, it can be said that schools are designed as factories. According to this design, parents are seen as determinants of student performance (Schlechty, 2009). This implies that the views that attribute most students' academic success to family characteristics (Carneiro, 2008; Coleman et al., 1966), which are accepted by policymakers and teachers, overshadow what can be done at school. This acceptance can be observed in the statement of the participant Edip, "We have parents who are incapable of fulfilling 424 Gölezlioğlu, Ünal / Parent attitudes and behaviors affecting teacher motivation their parental duties. When parents become aware, our motivation will also increase." This situation may lead teachers to value and invest less in parent-teacher relationships with socioeconomically disadvantaged parents (Bellibas & Gumus, 2013), as evidenced by their lower investment in the relationship (Mann & Gilmore, 2021; Ünal, 2012b). In other words, teachers who claim that their motivation decreases due to the lack of parental education and understanding may actually be igniting the fuze of parental behaviors that decrease their own motivation by not sufficiently investing in their parent-teacher relationships. Another assessment related to the theme of parental education and understanding is linked to teachers' academic optimism. Hoy (2008) highlighted the importance of academic optimism in teacher motivation. Teacher academic optimism refers to a self-directed positive belief system regarding the capacity to teach all students despite difficulties, establishing trusting relationships with students and parents, and prioritizing academic tasks (Woolfolk Hoy et al., 2008). RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) In their study, Üzüm and Ünal (2023) found that the fundamental determinant of teacher academic optimism is the teacher's experience of success or failure in their profession. It is natural for teachers who perceive schools and parents as determinants of performance (Schlechty, 2009) to experience failure or worry about potential failure, which can lead to a decrease in motivation. What is unnatural is accepting this situation without trying to create a new school design and provide teacher training on the parental involvement. In fact, researchers such as Swap (1990), Grolnick and Slowiaczek (1994), and Epstein (1995) have proposed models to ensure that parental involvement is not left to chance and that schools take specific measures to guarantee parental participation. For example, Epstein's (1995) model highlights the importance of parent education in eliminating the illiterature and understanding that teachers complain about. In other words, schools and teachers should not only complain about the illiterature and understanding of parents or mention how these factors affect their motivation but also implement practices aimed at addressing these issues. It is expected that policymakers and school administrators design schools as learning organizations and develop training programs that make parents equal partners according to this design. The contribution of the research to practitioners lies precisely at this point. Schools should be designed in a way that views parents as equal partners. Schools should establish parental involvement policies that implement this design without waiting for appropriate behavior from parents, aiming to both enhance teacher motivation and promote parental participation. Teacher training on the parental involvement also appears necessary for the effective implementation of these policies. The research has four notable limitations. Firstly, the small sample size of just 19 teachers from a specific region in Turkey limits the generalizability of the findings to different regions and school types. Secondly, relying solely on teacher perspectives may not fully capture the actual attitudes and behaviors of parents affecting teacher motivation, warranting a more comprehensive approach involving parents, students, and school administrators. Thirdly, purposeful sampling with a focus on specific factors may introduce sampling bias, potentially undermining the study's representativeness of parent attitudes and behaviors. Lastly, the exclusive use of semi-structured interviews as the data collection method, conducted in person, could lead to response bias and omitted topics due to time constraints, possibly introducing social desirability bias. 425 RESEARCH ON EDUCATION AND PSYCHOLOGY (REP) Ethic This study was ethically approved by the decision of the Necmettin Erbakan University Scientific Research Ethics Committee dates 11/02/2022 and numbers 2022–54. Author Contributions This study was produced from the master thesis prepared by the first author under the supervision of the second author. Conflicts of Interest There are no conflicts of interest in the research Funding The authors received no financial support for the authorship, research, and/or publication of this article. 426 Gölezlioğlu, Ünal / Parent attitudes and behaviors affecting teacher motivation References Abazaoğlu, İ., & Aztekin, S. (2016). 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W4375853407.txt	https://www.qeios.com/read/ZLDWAU/pdf	en		Another rate view on autocatalytic reactions	null	2,023	cc-by	5,243	Another rate view on autocatalytic reactions Miloslav Pekař Faculty of Chemistry, Brno University of Technology, Purkyňova 118, 612 00 Brno, Czech Republic E-mail: pekar@fch.vut.cz Abstract. Discussions on the proper conceptualization and definition of autocatalysis are still lively. Several examples of autocatalytic reaction schemes are analyzed for their concentration and rate time profiles using computer modeling. Autocatalytic features should be inherent in a relevant scheme and its real demonstration further depends on “proper parametrization”, i.e., proper values for the rate parameters (their ratios) and relevant concentrations. Flow-through reactors bring a new complication due to the effects of inlet feed on reaction rates. When discussing kinetic features, one should take into account both concentration and rate profiles. 1. Introduction Even more than 150 years after the first appearance of the autocatalysis concept (Peng et al., 2022) discussions on its proper conceptualization and definition are still lively; see for recent examples, Peng et al. (2022), Horváth (2020, 2021). Schuster (2019) reminds us that the term autocatalysis was introduced by Wilhelm Ostwald in 1890 “for the characterization of reactions that show an acceleration of the rate as a function of time”. Peng et al. (2022) point out earlier experimental work on autocatalysis and especially Ostwald’s investigations in which the reactant was supposed to be an autocatalytic substance. This contrasts with today’s views, in which a product is mostly considered to have an autocatalytic effect; examples of such textbook definitions were presented in an earlier work (Pekař, 2021). Peng et al. (2022) extended Ostwald’s original view of an autocatalytic reaction to the definition: “a reaction that can be written as a net reaction equation where reactants and products are non-overlapping sets with at least one reactant or product that has a catalytic effect on the process represented by that equation.” Now, the IUPAC’s definition is as follows (IUPAC, 1997a): “A chemical reaction in which a product (or a reaction intermediate) also functions as a catalyst. In such a reaction the observed rate of reaction is often found to increase with time from its initial value.” The first part of this definition rests on the definition of catalyst, from which only the first two phrases are cited: “A substance that increases the rate of a reaction without modifying the overall standard Gibbs energy change in the reaction; the process is called catalysis. The catalyst is both a reactant and product of the reaction.” (IUPAC, 1997b). Peng et al. (2022) even distinguish recessive and expansive autocatalysis; the former refers to cases in which a reactant provides a catalytic effect whereas in the latter case at least one of the products is the autocatalytic agent. The situation, therefore, still seems to be confusing. The IUPAC catalyst definition requires the presence of a catalyst among both reactants and products, whereas the IUPAC autocatalysis definition speaks only about a product being a catalyst (that is, autocatalyst). In contrast, Peng et al. (2022) require non-overlapping reactants and products. Evidently, the IUPAC autocatalysis definition tacitly assumes non-equal stoichiometric coefficients for an autocatalyst on both sides of the stoichiometric equation, in contrast to the catalyst definition. More importantly, the IUPAC’s definitions speak (also) about rates of reactions and this is essential, because any catalysis is a kinetic phenomenon. It is not clear why the IUPAC catalyst (and catalysis, in fact) definition requires an https://doi.org/10.32388/ZLDWAU 1 increase in the rate of a reaction while the IUPAC autocatalysis definition states that the rate increase is observed only frequently, and, therefore, not always. Recently, I published a brief note on the fruitfulness of not ignoring the inspection of rates when discussing autocatalysis as a part of chemical kinetics (Pekař, 2022). That note extended an example analyzed in papers by Horváth (2020, 2021), who tried to provide a proper classification of autocatalysis. Horváth, in essence, follows the product-based view. For example: “A reaction is called autocatalytic when one of its products enhances the rate of its own formation.” (Horváth, 2020) or “autocatalyst (...) [is] a species (...) that increases the rate at which the given process occurs and at the same time it functions as a product of the overall process.“ Horváth (2021) also claims that if “the addition of a product speeds up its own formation, then the corresponding (...) is indeed autocatalytic.” In this work, I further elaborate on the rate view of autocatalysis and extend the previous, brief note (Pekař, 2022). Owing to some responses to that previous text I also try to apply a pedagogical approach. Besides the traditional batch system, also a flow-through arrangement is included. The 2 tool is, again, computer modeling, which was performed with the Chemical Reaction Engineering Module of the COMSOL Multiphysics package, version 6.0 or 6.1. 2. Results 2.1 Simple reaction The simplest autocatalytic reaction is probably (Schuster, 2019): A + X → 2X. (R1) It can be rewritten into an atom-conserving (the conservative approach (Érdi and Tóth, 1989)) but still general form as (Pekař, 2021): BX + X → 2X + B. (R2) The autocatalytic specie should be the component X, which is consumed (as a reactant) and at the same time produced (as a product) but in a larger amount. Examples of the behavior of (R2) in a batch reactor are given in Fig. 1 for an increased initial concentration of X (𝑐!" ) at a constant initial " concentration of BX (orA) (𝑐#! = 1000 mol m–% ) and with the rate constant (𝑘) equal to 2×10–5 mol– 1 3 –1 m s . For the lower X initial concentration, we see a typical S-shaped concentration-time profile for both products (Fig. 1 upper). This shape is “smoothed” with increasing X initial concentration to a parabolic-like shape (cf. Fig. 1 lower). The reaction rate for the lower X initial concentration has a time profile with a clear and high maximum – up to this maximum, the reaction really accelerates 3 throughout its course. An increased X initial concentration decreased the maximum and shortened the duration of the increasing reaction rate. For the highest used (and the other shown) X initial concentration (equal initial concentrations of both reactants), no maximum is seen and the rate decreases continuously throughout the whole course of the reaction (Fig. 1 lower) – i.e., no acceleration is observed. It should be noted that the inflection point on the S-shape corresponds to the maximum on the reaction rate profile. Table 1. Parameters used to simulate reaction (R2) in a batch reactor Figure 1 upper lower 𝑘/(mol–1m3s–1) 2×10–5 2×10–5 𝑐!" /(mol m-3) 50 1000 Table 2. Parameters used to simulate reversible reaction (R1) in a batch reactor Figure 2 upper lower 𝑘 & /(mol–1m3s–1) 2×10–5 2×10–5 𝐾 1 1 " 𝑐' /(mol m-3) 50 1500 𝑐(" /(mol m-3) 1000 1000 The simple reaction (R1) or (R2) is identical to that given by Peng et al. (2022), except that Peng et al. also considered the reversed direction and used different symbols (M ≡ X, F ≡ A ≡ BX; M + F 2M). Here, we retain the original symbols of Peng et al. (2022). The reversed direction did not bring anything substantially new. Of course, both concentration profiles end at equilibrium concentrations; the reactant (F) is not fully consumed, as in the irreversible case. S-shaped concentration profiles can be found for both components (see the example in Fig. 2) as well as a maximum on the (overall) rate profile (Fig. 2). What is new and specific is the possibility to decompose the overall rate into forward and reversed rates (remember that 𝑟 = 𝑟 & − 𝑟 – ). In the same example, both rates increased with time and no maximum was observed for either of them. This is quite standard for the reversed rate, because the amount of its reactant (the overall product M) continuously increases by means of the forward reaction. This is less expected for the forward direction because its reactants (M and F) are consumed in this direction. However, just due to the autocatalytic feature, more M is produced than consumed at the same time. Increasing the initial concentration of M changes the shape of the concentration profiles to a parabolic-like form and erases the increasing part of the overall rate profile, i.e., its maximum is at time zero and the rate continuously decreases. This means that the autocatalytic behavior is eliminated. When the initial concentration of M exceeds that of F, the sign of the overall rate is reversed; i.e., the reaction runs, in fact, in the reversed direction and the absolute value of the overall rate continuously decreases (Fig. 2 lower). No autocatalytic behavior is thus seen. The reversed rate continuously decreases due to the 4 continuous depletion of its reactant (M), whereas the forward rate increases because the production of F exceeds the consumption of M. The concentration profiles are parabolic-like (Fig. 2 lower). The magnitude of the equilibrium constant has no significant qualitative effect relevant to this discussion; the interested reader can check it in his own simulations. Similar comments can be ascribed to a slightly more complex reaction found in Peng et al. (2022): P + 2Q 2P + Q. 2.2 Ostwald’s lactonization example Ostwald’s valerolactone example (Ostwald, 1890) can be formally written as HA → L + H) O, (R3.1) * & HA → A + H , (R3.2) where HA is g-hydroxyvaleric acid and L is g-valerolactone. The autocatalyzed reaction should be (R3.1), which is known to be catalyzed by hydrogen ions. Ostwald gives no rate equation; perhaps the simplest one should be: 𝑟+, = 𝑘+, 𝑐-. 𝑐-! . (1) However, this form means no reaction without the presence of hydrogen ions, that is, there is no non-(auto)catalytic path. A more appropriate general form could thus be 𝑟+, = 𝑘+," 𝑐-. + 𝑘+, 𝑐-. 𝑐-! . (2) The first term in (2) represents the “ordinary”, non-autocatalytic path, while the second term expresses the (auto)catalytic action of protons formed in (R3.2). 5 There is a problem with the dissociation of g-hydroxyvaleric acid as a weak acid. SciFinder1 estimates its pKa as 4.61 (Henry (1892) published a value which gives pKa = 4.69). The (equilibrium) concentration of protons is thus very low (cf Ostwald´s: “...der dissociirte Antheil der Säure nicht gross ist...”2 (Ostwald, 1890)) and the reaction (R3.2) quickly attains equilibrium. Thus, unless the value of 𝑘+, is really very high, the catalytic effect of H& is hardly seen. This accords with Ostwald’s qualitative statement: “...bei Gegenwart des Neutralsalzes behält die g-Oxyvaleriansäure ihren Säuretiter tagelang fast unverändert bei und lässt nur einen äusserst langsamen Uebergang in das Lacton erkennen.”2 (Ostwald, 1890). The first example involving only the autocatalytic rate contribution, i.e. with 𝑘+," = 0, (Fig. 3) shows a very fast increase in the dissociation rate and the rapid attainment of dissociation equilibrium. The former is accompanied by a very fast increase also in the lactonization step rate, which demonstrates (auto)catalysis. However, the lactonization rate quickly reaches a maximum and continuously decreases due to the depletion of the acid supply, as in the case of a general, non-catalyzed reaction in a batch system. The concentration profiles of the acid and lacton do not show any specific (autocatalytic) features. Both change smoothly and parabolically, the acid decreasing, the lactone increasing (not shown). The concentration of H& is very low (and equal to the acid anion concentration) due to the low value of the dissociation constant. Its time profile also has a maximum as a result of equilibrium (R3.2) responding to the depletion of the acid concentration. Adding the direct rate step (𝑘+," > 0) increased the initial rate of lactone formation while not depressing the height of the maximum (measured as the difference to the value at time zero) in the autocatalytic phase (Fig. 4), at least for the (low) value of 𝑘+," used in the simulation. The effect of adding hydrogen ions to the initial mixture on the lactone formation rate is clear (Fig. 5) when compared with the same situation with no added hydrogen ions. However, even in the former case, the rate can continuously decrease throughout the course of time (Fig. 5), as in the case of a nonautocatalytic reaction. Perhaps surprisingly, the increased rate of formation of the autocatalytic species (the increased rate constant of hydroxyvaleric acid dissociation) barely changed the rate of lactone formation; on the other hand, the rate of acid dissociation was increased by several orders (data not shown). Yet, the concentration of H& was determined mainly by the equilibrium constant, which remained unchanged in this example. A clearly visible S-shaped lactone concentration profile can be obtained for sufficiently high values of 𝑘+, (Fig. 6). The lactonization rate had, here, a maximum at the time corresponding to the inflection point on the S-shape; the dissociation rate decreased up to the 1 2 https://www.cas.org/solutions/cas-scifinder-discovery-platform/cas-scifinder English translation can be found in supplementary to Peng et al. 2022 6 reversed direction and then attained a minimum, these results indicating responses of the dissociation equilibrium to the consumption of the acid in the lactonization step. ± Table 3. Parameters used to simulate reaction (R3) in a batch reactor. Rate constants 𝑘-. refer to the dissociation step (R3.2) Fig. 3 4 5 6 7a a 𝑘+," /s–1 0 5×10–3 5×10–3 1×10–9 0 𝑘+, /(mol–1m3s–1) 10–1 10–1 10–1 10–1 12×10–5 & 𝑘-. / s–1 2×10–3 2×10–3 2×10–3 2×10–4 22×10–7 * 𝑘-. /(mol–1m3s–1) 80 80 80 8 88×10–3 " 𝑐-. /(mol m-3) 100 100 100 100 37 " 𝑐-∗ /(mol m-3) 0 0 20 0 0 units as in original source, Henry (1892) Ostwald´s note (1890) is reflective not experimental; he refers to experimental work by Henry, published (later) in Henry (1892). Only a minor part of Henry´s paper deals with autocatalysis; as an example of the fitting of his data by model (R3) and (2) ((1), in fact, cf. Table 3), Fig. 7 is presented (𝑥 is the lactone amount per cm3). Henry used the following equation to describe the kinetics of autocatalytic lactonization: 𝑑𝑥 ⁄𝑑𝑡 = 𝐶𝑦(𝐴 − 𝑥)) . (3) In it, 𝑥 is the amount of lactone formed in time 𝑡, 𝐶 is the rate coefficient (determined from experimental data), 𝐴 is the initial amount of hydroxyacid, and 𝑦 denotes a function which gives the amount of hydrogen ions. Henry derived the function 𝑦 from considerations on the hydroxyacid dissociation equilibrium. Because 𝑦 is then proportional to (𝐴 − 𝑥)*1 and 𝐴 − 𝑥 is proportional to the amount of hydroxyacid reacted to lacton, Henry´s equation is not far from (1). 2.3 Landolt reaction scheme Horváth suggested using (the visualization of) “the effect of the initially added product on the concentration–time profiles” as “a simple tool to decide whether the experimental system or the theoretical model has an autocatalytic feature or not” (Horváth, 2021). He presented an example of a parabolic-like profile in the Landolt mechanism fulfilling this criterion (Figure 1a in ref. Horváth (2021)). Let us look at a very similar 7 example from the viewpoint of rates – that of the cyan curve of Figure 1B in Horváth‘s other work with the same mechanism (Horváth, 2020) and probably demonstrating the suppression of the autocatalytic feature by “improper parametrization”. Because the reference Horváth (2020) was commented in the previous note (Pekař, 2022), only the reaction scheme is given here: A + B → C, (R4.1) B + C → D, (R4.2) A + D → 2C. (R4.3) When no product C is initially present in a batch system, the rate of its formation continuously decreases during the course of the reaction and no autocatalytic behavior is observed (Pekař, 2022). If the product is present initially, its formation rate increases over a very short initial time interval and then decreases continuously (Fig. 8). That very short interval can be considered as an autocatalytic feature evoked by the non-zero initial concentration of the product. If the initial product concentration is sufficiently high, its initial formation rate can even be negative, i.e., its destruction outweighs its formation (Fig. 9). Paradoxically, the initial rate of C formation is thus decreased by adding this autocatalyzing product into the initial mixture, because it is consumed in the second step at the same time. Apparently, the product concentration profiles remain parabolic-like (data not shown). The example in ref. Horváth (2021) is suitable also for demonstrating the role of the inspection of rates in the discussion of S-shaped/parabolic concentration profiles. The curves in Fig. 1a of that reference are of parabolic shape and show increasing order with the increasing initial concentration of product C, similarly to the curves in Fig. 1b of Horváth (2021), which are, however, S-shaped. The S-shape of the concentration profile actually means the existence of an inflection point at which the first derivative possesses an extreme. In this example, this means an extreme on the rate profile, because here 𝑟2 = 𝑑𝑐2 /𝑑𝑡. A maximum on the rate profile is observed already in the case of a zero initial concentration of C and for the (black) curve from Fig. 1b of Horváth (2021); see Fig. 10 here. However, the corresponding inflex point on the concentration profile is featureless and impossible to see in the small drawing presented in Fig. 1b of ref. Horváth (2021). Even an enlarged figure requires careful inspection, cf. Fig. 10 (lower). The S-shape thus need not be attributed only to curves on which it is easily recognizable. Table 4. Parameters used to simulate reaction (R4) in a batch reactor; 𝑐." = 𝑐#" = 1000 mol m-3, no D at 𝑡 = 0. Fig. 8 9 10 𝑘1 /(mol–1m3s–1) 32×10–7 32×10–7 6×10–6 𝑘) /(mol–1m3s–1) 32×10–7 32×10–7 2×10–5 𝑘% /(mol–1m3s–1) 104 104 104 𝑐2" /(mol m-3) 25 1200 0 8 2.4 CSTR as an example of flow system CSTR (continuous stirred tank reactor) systems bring new aspects to (not only) autocatalysis. Reaction rates are affected by the continuous feed into the reactor. For example, in a batch system, the concentration profiles of the Landolt scheme (R4), the cyan curve in Fig. 1 of Horváth (2020), are parabolic-like and 𝑟2 is decreasing (Pekař, 2022), whereas in CSTR the former profile is S-shaped and the latter is increasing; 𝑟2 is increasing also when plotted against 𝑐2 – thus apparently indicating autocatalysis (data not shown). In a batch system, the 𝑟1 profile is decreasing and the 𝑟) and 𝑟% profiles are almost equal and with a maximum. In contrast, in CSTR, all rate profiles are increasing (and S-shaped) and 𝑟1 is evidently higher than the other two rates (which are also almost equal and thus causing an extremely low concentration of intermediate D), including their steady state values. When the product C is also present in the inlet stream, its concentration profile changes from Sshaped to parabolic-like (Fig. 11), while the autocatalytic effect of the product is demonstrated by its formation rate increasing in time and being higher than when no product is injected (Fig. 12). It should be noted that the corresponding rate parameters ratio (𝑘) /𝑘1 ) in the original work (Horváth, 2020) was probably considered as an example of non-autocatalytic parametrization. In the standard CSTR model, it is impossible to separate the effect of continuous feeding from the pure autocatalytic effect on reaction rates. To achieve such separation, we can explore the ideas published by Rodrigues et al. (2017). The standard molar balance of a constant volume CSTR is (Scott Fogler, 1997): 𝜏 𝑑𝑐3 ⁄𝑑𝑡 = 𝑐34 − 𝑐3 + 𝜏𝑟3 ; 𝜏 = 𝑉 ⁄𝑣, (4) where 𝜏 is the space time (𝑉 is the constant volume and 𝑣 is the constant volumetric flow rate) and 𝑐34 is the component concentration at the reactor inlet. The concentration of component 𝑖 (in fact, its change in time) is divided into two parts – the “purely flowing” and the “reacting”: 𝑐3 = 𝑐35 + 𝑐36 . The balances for these two parts are then: 𝜏 𝑑𝑐35 ⁄𝑑𝑡 = 𝑐34 − 𝑐35 , (5) 𝜏 𝑑𝑐36 ⁄𝑑𝑡 = −𝑐36 + 𝜏𝑟3 . (6) 4 4 4 Note that 𝑐35 = 𝑐3 , 𝑐36 = 0 and the sum of (5) and (6) is (4). Rate equations for 𝑟3 are formulated in 𝑐3 ´s, of course. Decomposition of the concentration of product C for the previous example is shown in Fig. 13. The 𝑐25 profile is parabolic, as is the overall 𝑐2 profile, whereas the 𝑐26 is sigmoidal, as in the case when there is no C at the inlet. The rate profiles remain unchanged, of course. 5. Discussion Autocatalysis is a kinetic phenomenon and as such is very tightly linked to the rate of reaction. Autocatalytic analyses thus should also include the inspection of rates. This does not mean that concentrations and their (time) profiles can then be ignored. On the contrary, none of these two 9 insights should be ignored, but they have to be combined. This, hopefully, was clearly illustrated by examples in the previous note (Pekař, 2022) and in the Results section here. In a discussion of autocatalysis, we should clearly state which reaction we have in mind and what its rate is. In the case of a single(-step) reaction, the reaction in mind is clear and unambiguous. In the case of multi-step reactions, the individual steps generally have different rates, and we should clearly state which one we are discussing. For example, the simple reaction from section 2.1 is a single reaction and its rate is considered. The Landolt scheme from section 2.3 is an example of a multi-step reaction (with three steps) and we discussed the product reaction (formation) rate. It should be noted that that rate is a combination of all three step rates: 𝑟2 = 𝑟1 − 𝑟) + 2𝑟% . In the case of lactonization, we focused on the rate of (R3.1), while (R3.2) was a side reaction producing the autocatalytic specie in the system as a whole. A catalyst affects the reaction rate and thus has to occur in the rate equation. Rate equations generally describe the effects of two principal factors determining the reaction rate: temperature and concentrations. The temperature effect is described by the rate constant (coefficient) and is outside the scope of this discussion (it is sufficient to restrict the discussion to isothermal cases). Concentrations are present in the rate equation per se, often raised to a power (the reaction order). The catalyst concentration thus appears in the rate equation. An “ordinary” catalyst is a substance “external” to the catalyzed reaction, not among its “ordinary” reactants. The reaction can proceed without its presence, though possibly at a very small (negligible) rate. When the catalyst is added (externally), the reaction rate increases (appreciably) and the reaction usually follows another, faster pathway. For example, a slow synthesis A + B → AB may be switched to a faster, catalytic route A + cat → A ∙ cat; A ∙ cat + B → AB + cat, at the end, from which the catalyst is released “unchanged”. The rate of AB formation can be expressed in the former case as 10 𝑟.# = 𝑘𝑐. 𝑐# , in the latter as 𝑟.# = 𝑘) 𝑐.∙89: 𝑐# , where the catalyst amount is hidden in 𝑐.∙89: (the formation rate of A ∙ cat in the first step is given by 𝑘1 𝑐. 𝑐89: ). Autocatalysis is specific by the fact that the catalyst is not an “external” but an “internal, intrinsic” substance, one of the “ordinary” reactants or products. The occurrence of an autocatalyst in the rate equation must be inspected carefully, because “ordinary” reactants (and products in the case of reversible reactions) are normally present in rate equations. In other words, an (increased) reactant concentration always has an accelerating effect on the rate of reaction, providing it is of the form 𝑟 = 𝑐6;98:9<: 𝑓(𝑇, 𝑐), where 𝑓(𝑇, 𝑐) is a function of temperature and (other) concentrations. Thus, recessive autocatalysis (Peng et al. 2022) remains unclear unless the reactant, the accelerated reaction, and its rate equation are specified precisely. Further, we should clearly state the aspect or thing, relative to which the increased, catalyzed, rate is considered. In the case of standard, “external” catalysis, it is relative to the uncatalyzed reaction; that is, the reaction without the addition of a catalyst. See, for example, the discussion of the lactonization example in sec. 2.3. In the case of autocatalysis, the situation is more intricate because the catalyst is inherently present in the reacting mixture. We can play with its concentration as Henry did in the lactonization experiments or Horváth, in the simulation of Landolt scheme, and compare the relevant time profiles. We can thus make a relative comparison of the profiles detected for the varying concentration. In all cases, we can and should simply watch the time profile of the relevant reaction rate and its relation to the (auto)catalyst concentration. Playing with the (initial) concentration can be ambiguous in revealing autocatalytic behavior, as noted in (the middle of) section 2.3. Briefly, an autocatalytic reaction scheme should inherently contain an autocatalytic feature – the formation of a specie which could really increase the rate of the relevant reaction under consideration. This is a necessary condition determining autocatalytic reactions, though it may not be a sufficient one, as pointed out, for example, by Horváth in his note on proper parametrization (Horváth, 2020). However, as the rate equation teaches us, parametrization includes not only the rate 11 coefficients but also the concentrations of the constituents of the reacting mixture (and, probably, also the temperature, especially in non-isothermal reacting mixtures). In the simple reaction, the autocatalytic feature is the formation of 2X when one X is consumed at the same time. Similarly, in the Landolt scheme, the autocatalytic feature is the formation of 2C when one C is consumed – but in two different steps. In these two examples, a product is a reactant at the same time. In the lactonization example, the autocatalytic feature is the accelerating effect of hydrogen ions (on lactone formation) which are formed from the reactant by a side reaction; i.e., one (side) product, not (necessarily) a reactant, is the catalyst. This catalyst, hydrogen ions, can also be added externally, as in most experiments described by Henry (1892). The role of concentrations in the “proper parametrization” was illustrated by Fig. 1 (simple reaction) and Fig. 2 (reversible simple reaction). A note to the paper by Peng et al. (2022): the reaction M + F 2 M is rewritten there as F M, said to be catalyzed by M and then decomposed in two steps: F → M, catalyzed by M, and M → F, catalyzed by M. No more specification is given. The rate of the catalyzed first step would probably be 𝑘( 𝑐( 𝑐' , of the catalyzed second step, probably (𝑘' + 𝑘'9 𝑐' )𝑐' . The kinetic equilibrium constant of the summary reaction, obtained according to the kinetic equilibrium condition 𝑟⃗;= = 𝑟⃖;= , is then 𝐾 = 𝑘( 𝑐',;= ⁄Y𝑘' + 𝑘'9 𝑐',;= Z = 𝑐',;= ⁄𝑐(,;= . This expression was written to be basically consistent with the expression for the thermodynamic equilibrium constant (𝑎',;= ⁄𝑎(,;= ; 𝑎 denotes the activity). The thermodynamic equilibrium constant is “hidden” in the standard reaction Gibbs energy mentioned in the IUPAC definition of catalyst (see Introduction). The standard mass-action kinetics used for this reaction in the Results section seems to be fully adequate and sufficient for describing this reaction, including its autocatalytic behavior. The statement on the permanent reaction rate increase, cited in Peng et al. (2022), is evidently not valid, particularly in batch systems, cf., as examples, Figs. 1 and 6. The S-shaped product concentration time profile can be a strong indicator of autocatalysis but is not unambiguous, as pointed out, for example, by Horváth (2021); see also the discussion of the lactonization example in sec. 2.3. In the case of a simple, single reaction in a batch system, it is a result of autocatalysis, because it reflects the (temporarily) accelerated product formation. At the same time, it (its inflection point) indicates the existence of a rate maximum, i. e., not a continuous reaction rate increase. In the case of a multi-step reaction scheme, the S-shaped product concentration time profile can be a consequence also of other kinetic features like the consecutiveness of reactions leading to that product formation. A typical example of the latter is the consecutive transformation of A to C: A → B → C. Here, the S-shape of the 𝑐2 time profile in the batch reactor is accompanied by a maximum on the 𝑐# time profile. Flow-through systems like CSTR add new complications – instantaneous feed in (and out) affect the rates of reaction steps and can interfere with “pure” autocatalysis and suppress it regardless of the presence of autocatalytic features in the underlying reaction scheme. In other words, the reactor 12 inlet and outlet should be considered in the “proper parametrization”. Flow-through systems are often operated under steady state (ss) conditions where rates and concentrations do not change in time. This special case is not discussed here; let it only be noted that CSTR steady state balance 𝑟3?? = (𝑐3?? − 𝑐34 )⁄𝜏 shows that a stationary rate depends on both inlet and initial concentrations, generally. Conclusions The IUPAC definition of a catalyst is not bad but should be used carefully in the case of autocatalysts and in discussions of autocatalysis. An autocatalytic feature should be present in the reaction scheme – the formation of a specie which acts, at the same time, as a reactant in a relevant step and whose formation outweighs (temporarily) its consumption, or which participates in the rate equation of some step (while not necessarily being a standard reactant in the stoichiometric equation of that step). This still may not be sufficient, as proper values of relevant rate coefficients and concentrations are important for autocatalysis to really appear. Special attention should be devoted to flow-through systems in which reaction rates are affected by a continuous inlet of standard reactant(s). References Érdi P., Tóth J. (1989). Mathematical models of chemical reactions. Princeton: Princeton University Press. Henry P. (1892). Über die wechselseitige Umwandlung der Laktone und der Oxysäuren. Zeitschrift für Physikalische Chemie 10U(1), 96-129. Horváth A. K. (2020) Law of Mass Action Type Chemical Mechanisms for Modeling Autocatalysis and Hypercycles: Their Role in the Evolutionary Race, ChemPhysChem 21(15), 1703-1710. Horváth A. K. (2021). Correct classification and identification of autocatalysis, Physical Chemistry Chemical Physics 23(12), 7178-7189. IUPAC (1997a) Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford. Online version (2019-) created by S. J. Chalk. https://goldbook.iupac.org/terms/view/A00525. IUPAC (1997b) Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford. Online version (2019-) created by S. J. Chalk. https://goldbook.iupac.org/terms/view/C00876. Ostwald W. (1890). Ueber Autokatalyse. Berichte über die Verhandlungen der Königlich Sächsischen Gesellschaft der Wissenschaften zu Leipzig, Mathematisch-Physische Classe 42, 189–191. Pekař M. (2021). Non-Equilibrium Thermodynamics View on Kinetics of Autocatalytic Reactions—Two Illustrative Examples. Molecules 26(3), article 585. Pekař M. (2022). Reaction rate view on autocatalysis. Qeios, https://doi.org/10.32388/IR2WXP. Peng Z., Paschek K., Xavier J.C. (2022). What Wilhelm Ostwald meant by “Autokatalyse” and its significance to origins-of-life research. BioEssays 44, 2200098. Rodrigues D., Billeter J., Bonvin D. (2017). Generalization of the concept of extents to distributed reaction systems. Chemical Engineering Science 171, 558-575. Schuster P. (2019). What is special about autocatalysis? Monatshefte für Chemie - Chemical Monthly 150(5),763–775. Scott Fogler H. (1992). Elements of Chemical Reaction Engineering, 2nd ed. Englewood Cliffs: PrenticeHall. 13
https://openalex.org/W4322211321	https://threatenedtaxa.org/index.php/JoTT/article/download/8172/9071	English	null	Differential kleptoparasitic interactions of Himalayan Vulture Gyps himalayensis with conspecifics and heterospecifics during various stages of breeding	Journal of threatened taxa	2,023	cc-by	8,853	Zoo Outreach Organization www.zooreach.org Zoo Outreach Organization www.zooreach.org 43/2 Varadarajulu Nagar, 5th Street West, Ganapathy, Coimbatore, Tamil Nadu 641006, India egistered Office: 3A2 Varadarajulu Nagar, FCI Road, Ganapathy, Coimbatore, Tamil Nadu 641006, India Ph: +91 9385339863 \| www.threatenedtaxa.org Email: sanjay@threatenedtaxa.org 43/2 Varadarajulu Nagar, 5th Street West, Ganapathy, Coimbatore, Tamil Nadu 641006, India Registered Office: 3A2 Varadarajulu Nagar, FCI Road, Ganapathy, Coimbatore, Tamil Nadu 641006, India Ph: +91 9385339863 \| www.threatenedtaxa.org Email: sanjay@threatenedtaxa.org Fundraising/Communications Mrs. Payal B. 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Boufford, Harvard University Herbaria, Cambridge, MA 02138-2020, USA Dr. Ritesh Kumar Choudhary, Agharkar Research Institute, Pune, Maharashtra, India Dr. Navendu Page, Wildlife Institute of India, Chandrabani, Dehradun, Uttarakhand, India Dr. Kannan C.S. Warrier, Institute of Forest Genetics and Tree Breeding, Tamil Nadu, India Editorial Board Dr. Russel Mittermeier Executive Vice Chair, Conservation International, Arlington, Virginia 22202, USA Prof. Mewa Singh Ph.D., FASc, FNA, FNASc, FNAPsy Ramanna Fellow and Life-Long Distinguished Professor, Biopsychology Laboratory, and Institute of Excellence, University of Mysore, Mysuru, Karnataka 570006, India; Honorary Professor, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore; and Adjunct Professor, National Institute of Advanced Studies, Bangalore Stephen D. Nash Scientific Illustrator, Conservation International, Dept. of Anatomical Sciences, Health Sciences Center, T-8, Room 045, Stony Brook University, Stony Brook, NY 11794-8081, USA Dr. Invertebrates Dr. R.K. Avasthi, Rohtak University, Haryana, India Dr. D.B. Bastawade, Maharashtra, India Dr. Partha Pratim Bhattacharjee, Tripura University, Suryamaninagar, India Dr. Kailash Chandra, Zoological Survey of India, Jabalpur, Madhya Pradesh, India Dr. Ansie Dippenaar-Schoeman, University of Pretoria, Queenswood, South Africa Dr. Rory Dow, National Museum of natural History Naturalis, The Netherlands Dr. Brian Fisher, California Academy of Sciences, USA Dr. Richard Gallon, llandudno, North Wales, LL30 1UP Dr. Hemant V. Ghate, Modern College, Pune, India Dr. M. Monwar Hossain, Jahangirnagar University, Dhaka, Bangladesh Mr. Jatishwor Singh Irungbam, Biology Centre CAS, Branišovská, Czech Republic. Dr. Ian J. Kitching, Natural History Museum, Cromwell Road, UK Dr. R.K. Avasthi, Rohtak University, Haryana, India Dr. D.B. Bastawade, Maharashtra, India Dr. Partha Pratim Bhattacharjee, Tripura University, Suryamaninagar, India Dr. Kailash Chandra, Zoological Survey of India, Jabalpur, Madhya Pradesh, India Dr. Ansie Dippenaar-Schoeman, University of Pretoria, Queenswood, South Africa Dr. Rory Dow, National Museum of natural History Naturalis, The Netherlands Dr. Brian Fisher, California Academy of Sciences, USA Dr. Richard Gallon, llandudno, North Wales, LL30 1UP Dr. Hemant V. Ghate, Modern College, Pune, India Dr. M. Monwar Hossain, Jahangirnagar University, Dhaka, Bangladesh Mr. Jatishwor Singh Irungbam, Biology Centre CAS, Branišovská, Czech Republic. Dr. Ian J. Kitching, Natural History Museum, Cromwell Road, UK continued on the back inside cover Cover: Pseudo-flying animals and wind-dependent seed & spore dispersers – made with digital painting in Krita. © Melito Prinson Pinto For Focus, Scope, Aims, and Policies, visit https://threatenedtaxa.org/index.php/JoTT/aims_scope For Article Submission Guidelines, visit https://threatenedtaxa.org/index.php/JoTT/about/submissions For Policies against Scientific Misconduct, visit https://threatenedtaxa.org/index.php/JoTT/policies_various continued on the back inside cover Cover: Pseudo-flying animals and wind-dependent seed & spore dispersers – made with digital painting in Krita. © Melito Prinson Pinto For Focus, Scope, Aims, and Policies, visit https://threatenedtaxa.org/index.php/JoTT/aims_scope For Article Submission Guidelines, visit https://threatenedtaxa.org/index.php/JoTT/about/submissions For Policies against Scientific Misconduct, visit https://threatenedtaxa.org/index.php/JoTT/policies_various Differential kleptoparasitic interactions of Himalayan Vulture Gyps himalayensis with conspecifics and heterospecifics during various stages of breeding Hameem Mushtaq Wani Department of Zoology, Central University of Kashmir, Ganderbal, Jammu and Kashmir 191201, India. Invertebrates hameemwani@gmail.com ISSN 0974-7907 (Online) \| ISSN 0974-7893 (Print) https://doi.org/10.11609/jott.8172.15.2.22606-22610 #8172 \| Received 02 September 2022 \| Final received 31 January 2023 \| Finally accepted 07 February 2023 OPEN ACCESS COMMUNICATION 200th Issue JoTT Journal of Threatened Taxa \| www.threatenedtaxa.org \| 26 February 2023 \| 15(2): 22606–22610 ISSN 0974-7907 (Online) \| ISSN 0974-7893 (Print) https://doi.org/10.11609/jott.8172.15.2.22606-22610 #8172 \| Received 02 September 2022 \| Final received 31 January 2023 \| Finally accepted 07 February 2023 OPEN ACCESS COMMUNICATION 200th Issue JoTT Journal of Threatened Taxa \| www.threatenedtaxa.org \| 26 February 2023 \| 15(2): 22606–22610 ISSN 0974-7907 (Online) \| ISSN 0974-7893 (Print) https://doi.org/10.11609/jott.8172.15.2.22606-22610 #8172 \| Received 02 September 2022 \| Final received 31 January 2023 \| Finally accepted 07 February 2023 OPEN ACCESS COMMUNICATION 2 Issue JoTT Journal of Threatened Taxa \| www.threatenedtaxa.org \| 26 February 2023 \| 15(2): 22606–22610 Differential kleptoparasitic interactions of Himalayan Vulture Gyps himalayensis with conspecifics and heterospecifics during various stages of breeding Hameem Mushtaq Wani Department of Zoology, Central University of Kashmir, Ganderbal, Jammu and Kashmir 191201, India. hameemwani@gmail.com Differential kleptoparasitic interactions of Himalayan Vulture Gyps himalayensis with conspecifics and heterospecifics during various stages of breeding Department of Zoology, Central University of Kashmir, Ganderbal, Jammu and Kashmir 191201, India. hameemwani@gmail.com Department of Zoology, Central University of Kashmir, Ganderbal, Jammu and Kashmir 191201, India hameemwani@gmail.com Abstract: Reports of kleptoparasitic events involving Gyps himalayensis (Himalayan Vulture) are limited. In this article we document intraspecific and interspecific kleptoparasitic interactions at nesting sites, and analyse factors influencing this behaviour. The study was carried out at Hirpora Wildlife Sanctuary of Kashmir Himalaya, at an elevation of about 2,546 m. We observed 61 instances of food theft involving conspecifics (n = 12) and heterospecifics (n = 49). The highest number of incidents were observed during the chick rearing period (n=40), followed by incubation (n = 10) and pre-laying periods (n = 5). We observed the highest number of attacks at nesting sites (n = 30) and the lowest in flight (n = 9). Keywords: Himalaya, Hirpora Wildlife Sanctuary, Kashmir, Kleptoparasitism, nest, vulture. Date of publication: 26 February 2023 (online & print) Date of publication: 26 February 2023 (online & print) Keywords: Himalaya, Hirpora Wildlife Sanctuary, Kashmir, Kleptoparasitism, nest, vulture. INTRODUCTION INTRODUCTION Kleptoparasitism is the acquisition of resources by theft (Brockmann & Barnard 1979; Hadjichrysanthou et al. 2018) such as prey or other materials that require time and effort to obtain. The practice is not without risk, since a kleptoparasite might be injured by its victim if it defends its prey (Iyengar 2008; Hadjichrysanthou et al. 2018). This behaviour is relatively widespread among birds, particularly sea birds. Kleptoparasitic interactions involving vultures, for example the Lammergeier Gypaetus barbatus and Black Vulture Aegypius monachus, have been reported in the literature (Margalida & Heredia 2002). Data on this behaviour at nesting zones, however, is limited. This may be due to the fact that while vultures congregate at carcasses (Mundy et al. 1992) they carry food in their crop to the nest where chicks are fed via regurgitation (Mushtaq 2020), making theft by other birds difficult. Himalayan Vultures feed on carcasses of dead animals (Image 2) (Wani et al. 2021) along with other scavengers including large billed crows and raven (Navaneethan et al. 2015). The availability of carrion can vary spatially and seasonally, thereby playing an important part in movement and distribution of species feeding on it (Wani et al. 2020). Himalayan vultures show intensive parental care during chick rearing periods. In this article, we documented intraspecific and interspecific kleptoparasitic interactions of Himalayan vulture at nesting sites, and analysed the factors influencing this behaviour. Image 1. Hirpora Wildlife Sanctuary. the sanctuary is divided into mixed coniferous forests, deciduous subalpine scrub forests and subalpine pastures. The coniferous forests are dominated by Kail pine, the sub alpine forests are dominated by fir while the deciduous subalpine scrub forests are dominated by Himalayan Birch Betula utilis and Juniper Juniperus communis (Wani et al. 2021). Study area Hirpora Wildlife Sanctuary spreads over an area of 341 km2 in Shopian District, Kashmir. At an altitude of 2,546 m, the sanctuary is located between 33.3955 oN & 74.3940 oE. It has forests, pastures, scrub land, waste land water bodies. To the north, the sanctuary is bounded by Lake Gumsar, to the east by Rupri, to the south by Saransar, to the west by the Pir Panjal pass and to northeast by Hirpora village (Wani et al. 2020) (Image 1). The area is renowned for its rich floral and faunal diversity. The main faunal elements of the sanctuary include- Pir Panjal Markhor Capra falconeri, Himalayan Musk Deer Moschus leucogaster, Himalayan Black Bear Ursus thibetanus, Himalayan Brown Bear Ursus arctos, Leopard Panthera pardus, Red Fox Vulpes vulpes, and Tibetan Wolf Canis lupus. The vegetation of Methods Field work was undertaken in Hirpora Wildlife Sanctuary from June 2019 to May 2020. Observations on food stolen, species involved and situation in which they occurred (in flight, at nest and on feeding site) were made during pre-laying, incubation and chick rearing period with the help of 10X binocular. Observations were made from vantage points (at a distance of about 300–400 m) that allowed a good view of nesting and feeding sites. In all intraspecific interactions observed, we recorded the individuals’ age which was determined by Grimmett et al. (2016). Editor: Bahar S. Baviskar, Wild-CER, Nagpur, India. Editor: Bahar S. Baviskar, Wild-CER, Nagpur, India. Editor: Bahar S. Baviskar, Wild-CER, Nagpur, India. Competing interests: The author declares no competing interests. Author details: Hameem Mushtaq Wani did his PhD programme from University of Kashmir, Srinagar. His doctoral thesis majorly focussed on bioecology and conservation status of vultures in Hirpora Wildlife Sanctuary of Kashmir Himalaya. He is currently working as a teaching faculty in the Department of Animal Science (Zoology), Central University of Kashmir, India. Acknowledgements: Author is grateful to the Department of Wildlife Protection, Government of Jammu & Kashmir, for providing necessary permission to work in Hirpora Wildlife Sanctuary. Differential kleptoparasitic interactions of Himalayan Vulture Image 1. Hirpora Wildlife Sanctuary. v Interactions during different periodst Highest number of attacks from both conspecifics and heterospecifics were observed during chick rearing period (n = 40) followed by incubation period (n = 10) and pre-laying period (n = 5). In chick rearing period, 90% attacks were defended successfully whereas in incubation period, only 62.5% of the attacks were defended successfully. However, during pre-laying period, all attacks from conspecifics and heterospecifics were defended successfully. The percentage of defended and non-defended attacks were statistically significant (H = 8.16; DF = 02; P <0.05) (Table 3). Table2. Kleptoparasitic interactions of Gyps himalayensis with conspecifics in Hirpora Wildlife Sanctuary. Table2. Kleptoparasitic interactions of Gyps himalayensis with conspecifics in Hirpora Wildlife Sanctuary. Host Kleptoparasite Gyps himalayensis Subadult Adult Kleptoparasite Sub-adult (04) 02 02 Adult (08) 06 02 Kruskal-Wallis ANOVA H = 7.89; DF = 01; P <0.05 Table 1. Kleptoparasitic interactions of Gyps himalayensis with heterospecifics in Hirpora Wildlife Sanctuary. and MINITAB (Ryan et al. 1992). A non-parametric test, Kruskal-Wallis one way ANOVA was used for testing the null hypothesis at p <0.05. Kleptoparasite Host Gyps himalayensis (Subadult) Gyps himalayensis (Adult) Gypaetus barbatus 05 02 Corvus corax 15 07 Corvus macrorhynchos 08 07 Corvus splendens 03 02 Kruskal-Wallis one way ANOVA H = 7.32; DF = 03; P <0.05 Table 3. Percentage of Kleptoparasitic attacks defended and not defended by Gyps himalayensis during Pre-laying, Incubation and Chick rearing period in Hirpora Wildlife Sanctuary. Table 3. Percentage of Kleptoparasitic attacks defended and not defended by Gyps himalayensis during Pre-laying, Incubation and Chick rearing period in Hirpora Wildlife Sanctuary. Table 3. Percentage of Kleptoparasitic attacks defended and not defended by Gyps himalayensis during Pre-laying, Incubation and Chick rearing period in Hirpora Wildlife Sanctuary. Period No. of attacks Percentage of attacks Defended (%) Non- defended (%) Pre-laying 05 5(100) 0(0.0) Incubation 16 10(62.5) 6(37.5) Chick rearing 40 36(90) 4(10.0) Kruskal-Wallis one way ANOVA H = 8.16; DF = 02; P <0.05 Interactions with heterospecifics We observed a total of 49 interactions of Himalayan Vulture with heterospecifics. All these interactions were statistically significant (H = 7.32; DF = 03; P <0.05). In 07 of these interactions, Gypaetus barbatus acted as kleptoparasite with 05 such interactions in which sub- adult Himalayan vulture acted as host. In rest of the two interactions, adult Himalayan vulture acted as host. Rest of the interactions involved different corvid species including- Common Raven Corvus corax (n = 22), Large- billed Crow Corvus macrorhynchos (n = 15), and House Crow Corvus splendens (n = 05). In 15 interactions with Common Raven, sub-adult Himalayan vulture individuals acted as hosts and in seven such interactions, adult Himalayan Vulture individuals acted as hosts. Similarly, in eight interactions with Corvus macrorhynchos, sub-adult Himalayan Vulture and in seven such interactions, adult Himalayan vulture acted as hosts. Among interactions with Corvus splendens, three interactions involve sub- adult Himalayan Vulture, and two interactions involve adult individuals as hosts (Table 1). RESULTS We observed 61 Himalayan Vulture interactions of food theft, 12 with conspecifics and 49 with heterospecifics. These interactions varied among different sites and seasons (Table 4, Table 5). The various heterospecifics involved in these interactions included Bearded Vulture Gypaetus barbatus (n = 7), Common Raven Corvus corax (n = 22), Large-billed Crow Corvus macrorhynchos (n = 15), and House Crow Corvus splendens (n = 5). Table2. Kleptoparasitic interactions of Gyps himalayensis with conspecifics in Hirpora Wildlife Sanctuary. Data analysisii Basic statistics such as, mean and standard deviation were calculated for all the variables and were given as X±SD. Statistical analysis were performed by using Windows based statistical packages- Micorsoft Excel Journal of Threatened Taxa \| www.threatenedtaxa.org \| 26 February 2023 \| 15(2): 22606–22610 22607 Differential kleptoparasitic interactions of Himalayan Vulture Table 1. Kleptoparasitic interactions of Gyps himalayensis with heterospecifics in Hirpora Wildlife Sanctuary. Interactions with conspecifics We observed a total of 12 interactions of Himalayan Vulture with conspecifics (Table 2). In eight interactions adult Himalayan vultures acted as kleptoparasites, and in four interactions sub-adult vultures acted as kleptoparasites. During the former case, four sub-adults and two adults acted as hosts whereas in the latter case, one adult and one sub-adult acted as hosts. All these interactions with conspecifics were statistically significant (H = 7.89; DF = 01; P <0.05) (Table 2). DISCUSSION Kleptoparasitism occurs when there is an association between species. However, it is equally obvious, that kleptoparasitism does not always occur when two species are found together. Rather, there are various ecological and behavioural conditions that make kleptoparasitism particularly likely. These include- large concentration of host (John & Lee 2019), large quantities of food (Mullers & Amar 2015) large and high quality food items (Iyengar 2008), predictable food supply (Dekker et al. 2012), visibility to food items (John & Lee 2019), food shortage behaviour of parasite (Mullers & Amar 2015), behaviour and habitat of host (Hamilton 2002). Dominance of adults over immature is a well- documented phenomenon in raptors (Moreno-Opo et al. 2020), but a reverse dominance pattern also has been observed (Rodríguez-Estrella & Rivera-Rodriguez 1992). In the case of conspecifics, plumage colouration of Himalayan vulture adults could act as a status signal (Negro et al. 1999). This signal could be used by territorial adults to displace other immature Himalayan Vultures not by attacking them, but simply by signalling their status while approaching them (Bautista et al. 1998). Our results suggested that the Corvus corax, C. macrorhynchos and C. splendens due to their little chance for foraging at carcass as compared to vultures, are making use of the spatial and temporal predictability of food resources by becoming kleptoparasites (Fisher 1985). Most of the thefts suffered at the nest by kleptoparasites took place during chick rearing, a period when food items often accumulate at the nest sites. Thefts in flight occurred during pre-laying and incubation period, a time when food availability is reduced and when weather may greatly limit the activities of foraging and locating food. For those age groups (principally <3 years, i.e., sub-adults) that are more dependent On the other hand, the Himalayan Vulture having low wing loading and its large wingspan give this species great dominance in flight (Donázar et al. 1993) and make it difficult for an opponent to steal food successfully. In the case of conspecifics, the fact that younger birds are less skilful in flight would mean that they would be less successful in actions of direct piracy, so that the energetic cost of those attempts might be greater than the likely benefits obtained from those actions (Fisher 1985; Moreno-Opo et al. 2020). Table 4. Kleptoparasitic interactions of Gyps himalayensis with conspecifics and heterospecifics in flight, at nest and at feeding site. Place/Site Thefts Defended Non- defended Flight 09 07 02 Nest 30 29 01 Feeding site 22 15 07 Kruskal-Wallis one way ANOVA H = 8.14; DF = 02; P <0.05 Season Attacks Attacks defended (%) Attacks not defended (%) Winter 28 92.85 7.15 Spring 12 83.33 16.67 Summer 14 57.14 42.86 Autumn 07 71.42 28.58 on predictable food sources such as feeding stations (Heredia 1991), this might be a foraging strategy used much more regularly. These results are in agreement with the idea that immature or inexperienced birds may compensate for their less effective foraging abilities by kleptoparasitism (Margalida & Bertran 2003). To the contrary, kleptoparasitism by adults could be an opportunistic foraging behaviour. Our observations were done in flight, in addition to nests and feeding sites. This accounts for the fact that breeding adults were the host bird in 79% of all observed events. sites were statistically significant (H = 8.14; DF = 02; P <0.05). We observed highest number of attacks at nesting site (n = 30) and lowest number of attacks in flight (n = 09). A total of 22 attacks were observed at feeding sites. Among 30 attacks, at nest site, 29 were defended successfully. On the other hand, among 22 attacks at feeding sites, only 15 were defended and rest (31.81%) were not defended (Image 2). Out of nine attacks in flight, seven were defended and in two attacks, kleptoparasite remained successful in taking away the food from Himalayan Vulture (Table 4). As a result of the cost/benefit rate, two factors would determine that the species that attempted stealing would resort to this indirect strategy: the territorial behaviour of the host species (Margalida & Bertran 2000) and the accumulation of food resources in nesting area. Differential kleptoparasitic interactions of Himalayan Vulture Differential kleptoparasitic interactions of Himalayan Vulture Wani Interactions at different sitesi The number of interactions of Himalayan Vulture with its conspecifics and heterospecifics at different Journal of Threatened Taxa \| www.threatenedtaxa.org \| 26 February 2023 \| 15(2): 22606–22610 22608 DISCUSSION The Himalayan Vulture’s attacks of intruders in the vicinity of the nest throughout the breeding season (Margalida & Bertran 2000) would act as deterrent Journal of Threatened Taxa \| www.threatenedtaxa.org \| 26 February 2023 \| 15(2): 22606–22610 22609 Differential kleptoparasitic interactions of Himalayan Vulture Wani Image 2. Himalayan Vulture feeding on carcass of a buffalo. © Wani Hameem Grimmett, R., C. Inskipp & T. Inskipp (2016). Birds of Indian Subcontinent. Bloomsbury publishing India, 528 pp. Hadjichrysanthou, C., M. Broom & J. Rychtář (2018). Models of kleptoparasitism on networks: the effect of population structure on food stealing behaviour. Journal of Mathematical Biology 76: 1465– 1488. https://doi.org/10.1007/s00285-017-1177-7 t Hamilton, I.M. (2002). Kleptoparasitism and the distribution of unequal competitors. Behavioural Ecology 13(2): 260–267 Heredia, B. (Eds.) (1991). El plan coordinado de actuaciones para la protecci6n del quebrantahuesos. El quebrantahuesos (Gypaetus barbatus) en los Pirineos, 126pp. Iyengar (2008). Kleptoparasitic interactions throughout the animal kingdom and a-re-evaluation, based on participant mobility, of the conditions promoting the evolution of kleptoparasitism. Biological Journal of the Linnean Society 93: 745–762. i John, J.R.M & W.S. Lee (2019). Kleptoparasitism of Shoebills Balaeniceps rex by African Fish Eagles Haliaeetus vocifer in Western Tanzania. Tanzania Journal of Science 45(2): 131–143. Margalida, A. & J. Bertran (2000). Breeding behaviour of the Bearded Vulture Gypaetus barbatus- minimal sexual differences in parental activities. Ibis 142: 225 ii Margalida, A. & J. Bertran (2001). Function and temporal variation in use of ossuaries by Bearded Vultures (Gypaetus barbatus) during the nestling period. Auk 118: 785–789.ii Image 2. Himalayan Vulture feeding on carcass of a buffalo. Margalida, A. & J. Bertran (2003). Interspecific and intraspecific kleptoparasitic interactions of the bearded vulture (Gypaetus barbatus) at nesting areas. Journal of Raptor Research 37: 157–160. and would make food at nest the least convenient for stealing. The success in aggressive encounters appears determined by the body size and condition, and the previous possession of the disputed resource (Bautista et al. 1998). In contrast, those species with higher aerial maneuverability but with smaller size, such as ravens, would have to focus their actions at the nest, where prey remains also accumulate. DISCUSSION Obtaining prey remains there may be less costly for those birds: (1) adults are gradually less often present at the nest as the breeding season progress (Margalida & Bertran 2000) and (2) prey items present in the nest have a higher meat content as consequence of differential requirements in nutrients for the chick (Margalida & Bertran 2001). i Margalilda, A. & R. Heredia (2002). Interspecific interaction between Lammergeier Gypaetus barbatus and Black Vulture Aegypius monachus: predation or kleptoparasitism? Sandgrouse 24: 138–139 ii Moreno-Opo, R., A. Trujillano & A. Margalida (2020). Larger size and older age confer competitive advantage: dominance hierarchy within European vulture guild. Scientific Reports 10, 2430 (2020). https://doi.org/10.1038/s41598-020-59387-4 t Mullers, R.H.E. & A. Amar (2015). Shoebill Balaenicpes rex foraging behaviour in the Bangweulu Wetlands, Zambia. Ostrich 86(1&2): 113–118 Mundy, P., D. Butchart, J. Ledger & S. Piper (1992).The vultures of Africa. Acorn Books & Russel Friedman Books, Randburg and Halfway, 100 pp.i Mushtaq, H. (2020). Bioecology and conservation status of vultures in Hirpora Wildlife Sanctuary of Kashmir Himalaya. Ph.D. Thesis. Department of Zoology, University of Kashmir, 121 pp. Navaneethan, B., K. Kalyansundaram Sankar, Q. Qureshi & M. Manjrekar (2015). The Status of Vultures in Bandhavgarh Tiger Reserve, Madhya Pradesh, Central India. Journal of Threatened Taxa 7(14): 8134–8138. https://doi.org/10.11609/jott.2428.7.14.8134- 8138 REFERENCES Grant Connette, Smithsonian Institution, Royal, VA, USA Dr. M. Zafar-ul Islam, Prince Saud Al Faisal Wildlife Research Center, Taif, Saudi Arabia Dr. Hem Sagar Baral, Charles Sturt University, NSW Australia Mr. H. Byju, Coimbatore, Tamil Nadu, India Dr. Chris Bowden, Royal Society for the Protection of Birds, Sandy, UK Dr. Priya Davidar, Pondicherry University, Kalapet, Puducherry, India Dr. J.W. 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Wood, Landcare Research, Canterbury, New Zealand Dr. Wendy Collinson-Jonker, Endangered Wildlife Trust, Gauteng, South Africa Dr. Rajeshkumar G. Jani, Anand Agricultural University, Anand, Gujarat, India Dr. O.N. Tiwari, Senior Scientist, ICAR-Indian Agricultural Research Institute (IARI), New Delhi, India Dr. J.A. Johnson, Wildlife Institute of India, Dehradun, Uttarakhand, India Dr. R. Ravinesh, Gujarat Institute of Desert Ecology, Gujarat, India Fishes Dr. Neelesh Dahanukar, IISER, Pune, Maharashtra, India Dr. Topiltzin Contreras MacBeath, Universidad Autónoma del estado de Morelos, México Dr. Heok Hee Ng, National University of Singapore, Science Drive, Singapore Dr. Rajeev Raghavan, St. Albert’s College, Kochi, Kerala, India Dr. Robert D. Sluka, Chiltern Gateway Project, A Rocha UK, Southall, Middlesex, UK Dr. E. Vivekanandan, Central Marine Fisheries Research Institute, Chennai, India Dr. Davor Zanella, University of Zagreb, Zagreb, Croatia Dr. A. Biju Kumar, University of Kerala, Thiruvananthapuram, Kerala, India Dr. Akhilesh K.V., ICAR-Central Marine Fisheries Research Institute, Mumbai Research Centre, Mumbai, Maharashtra, India Dr J A Johnson Wildlife Institute of India Dehradun Uttarakhand India Dr. Neelesh Dahanukar, IISER, Pune, Maharashtra, India Dr. Topiltzin Contreras MacBeath, Universidad Autónoma del estado de Morelos, México Dr. Heok Hee Ng, National University of Singapore, Science Drive, Singapore Dr. Rajeev Raghavan, St. Albert’s College, Kochi, Kerala, India Dr. Robert D. Sluka, Chiltern Gateway Project, A Rocha UK, Southall, Middlesex, UK Dr. E. Vivekanandan, Central Marine Fisheries Research Institute, Chennai, India Dr. Davor Zanella, University of Zagreb, Zagreb, Croatia Dr. A. Biju Kumar, University of Kerala, Thiruvananthapuram, Kerala, India Dr. Akhilesh K.V., ICAR-Central Marine Fisheries Research Institute, Mumbai Research Centre, Mumbai, Maharashtra, India Dr. J.A. Johnson, Wildlife Institute of India, Dehradun, Uttarakhand, India Dr. R. Ravinesh, Gujarat Institute of Desert Ecology, Gujarat, India Amphibians Dr. Sushil K. Dutta, Indian Institute of Science, Bengaluru, Karnataka, India Dr. Annemarie Ohler, Muséum national d’Histoire naturelle, Paris, France Dr. L.D. Singla, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India Dr. Rupika S. Rajakaruna, University of Peradeniya, Peradeniya, Sri Lanka Dr. Bahar Baviskar, Wild-CER, Nagpur, Maharashtra 440013, India Mammals Uniyal, Wildlife Institute of India, Dehradun, Uttarakhand 248001, India Dr. John T.D. Caleb, Zoological Survey of India, Kolkata, West Bengal, India Dr. Priyadarsanan Dharma Rajan, Ashoka Trust for Research in Ecology and the Environment (ATREE), Royal Enclave, Bangalore, Karnataka, India Mammals Dr. Giovanni Amori, CNR - Institute of Ecosystem Studies, Rome, Italy Dr. Anwaruddin Chowdhury, Guwahati, India Dr. David Mallon, Zoological Society of London, UK Dr. Shomita Mukherjee, SACON, Coimbatore, Tamil Nadu, India Dr. Angie Appel, Wild Cat Network, Germany Dr. P.O. Nameer, Kerala Agricultural University, Thrissur, Kerala, India Dr. Ian Redmond, UNEP Convention on Migratory Species, Lansdown, UK Dr. Heidi S. Riddle, Riddle’s Elephant and Wildlife Sanctuary, Arkansas, USA Dr. Karin Schwartz, George Mason University, Fairfax, Virginia. Dr. Lala A.K. Singh, Bhubaneswar, Orissa, India Dr. Mewa Singh, Mysore University, Mysore, India Dr. Paul Racey, University of Exeter, Devon, UK Dr. Honnavalli N. Kumara, SACON, Anaikatty P.O., Coimbatore, Tamil Nadu, India Dr. Nishith Dharaiya, HNG University, Patan, Gujarat, India Dr. Spartaco Gippoliti, Socio Onorario Società Italiana per la Storia della Fauna “Giuseppe Altobello”, Rome, Italy Dr. Justus Joshua, Green Future Foundation, Tiruchirapalli, Tamil Nadu, India Dr. H. Raghuram, The American College, Madurai, Tamil Nadu, India Dr. Paul Bates, Harison Institute, Kent, UK Dr. Jim Sanderson, Small Wild Cat Conservation Foundation, Hartford, USA Dr. Dan Challender, University of Kent, Canterbury, UK Dr. David Mallon, Manchester Metropolitan University, Derbyshire, UK Dr. Brian L. Cypher, California State University-Stanislaus, Bakersfield, CA Dr. S.S. Talmale, Zoological Survey of India, Pune, Maharashtra, India Prof. Karan Bahadur Shah, Budhanilakantha Municipality, Kathmandu, Nepal Dr. Susan Cheyne, Borneo Nature Foundation International, Palangkaraja, Indonesia Dr. Hemanta Kafley, Wildlife Sciences, Tarleton State University, Texas, USA Dr. Giovanni Amori, CNR - Institute of Ecosystem Studies, Rome, i Dr. Kareen Schnabel, Marine Biologist, Wellington, New Zealand Dr. Anwaruddin Chowdhury, Guwahati, India Dr. Amazonas Chagas Júnior, Universidade Federal de Mato Gross Dr. R.J. Shiel, University of Adelaide, SA 5005, Australia Dr. Siddharth Kulkarni, The George Washington University, Washington, USA Dr. Priyadarsanan Dharma Rajan, ATREE, Bengaluru, India Dr. Phil Alderslade, CSIRO Marine And Atmospheric Research, Hobart, Austra Dr. John E.N. Veron, Coral Reef Research, Townsville, Australia Dr. Daniel Whitmore, State Museum of Natural History Stuttgart, Rosenstein, Germany.i Dr. Daniel Whitmore, State Museum of Natural History Stuttgar Dr. Yu-Feng Hsu, National Taiwan Normal Uni Dr. Yu-Feng Hsu, National Taiwan Normal University, Taipei City, Taiwan Dr. Keith V. Wolfe, Antioch, California, USA Dr. Tomas Ditrich, Faculty of Education, University of South Bohemia in Ceske Dr. Tomas Ditrich, Faculty of Education, University of South Bohemia in Ceske Budejovice, Czech Republic Dr. Mihaly Foldvari, Natural History Museum, University of Oslo, Norway Dr. V.P. REFERENCES K.S.C., Kuwait Dr. Himender Bharti, Punjabi University, Punjab, India Mr. Purnendu Roy, London, UK Dr. Saito Motoki, The Butterfly Society of Japan, Tokyo, Japan Dr. Sanjay Sondhi, TITLI TRUST, Kalpavriksh, Dehradun, India Dr. Nguyen Thi Phuong Lien, Vietnam Academy of Science and Technology, Hanoi, Vietnam Dr. Nitin Kulkarni, Tropical Research Institute, Jabalpur, India Dr. Robin Wen Jiang Ngiam, National Parks Board, Singapore Dr. Lional Monod, Natural History Museum of Geneva, Genève, Switzerland. Dr. Asheesh Shivam, Nehru Gram Bharti University, Allahabad, India Dr. Rosana Moreira da Rocha, Universidade Federal do Paraná, Curitiba, Brasil Dr. Kurt R. Arnold, North Dakota State University, Saxony, Germany Dr. James M. Carpenter, American Museum of Natural History, New York, USA Dr. David M. 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Scientist, Zoological Survey of India, Pune, India Dr. Manju Siliwal, WILD, Coimbatore, Tamil Nadu, India Dr. G.P. Sinha, Botanical Survey of India, Allahabad, India Dr. K.A. Subramanian, Zoological Survey of India, New Alipore, Kolkata, India Dr. REFERENCES P.M. Sureshan, Zoological Survey of India, Kozhikode, Kerala, India Dr. R. Varatharajan, Manipur University, Imphal, Manipur, India Dr. Eduard Vives, Museu de Ciències Naturals de Barcelona, Terrassa, Spain Dr. James Young, Hong Kong Lepidopterists’ Society, Hong Kong Dr. R. Sundararaj, Institute of Wood Science & Technology, Bengaluru, India Dr. M. Nithyanandan, Environmental Department, La Ala Al Kuwait Real Estate. Co. 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Claborn, Missouri State University, Springfield, USA Dr. Kareen Schnabel, Marine Biologist, Wellington, New Zealand Dr. Amazonas Chagas Júnior, Universidade Federal de Mato Grosso, Cuiabá, Brasil Mr. Monsoon Jyoti Gogoi, Assam University, Silchar, Assam, India Dr. Heo Chong Chin, Universiti Teknologi MARA (UiTM), Selangor, Malaysia Dr. R.J. Shiel, University of Adelaide, SA 5005, Australia Dr. Siddharth Kulkarni, The George Washington University, Washington, USA Dr. Priyadarsanan Dharma Rajan, ATREE, Bengaluru, India Dr. Phil Alderslade, CSIRO Marine And Atmospheric Research, Hobart, Australia Dr. John E.N. Veron, Coral Reef Research, Townsville, Australia Dr. Daniel Whitmore, State Museum of Natural History Stuttgart, Rosenstein, Germany. Dr. Yu-Feng Hsu, National Taiwan Normal University, Taipei City, Taiwan Dr. Keith V. Wolfe, Antioch, California, USA Dr. Siddharth Kulkarni, The Hormiga Lab, The George Washington University, Washington, D.C., USA Dr. Tomas Ditrich, Faculty of Education, University of South Bohemia in Ceske Budejovice, Czech Republic Dr. Mihaly Foldvari, Natural History Museum, University of Oslo, Norway Dr. V.P. Uniyal, Wildlife Institute of India, Dehradun, Uttarakhand 248001, India Dr. John T.D. Caleb, Zoological Survey of India, Kolkata, West Bengal, India Dr. Priyadarsanan Dharma Rajan, Ashoka Trust for Research in Ecology and the Environment (ATREE), Royal Enclave, Bangalore, Karnataka, India Fishes Dr. Neelesh Dahanukar, IISER, Pune, Maharashtra, India il i h i id d ó d l d d l é i Birds Dr. REFERENCES Hem Sagar Baral, Charles Sturt University, NSW Australia Mr. H. Byju, Coimbatore, Tamil Nadu, India Dr. Chris Bowden, Royal Society for the Protection of Birds, Sandy, UK Dr. Priya Davidar, Pondicherry University, Kalapet, Puducherry, India Dr. J.W. Duckworth, IUCN SSC, Bath, UK Dr. Rajah Jayapal, SACON, Coimbatore, Tamil Nadu, India Dr. Rajiv S. Kalsi, M.L.N. College, Yamuna Nagar, Haryana, India Dr. V. Santharam, Rishi Valley Education Centre, Chittoor Dt., Andhra Pradesh, India Dr. S. Balachandran, Bombay Natural History Society, Mumbai, India Mr. J. Praveen, Bengaluru, India Dr. C. Srinivasulu, Osmania University, Hyderabad, India Dr. K.S. Gopi Sundar, International Crane Foundation, Baraboo, USA Dr. Gombobaatar Sundev, Professor of Ornithology, Ulaanbaatar, Mongolia Prof. Reuven Yosef, International Birding & Research Centre, Eilat, Israel Dr. Taej Mundkur, Wetlands International, Wageningen, The Netherlands Dr. Carol Inskipp, Bishop Auckland Co., Durham, UK Dr. Tim Inskipp, Bishop Auckland Co., Durham, UK Dr. V. Gokula, National College, Tiruchirappalli, Tamil Nadu, India Dr. Arkady Lelej, Russian Academy of Sciences, Vladivostok, Russia Dr. Simon Dowell, Science Director, Chester Zoo, UK Dr. Mário Gabriel Santiago dos Santos, Universidade de Trás-os-Montes e Alto Douro, Quinta de Prados, Vila Real, Portugal Dr. Grant Connette, Smithsonian Institution, Royal, VA, USA Dr. M. Zafar-ul Islam, Prince Saud Al Faisal Wildlife Research Center, Taif, Saudi Arabia Dr. Hem Sagar Baral, Charles Sturt University, NSW Australia Mr. H. Byju, Coimbatore, Tamil Nadu, India Dr. Chris Bowden, Royal Society for the Protection of Birds, Sandy, UK Dr. Priya Davidar, Pondicherry University, Kalapet, Puducherry, India Dr. J.W. Duckworth, IUCN SSC, Bath, UK Dr. Rajah Jayapal, SACON, Coimbatore, Tamil Nadu, India Dr. Rajiv S. Kalsi, M.L.N. College, Yamuna Nagar, Haryana, India Dr. V. Santharam, Rishi Valley Education Centre, Chittoor Dt., Andhra Pradesh, India Dr. S. Balachandran, Bombay Natural History Society, Mumbai, India Mr. J. Praveen, Bengaluru, India Dr. C. Srinivasulu, Osmania University, Hyderabad, India Dr. K.S. Gopi Sundar, International Crane Foundation, Baraboo, USA Dr. Gombobaatar Sundev, Professor of Ornithology, Ulaanbaatar, Mongolia Prof. Reuven Yosef, International Birding & Research Centre, Eilat, Israel Dr. Taej Mundkur, Wetlands International, Wageningen, The Netherlands Dr. Carol Inskipp, Bishop Auckland Co., Durham, UK Dr. Tim Inskipp, Bishop Auckland Co., Durham, UK Dr. V. Gokula, National College, Tiruchirappalli, Tamil Nadu, India Dr. Arkady Lelej, Russian Academy of Sciences, Vladivostok, Russia Dr. Simon Dowell, Science Director, Chester Zoo, UK Dr. Mário Gabriel Santiago dos Santos, Universidade de Trás-os-Montes e Alto Douro, Quinta de Prados, Vila Real, Portugal Dr. REFERENCES Negro, J.J., A. Margalida, F. Hiraldo, & R. Heredia (1999). The function of cosmetic coloration of Bearded Vultures: when art imitates life. Animal Behaviour 58: F14–F17i Bautista, L.M., J.C. Alonso & J.A. Alonso (1998). Foraging site displacement in Common Crane flocks. Animal Behaviour 56: 1237– 1243.i Rodríguez-Estrella, R. & L.B. Rivera-Rodríguez (1992). Kleptoparasitism and other interactions of Crested Caracara in the Cape region, Baja California, Mexico. Journal of Field Ornithology 63: 177–180. Brockmann, H.J. & C.J. Barnard (1979). Kleptoparasitism in birds. Animal Behaviour 27: 487–514. f California, Mexico. Journal of Field Ornithology 63: 177–18 Ryan, F.B., B.L. Joiner & A.T. Ryan (1992).MINITAB Handbook. Boston PWS-KENT Publishing Company, 376 pp.i Dekker, D., M. Out, M. Tabak & R. Ydenberg (2012).The effect of kleptoparasitic bald eagles and gyrfalcons on the kill rate of peregrine falcons hunting dunlins wintering in British Columbia. Condor 114(2): 290–294.l Wani, H.M., M.F. Fazili & R. Ahmad (2021). Seasonal habitat selection of Gypshimalayensis in Hirpora Wildlife Sanctuary, Jammu and Kashmir. 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Orr, Griffith University, Nathan, Australia Dr. Sameer Padhye, Katholieke Universiteit Leuven, Belgium Dr. Nancy van der Poorten, Toronto, Canada Dr. Kareen Schnabel, NIWA, Wellington, New Zealand Dr. R.M. Sharma, (Retd.) Scientist, Zoological Survey of India, Pune, India Dr. Manju Siliwal, WILD, Coimbatore, Tamil Nadu, India Dr. G.P. Sinha, Botanical Survey of India, Allahabad, India Dr. K.A. Subramanian, Zoological Survey of India, New Alipore, Kolkata, India Dr. P.M. Sureshan, Zoological Survey of India, Kozhikode, Kerala, India Dr. R. Varatharajan, Manipur University, Imphal, Manipur, India Dr. Eduard Vives, Museu de Ciències Naturals de Barcelona, Terrassa, Spain Dr. James Young, Hong Kong Lepidopterists’ Society, Hong Kong Dr. R. Sundararaj, Institute of Wood Science & Technology, Bengaluru, India Dr. M. Nithyanandan, Environmental Department, La Ala Al Kuwait Real Estate. Co. Reptiles Reviewers 2019–2021 Due to pausity of space, the list of reviewers for 2018–2020 is available online. The opinions expressed by the authors do not reflect the views of the Journal of Threatened Taxa, Wildlife Information Liaison Development Society, Zoo Outreach Organization, or any of the partners. The journal, the publisher, the host, and the partners are not responsible for the accuracy of the political boundaries shown in the maps by the authors. Journal of Threatened Taxa is indexed/abstracted in Bibliography of Sys tematic Mycology, Biological Abstracts, BIOSIS Previews, CAB Abstracts, EBSCO, Google Scholar, Index Copernicus, Index Fungorum, JournalSeek, National Academy of Agricultural Sciences, NewJour, OCLC WorldCat, SCOPUS, Stanford University Libraries, Virtual Library of Biology, Zoologi cal Records. NAAS rating (India) 5.64 Print copies of the Journal are available at cost. Write to: The Managing Editor, JoTT, c/o Wildlife Information Liaison Development Society, 43/2 Varadarajulu Nagar, 5th Street West, Ganapathy, Coimbatore, Tamil Nadu 641006, India ravi@threatenedtaxa.org Print copies of the Journal are available at cost. Write to: The Managing Editor, JoTT, c/o Wildlife Information Liaison Development Society, 43/2 Varadarajulu Nagar, 5th Street West, Ganapathy, Coimbatore, Tamil Nadu 641006, India ravi@threatenedtaxa.org Print copies of the Journal are available at cost. Write to: The Managing Editor, JoTT, c/o Wildlife Information Liaison Development Society, 43/2 Varadarajulu Nagar, 5th Street West, Ganapathy, Coimbatore, Tamil Nadu 641006, India ravi@threatenedtaxa.org www.threatenedtaxa.org O The Journal of Threatened Taxa (JoTT) is dedicated to building evidence for conservation globally by publishing peer-reviewed articles online every month at a reasonably rapid rate at www.threatenedtaxa.org. All articles published in JoTT are registered under Creative Commons Attribution 4.0 International License unless otherwise mentioned. JoTT allows allows unrestricted use, reproduction, and distribution of articles in any medium by providing adequate credit to the author(s) and the source of publication. OPEN ACCESS ISSN 0974-7907 (Online) \| ISSN 0974-7893 (Print) ISSN 0974-7907 (Online) \| ISSN 0974-7893 (Print) Short Communications First record of the Great Seahorse Hippocampus kelloggi Jordan & Snyder, 1901 (Actinopterygii: Syngnathiformes: Syngnathidae) from the northwestern coast of Bay of Bengal – Anil Kumar Behera, Biswajit Mahari & Amrit Kumar Mishra, Pp. 22737–22740 Schoenoplectiella erecta (Poir.) Lye ssp. raynalii (Schuyler) Beentje (Cyperaceae) – a new record to India from Ossudu Bird Sanctuary, Villupuram District, Tamil Nadu – Chandrasegrane Pradeep, Paneerselvam Umamaheswari, Natesan Balachandran & Raphael Mathevet, Pp. 22741–22745 – H. Byju, N. Raveendran & S. Ravichandran, Pp. 22574–22585 Habitats of House Sparrow Passer domesticus (Linnaeus, 1758) in Rameswaram Island, Tamil Nadu, India – M. Pandian, Pp. 22586–22596 Schoenoplectiella erecta (Poir.) Lye ssp. raynalii (Schuyler) Beentje (Cyperaceae) – a new record to India from Ossudu Bird Sanctuary, Villupuram District, Tamil Nadu – Chandrasegrane Pradeep, Paneerselvam Umamaheswari, Natesan Balachandran & Raphael Mathevet, Pp. 22741–22745 Seasonal diversity and dietary guild structure of birds in two Vindhyan gorge forests of Rajasthan, India – Ashvini Kumar Joshi, Pp. 22597–22605 Differential kleptoparasitic interactions of Himalayan Vulture Gyps himalayensis with conspecifics and heterospecifics during various stages of breeding – Hameem Mushtaq Wani, Pp. 22606–22610 Notes Status of the Sumatran Striped Rabbit Nesolagus netscheri in Isau-Isau Wildlife Reserve, South Sumatra Province, Indonesia – Arum Setiawan, Muhammad Iqbal, Octavia Susilowati, Doni Setiawan, Martialis Puspito Khristy Maharsi & Indra Yustian, Pp. 22746–22748 Photographic record of the butterfly ray Gymnura cf. poecilura (Myliobatiformes: Gymnuridae) from the Bhagirathi-Hooghly River in West Bengal, eastern India – Priyankar Chakraborty, Pp. 22749–22751 First report of the fairyfly Schizophragma mitai Triapitsyn (Hymenoptera: Mymaridae) from India with notes on S. indica Rehmat & Anis – Anandhan Rameshkumar, Nazurius Anand, Sayan Sardar & Sarfrazul Islam Kazmi, Pp. 22752–22756 Occurrence of Ranunculus sceleratus L. (Ranunculaceae) from the Nilgiri District, Tamil Nadu, India – J. Shashikanth, S.Mugendhiran & Digvijay Verma, Pp. 22757–22760 First report of Meliola panici on Ottochloa nodosa (Kunth) Dandy (Poaceae) – Gopinathan Nair Gokul & Jacob Thomas, Pp. 22761–22763 New record of an usneoied lichen Usnea hirta (L.) Weber ex F.H.Wigg. from India – K.S. Vinayaka, Archana R. Mesta & N. Rajeshwari, Pp. 22764–22766 On the occurrence of two species of rare cyanobacterial genus Petalonema M.J.Berkeley ex Wolle, 1887 (Cyanophyceae: Nostocales: Scytonemataceae) from eastern Himalaya, India – Jai Prakash Keshri, Narendra Nath Koley & Jay Mal, Pp. 22767–22770 Status of the Sumatran Striped Rabbit Nesolagus netscheri in Isau-Isau Wildlife Reserve, South Sumatra Province, Indonesia – Arum Setiawan, Muhammad Iqbal, Octavia Susilowati, Doni Setiawan, Martialis Puspito Khristy Maharsi & Indra Yustian, Pp. 22746–22748 Status of the Sumatran Striped Rabbit Nesolagus netscheri in Isau-Isau Wildlife Reserve, South Sumatra Province, Indonesia – Arum Setiawan, Muhammad Iqbal, Octavia Susilowati, Doni Setiawan, Martialis Puspito Khristy Maharsi & Indra Yustian, Pp. 22746–22748 Range extension of Isthmoheros tuyrensis, a threatened species of fish (Cichlidae) in Panama: including new ecological and morphological data – Arturo Dominici-Arosemena, Arturo Angulo, Haydee Osorio-Ugarte, Quiriatjaryn Ortega-Samaniego, Andrés Fraiz, Arminda Guerrel, Edgar Araúz, Jennyfer Montiel, Beatriz Medina, Yehudi Rodríguez-Arriatti, Yessenia González, Javier Pardo, Karly Urriola & Adrián Ramos-Merchante, Pp. 22611–22622 Photographic record of the butterfly ray Gymnura cf. poecilura (Myliobatiformes: Gymnuridae) from the Bhagirathi-Hooghly River in West Bengal, eastern India – Priyankar Chakraborty, Pp. 22749–22751 Photographic record of the butterfly ray Gymnura cf. poecilura (Myliobatiformes: Gymnuridae) from the Bhagirathi-Hooghly River in West Bengal, eastern India – Priyankar Chakraborty, Pp. 22749–22751 Tadpole morphology of Jerdon’s Narrow-mouthed Frog Uperodon montanus (Jerdon, 1853) with a range and elevation extension report from Western Ghats, India – Amit Hegde, Girish Kadadevaru & K.P. Dinesh, Pp. Rediscovery of Sewardiella tuberifera Kash., a long-lost monotypic endemic Indian liverwort Rediscovery of Sewardiella tuberifera Kash., a long-lost monotypic endemic Indian liverwort Communications Notes 22623–22631 Tadpole morphology of Jerdon’s Narrow-mouthed Frog Uperodon montanus (Jerdon, 1853) with a range and elevation extension report from Western Ghats, India – Amit Hegde, Girish Kadadevaru & K.P. Dinesh, Pp. 22623–22631 First report of the fairyfly Schizophragma mitai Triapitsyn (Hymenoptera: Mymaridae) from India with notes on S. indica Rehmat & Anis – Anandhan Rameshkumar, Nazurius Anand, Sayan Sardar & Sarfrazul Islam Kazmi, Pp. 22752–22756 An annotated checklist of the economically important family of moths (Lepidoptera: Heterocera: Noctuidae) of the northern Western Ghats, India, with notes on their type species, diversity, distribution, host plants, and an unusual new faunistic record – Aparna Sureshchandra Kalawate, Prachee Surwade & S.N. Pawara, Pp. 22632– 22653 Report of a tussock moth genus Maeoproctis (Lepidoptera: Erebidae: Lymantriinae: Nygmiini) from India – Gagan Preet Kour Bali & Amritpal Singh Kaleka, Pp. 22654–22660 Butterflies of Silent Valley National Park and its environs, Western Ghats of Kerala, India – Kalesh Sadasivan, P.C. Sujitha, Toms Augustine, Edayillam Kunhikrishnan, Vinayan P. Nair, M. Divin Murukesh & Baiju Kochunarayanan, Pp. 22661–22676 Notes on morphology and bionomics of Urolabida histrionica (Westwood) (Heteroptera: Urostylididae) from Assam, India – Sachin Ranade & Hemant V. Ghate, Pp. 22677–22685 Andromonoecy functional through heterostyly and large carpenter bees as principal pollinators in Solanum carolinense L. (Solanaceae) – Suvarna Raju Palathoti & Aluri Jacob Solomon Raju, Pp. 22686–22694 An inventory of endemic and near endemic angiosperm flora of Biligiri Rangaswamy Temple Tiger Reserve, peninsular India – J. Jayanthi, Pp. 22695–22717 Multidimensional time-lapse of a relict species Canarium strictum Roxb. from a sacred landscape in Pune District, India – Mukul Mahabaleshwarkar, Nivedita Ghayal, Supriya Mahabaleshwarkar & Vinaya Ghate, Pp. 22718–22725 – Go New from – K. On M.J east – Ja Butterflies of Silent Valley National Park and its environs, Western Ghats of Kerala, Indiai – Kalesh Sadasivan, P.C. Sujitha, Toms Augustine, Edayillam Kunhikrishnan, Vinayan P. Nair, M. Divin Murukesh & Baiju Kochunarayanan, Pp. 22661–22676 Notes on morphology and bionomics of Urolabida histrionica (Westwood) (Heteroptera: Urostylididae) from Assam, India – Sachin Ranade & Hemant V. Ghate, Pp. 22677–22685 Andromonoecy functional through heterostyly and large carpenter bees as principal pollinators in Solanum carolinense L. (Solanaceae) – Suvarna Raju Palathoti & Aluri Jacob Solomon Raju, Pp. 22686–22694 Threatened Taxa Publisher & Host An inventory of endemic and near endemic angiosperm flora of Biligiri Rangaswamy Temple Tiger Reserve, peninsular India – J. Jayanthi, Pp. Communications Sunda Clouded Leopard Neofelis diardi (Cuvier, 1823) (Mammalia: Carnivora: Felidae) occupancy in Borneo: results of a pilot vehicle spotlight transect survey – Jephte Sompud, Sze Lue Kee, Kurtis Jai-Chyi Pei, Paul Liau, Collin Goh & Anthony J. Giordano, Pp. 22559–22566 Sunda Clouded Leopard Neofelis diardi (Cuvier, 1823) (Mammalia: Carnivora: Felidae) occupancy in Borneo: results of a pilot vehicle spotlight transect survey – Jephte Sompud, Sze Lue Kee, Kurtis Jai-Chyi Pei, Paul Liau, Collin Goh & Anthony J. Giordano, Pp. 22559–22566 – Sapana Pant, S.D. Tewari, Prachi Joshi, Manisha Bhandari & Richa Arya, Pp. 22726–22730 – Sapana Pant, S.D. Tewari, Prachi Joshi, Manisha Bhandari & Richa Arya, Pp. 22726–22730 Physcomitrium eurystomum Sendtn. (Funariaceae: Bryophyta) and Splachnobryum obtusum (Brid.) Müll. Hal. (Splachnobryaceae: Bryophyta), two rare moss species from the Western Ghats of Kerala – C. Nair Manju, P.M. Vineesha, B. Mufeed & K.P. Rajesh, Pp. 22731–22736 Physcomitrium eurystomum Sendtn. (Funariaceae: Bryophyta) and Splachnobryum obtusum (Brid.) Müll. Hal. (Splachnobryaceae: Bryophyta), two rare moss species from the Western Ghats of Kerala – C. Nair Manju, P.M. Vineesha, B. Mufeed & K.P. Rajesh, Pp. 22731–22736 On the occurrence of Eurasian Otter Lutra lutra (Carnivora: Mustelidae) in Neeru stream of Chenab catchment, Jammu & Kashmir, India – Dinesh Singh, Anil Thakar & Neeraj Sharma, Pp. 22567–22573 On the occurrence of Eurasian Otter Lutra lutra (Carnivora: Mustelidae) in Neeru stream of Chenab catchment, Jammu & Kashmir, India – Dinesh Singh, Anil Thakar & Neeraj Sharma, Pp. 22567–22573 Short Communications Distribution of avifauna on twenty-one islands of the Gulf of Mannar Biosphere Reserve, India Distribution of avifauna on twenty-one islands of the Gulf of Mannar Biosphere Reserve, India Notes 22695–22717 Multidimensional time-lapse of a relict species Canarium strictum Roxb. from a sacred landscape in Pune District, India – Mukul Mahabaleshwarkar, Nivedita Ghayal, Supriya Mahabaleshwarkar & Vinaya Ghate, Pp. 22718–22725
https://openalex.org/W2980503686	https://jneuroengrehab.biomedcentral.com/track/pdf/10.1186/s12984-019-0602-0	English	null	A novel wearable device to deliver unconstrained, unpredictable slip perturbations during gait	Journal of neuroengineering and rehabilitation	2,019	cc-by	9,173	Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 https://doi.org/10.1186/s12984-019-0602-0 A novel wearable device to deliver unconstrained, unpredictable slip perturbations during gait Corbin M. Rasmussen* and Nathaniel H. Hunt Abstract Keywords: Slips, Falls, Balance, Task-specific Training, Gait, Perturbations, Wearable Technology * Correspondence: cmrasmussen@unomaha.edu Department of Biomechanics, University of Nebraska at Omaha, 6160 University Drive South, Omaha, NE 68182, USA * Correspondence: cmrasmussen@unomaha.edu Department of Biomechanics, University of Nebraska at Omaha, 6160 University Drive South, Omaha, NE 68182, USA * Correspondence: cmrasmussen@unomaha.edu Department of Biomechanics, University of Nebraska at Omaha, 6160 University Drive South, Omaha, NE 68182, USA © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Task-specific perturbation training is a widely studied means of fall prevention, utilizing techniques that induce slips or slip-like perturbations during gait. Though effective, these methods only simulate narrow ranges within the larger space of possible slipping conditions encountered in daily life. Here we describe and test a novel, wearable apparatus designed to address these limitations and simulate a diverse range of slipping disturbances. Methods: The device consists of wireless triggering and detachable outsole components that provide adequate friction with the floor when secured to the wearer’s foot, but suddenly create a low-friction surface underfoot upon release. “Benchtop” tests were carried out to quantify device triggering characteristics (i.e. cutting temperature, release delay) and the resulting friction reduction. The device was also tested on six healthy young adults (3 female, age 23 ± 2.4 years), who walked with and without the device to observe how gait kinematics and spatiotemporal parameters were influenced, then performed 12 walking trials ending with a slip delivered by the device. Each participant also completed a survey to obtain opinions on device safety, device comfort, slip realism, and slip difficulty. A linear mixed effects analysis was employed to compare subject spatiotemporal parameters with and without the apparatus, as well as correlation coefficients and root mean square errors (RMSE) to assess the impact of the device on lower limb gait kinematics. Slip onset phases, distances, directions, velocities, and recovery step locations were also calculated. Results: This device rapidly diminishes available friction from static coefficients of 0.48 to 0.07, albeit after a substantial delay (0.482 ± 0.181 s) between signal reception and outsole release. Strong correlations (R > 0.93) and small RMSE between gait kinematics with and without the device indicate minimal effects on natural gait patterns, however some spatiotemporal parameters were significantly impacted. A diverse range of slip perturbations and recovery steps were successfully elicited by the device. Conclusions: Our results highlight the efficacy and utility of a wearable slipping device to deliver diverse slip conditions. Such an apparatus enables the study of unconstrained slips administered across the gait cycle, as well as during different locomotor behaviors like turning, negotiating slopes, and level changes. Background unique disturbances that can be applied. Specifically, emu- lated perturbations from sliding platforms and treadmills are limited entirely to the anterior-posterior direction, while actual slips possess a notable mediolateral component [26]. Slippery floor surfaces provide the most realistic distur- bances when encountered, but they suffer from a high de- gree of predictability with repeated exposure [6, 25]. These constraints are inherent to the devices and methods used to administer the perturbations. Task-specific training has gained popularity in rehabilita- tion as an effective intervention to regain [1, 2] and reinforce [3] motor skills. This methodology closely mimics the sensorimotor and environmental interactions of the target task, thereby forcing the patient or subject to repetitively execute the movements necessary to ac- complish the goal of the task [4]. This paradigm is widely utilized in studies of gait stability and fall preven- tion by administering repeated perturbations to an indi- vidual in the form of simulated trips and slips [5–7]. p In order to reduce or remove these restrictions and allow for more realistic slips to be imposed, a new type of apparatus is required. Our aim was to develop such a device. Namely, this apparatus would deliver repeated slipping perturbations that were unconstrained in direc- tion and magnitude, yet be spatially and temporally un- predictable to the wearer and controlled by an attending researcher or clinician. In addition, the slips delivered by this device could be delivered at any point during stance phase. We elected to pursue a wearable design, as this would remove the spatial predictability limitation while still allowing slips to be delivered during over-ground walking. Indeed, a previous exploration of the design space for variable-friction devices concluded that, com- pared to various environment-based approaches, a wear- able, shoe-based option was the most promising [27]. Following development of the initial prototype, subject and non-subject tests were performed to quantify the functional attributes of the device, as well as assess and validate its ability to deliver diverse perturbations and elicit recovery reactions. Based on the results of these tests, we outline improvements to be made for future device iterations. Finally, we also highlight a number of previously inaccessible gait modalities and research topics that are unlocked with the use of a wearable slip- ping device. Background p p Many successful task-specific techniques and appara- tuses have been reported in the literature to study slip- ping perturbations in particular, the cause of 22–25% of falls in the community [8, 9]. Of these, the most widely used are sudden, transient accelerations on a treadmill during otherwise steady-state walking [10–14], one or more unlockable sliding platforms embedded in a level walkway [15–17], and oiled areas encountered along a path [18, 19]. Important advances in our understanding of reactions to “slip-and-fall” events across age groups, as well as supporting evidence for the potency of per- turbation training on stability, have stemmed from the use of these perturbation methods. For example, arm swing in the sagittal plane mitigates deviations in trunk posture following a slip, an action that older adults are less able to utilize [19]. Individuals appear to transition from a reactive to anticipatory mechanism during per- turbation training, characterized by an anteriorly-shifted center of mass (CoM), altered lower limb kinematics, and reduced heel contact velocity [17, 20]. Increases in compensatory step lengths [14], improved measures of stability immediately after slip onset and recovery step touchdown, and spatial-temporal adaptations [11] have also been reported. The adjustments obtained from treadmill slip training in particular can be scaled to match the intensity of perturbation [14], and those from sliding platforms can be generalized to other tasks of varying context similarity [21–23]. While these results have only been shown for their respective perturbation methods to date, the findings illustrate the potential plasticity of learned slip recovery strategies. After partici- pating in a perturbation protocol, subjects have retained their acquired stabilization skills for months [22], and quickly readapt after periods over a year [23, 24]. © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Page 2 of 11 Page 2 of 11 Materials and Methods Device Design Our device (Fig. 1a, b), which hereafter will be called the Wearable Apparatus for Slipping Perturbations (WASP), consists of two interacting components: a detachable outsole (Fig. 1c) and a wirelessly controlled release mechanism (Fig. 1d). The key element of the detachable outsole is a modified rubber boot sole with seven, one- inch wide nylon webbing straps fastened around the per- imeter with screws. The free ends of these straps are threaded through custom 3D printed buckles (Fig. 1e) that enable the attachment of the outsole to the release mechanism. Adhered to the top of the boot sole is a layer of foam to cover the screw heads and provide a flat surface, followed by two thin sheets of polytetrafluoro- ethylene (PTFE) film. PTFE is a low-friction compound colloquially known as Teflon™. A 1:1 mixture of water and personal lubricant [28] is applied between the PTFE Clearly, available methods for administering task-specific slip perturbations are capable of eliciting valuable insight on slip attributes, response movements, and stability adap- tations. However, they have practical limitations associated with producing slips that are unexpected [6, 25], kinematic- ally unconstrained [26], and that represent the variety of slip conditions encountered in daily life. Foot velocity, sliding distance, slip location, and/or slip direction are restricted with these procedures, narrowing the range of Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Page 3 of 11 Fig. 1 a Color-coded diagram of WASP, individually highlighting the components. b The entire WASP assembly fitted to a shod prosthetic foot. c Superior and inferior views of the WASP outsole with PTFE sheets and straps. d The trigger mechanism, detailing the internal electronics and the nichrome interface. e An example of the custom 3D printed buckles used to connect the two components via the fluorocarbon filament In its attached state, WASP is intended to facilitate natural walking by providing adequate friction between the floor and plantar surface of the rubber outsole. Upon reception of a wireless trigger, the relay channels electri- city to the nichrome strand, rapidly heating it. The fluorocarbon filament loop in contact with the nichrome is severed by the intense heat, which relieves the tension on the loop from the 3D printed buckles and releases the detachable outsole [see Additional file 1]. Functional Testing We performed a number of benchtop analyses to quan- tify the key functional characteristics at play while using WASP. To assess the temperatures realized by the ni- chrome wire through repeated activation cycles, we re- corded the wire with an infrared thermal imaging camera (FLIR T540, FLIR Systems, Inc.; Wilsonville, OR) at 30 Hz through 20 consecutive cycles. Each trial was separated by a “rest period” between seven and ten min in length to closely mimic the protocol used in our hu- man data collections described later. The camera was calibrated to an emissivity of 0.65 [29], positioned 0.3 m away from the cutter box, and set to record within a temperature range between 150° and 3000° Celsius. This experiment began with a new battery, and its voltage was recorded immediately before each cycle to examine battery drain with repeated use. sheets. Two pieces of slip-resistant safety tape were at- tached to the dorsal surface of the top PTFE layer to provide friction with the wearer’s shoe, which in turn only allows sliding to occur between the lubricated PTFE layers. The release mechanism, contained entirely in a 3D printed housing, is affixed to the wearer’s shod foot via nylon strap and buckle. Within the housing is a Bluetooth-enabled relay that, when activated through a smartphone application (Adafruit Industries; New York City, NY), supplies power from a 9-V battery to a length of six 38 AWG nichrome wires twisted together (Fig. 1d). Nichrome is a high-resistance alloy of nickel and chro- mium, characterized by its ability to dissipate large amounts of energy as heat yet maintain a resistance to oxi- dation. This nichrome strand is housed in its own case apart from the battery and electronics. A 13.5-in. circum- ference loop of 0.028-in. thick fluorocarbon filament is passed through the nichrome case, perpendicular to and in contact with the wire. This filament loop provides the attachment point for the detachable outsole component via the 3D printed buckles (Fig. 1e). The entire apparatus is secured tightly to the wearer’s foot by pulling the excess nylon webbing through these buckles. Because WASP relies on the rapid heating of a ni- chrome strand to melt a fluorocarbon filament, there is an inherent delay between reception of the triggering signal and the instant of release. Materials and Methods Device Design Once re- leased, the wearer is exposed to the low-friction interface between the lubricated PTFE sheets, ultimately causing a slip perturbation [see Additional file 2]. In order to de- liver repeated slips, the outsole must be prepared and reattached to the wearer’s foot between trials. Additional lubricant is applied between the two PTFE film layers to replace what was lost during the previous trial and to ensure a low friction surface after the next outsole re- lease. To reattach the outsole, the severed fluorocarbon filament loop is removed and a new loop is placed in the housing across the nichrome strand. The buckles of the outsole are then hooked onto the new loop and the nylon straps tightened in the same way as described before. Fig. 1 a Color-coded diagram of WASP, individually highlighting the components. b The entire WASP assembly fitted to a shod prosthetic foot. c Superior and inferior views of the WASP outsole with PTFE sheets and straps. d The trigger mechanism, detailing the internal electronics and the nichrome interface. e An example of the custom 3D printed buckles used to connect the two components via the fluorocarbon filament Functional Testing To determine the length of this delay, we recorded 20 cutting trials with a high-speed camera (Fastec Imaging Corp.; San Diego, CA) recording at 1000 Hz. WASP was affixed to a shod prosthetic foot as in Fig. 1b and equipped with an LED programmed to illuminate when the triggering signal was received. The number of frames between the light- ing of the LED and the first observable movement of the filament were counted and converted to the correspond- ing amount of time. As in the previously described Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Page 4 of 11 Page 4 of 11 (2019) 16:118 the ramp and horizontal. The tangent of this angle was taken as the SCoF for each trial. temperature tests, seven to ten min separated each trial. Also, a new battery was used and its charge measured prior to each cut. Study Participants Slipping is facilitated by inadequate friction between an individual’s foot and the supporting surface they are standing or walking on. To imitate this mechanism, it is essential for WASP to instantly transition from provid- ing sufficient friction for walking to creating a low- friction surface underfoot. To discern the magnitude of this change, a ramp and weight carrying rig (Fig. 2) were assembled to test the static coefficients of friction (SCoF) of the three interfaces that may be encountered while using WASP: WASP-floor, unlubricated PTFE-PTFE, and PTFE-PTFE lubricated with the 1:1 mixture [28]. To simulate the lab floor, a spare flooring tile was used as the sliding surface for the WASP-floor condition. Weights of 75 lbs., 180 lbs., and 240 lbs. were tested in all conditions except WASP-floor to analyze the effect of varying body weights on the supplied friction (240 lbs. was not performed in the WASP-floor condition due to concerns about device safety). Each weight condition was performed ten times. Trials consisted of slowly ele- vating one end of the ramp to create a slope. A cam- corder (Canon Inc.; Tokyo, Japan) documented each trial at 30 Hz. An image was extracted from each video at the instant that the rig began to slide down the ramp. Ramp angles relative to a pendulum were measured using ImageJ [30] and used to derive the angle between Approval for the following protocol was obtained from the University of Nebraska Medical Center Institutional Review Board, and all procedures were in accordance with pertinent regulations and guidelines. We piloted our de- vice on six healthy, young adults (mean ± SD {range} age: 23 ± 2.4 {20–27} yrs., height: 1.76 ± 0.08 {1.71–1.91} m, mass: 73.33 ± 13.96 {56.25–90.27} kg, 3 females), who were all screened and consented to participate in this study. Exclusionary criteria included cardiopulmonary, musculoskeletal, and neurological conditions or injuries that may influence normal gait patterns. Anthropometric measurements (i.e. body weight, height, limb dominance) were collected from each participant. Experimental Protocol 2 Static friction testing setup used to obtain SCoF values, recorded from a similar perspective as seen in this photo with a video camera Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Page 5 of 11 Page 5 of 11 Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Following this time, WASP was triggered by an attend- ing researcher in an attempt to coincide with either heel strike, mid-stance, or toe-push. Because of the delay be- tween signal reception and detachment, the researcher was required to anticipate the desired gait phase. Trial duration and slipping phase were randomized using a Matlab script (Mathworks Inc.; Natick, MA). After trig- gering, subjects were allowed a five-minute rest period while WASP was reset. This protocol was repeated 12 times for each subject (Fig. 3a), after which a post-study questionnaire was completed to extract their perceptions of device comfort, slip realism, recovery difficulty, and safety while wearing WASP. Following this time, WASP was triggered by an attend- ing researcher in an attempt to coincide with either heel strike, mid-stance, or toe-push. Because of the delay be- tween signal reception and detachment, the researcher was required to anticipate the desired gait phase. Trial duration and slipping phase were randomized using a Matlab script (Mathworks Inc.; Natick, MA). After trig- gering, subjects were allowed a five-minute rest period while WASP was reset. This protocol was repeated 12 times for each subject (Fig. 3a), after which a post-study questionnaire was completed to extract their perceptions of device comfort, slip realism, recovery difficulty, and safety while wearing WASP. determine the effect of wearing WASP on gait speed (n = 6 without WASP, 61 with WASP), stride time, stride length, and stride width (n = 42 without WASP, 394 with WASP). The presence of WASP in a given trial was entered into the model as a fixed effect, while sub- jects were treated as a random effect to resolve non- independencies within the fixed effect (i.e. presence of WASP). The alpha level for these analyses was set at 0.05. In addition, root mean square error (RMSE) and correlation coefficients were calculated for lower limb kinematics observed while walking with and without WASP to quantify the impact of the device. Experimental Protocol Slip onset times were determined visually by three in- dividuals using the model videos built using Visual3D (C-Motion Inc.; Germantown, MD). Each individual la- beled the frame in the video they believed corresponded with the instant the slip began. Discrepancies between individuals were resolved in the following ways: 1) the frame identified by the majority was used, or 2) if none of the individuals were in agreement, then the midpoint between the two closest frames was used. If individuals could not distinguish a slip at any point in a trial, the Experimental Protocol Subjects were outfitted with a form-fitting compression suit, lab-provided athletic shoes, and a full-body retro- reflective marker set. A safety harness was also fitted to each subject, which was connected to a fall arresting sys- tem mounted to a rail on the ceiling. A load cell rated for 500 kg (HT Sensor Technology Co. LTD; Xi’an, China) was placed in series with the system to measure the forces exerted on the harness by each subject during slipping trials. If the measured force supported by the harness exceeded 30% of the subject’s body weight, the trial was classified as a fall [31]. Fig. 2 Static friction testing setup used to obtain SCoF values, recorded from a similar perspective as seen in this photo with a video camera To obtain a description of each participant’s natural walking kinematics, we asked them to walk across an eight meter walkway at a self-selected comfortable speed without wearing WASP. Marker trajectories were col- lected at 120 Hz by a 17-camera motion capture system (Motion Analysis Corp.; Santa Rosa, CA). To ensure that any observed biomechanical differences between walking with and without WASP could be attributed to the de- vice, all participants were connected to the ceiling- mounted safety harness during these trials. Gait speed was observed in real time using an electronic timing sys- tem (Dashr, LLC; Lincoln, NE). Following this, subjects were given a five-minute rest period, during which WASP was attached to their dominant foot. A WASP outsole was affixed to the non-dominant foot of each subject as well to prevent differences in limb length. While wearing WASP, each participant was asked to walk back and forth across the walkway at the same self- selected comfortable speed as in the previous non- WASP condition. Feedback was provided to the subjects during the trials when necessary to keep them as close to that speed as possible. All trials were between two and five min in length, before which subjects were in- formed that they may or may not experience a slip [15]. Fig. Data and Statistical Analysis Gait events were defined using the coordinate-based al- gorithm described by Zeni and colleagues [32]. Spatio- temporal gait parameters (i.e. stride time, stride length, stride width, and gait speed) were then calculated for unperturbed walking both with and without WASP. To assess the effect of the wearable device on natural gait patterns, a linear mixed effects analysis was used to Fig. 3 a An illustration of the protocol used during human subject data collections. b An illustration of the human subject trials included and excluded in data analysis Fig. 3 a An illustration of the protocol used during human subject data collections. b An illustration of the human subject trials included and excluded in data analysis Fig. 3 a An illustration of the protocol used during human subject data collections. b An illustration of the human subject trials included and excluded in data analysis Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Page 6 of 11 Page 6 of 11 trial was excluded from further analysis (n = 25, Fig. 3b). Due to the uncontrolled delay of the release mechanism, 14 trials ended with the WASP outsole releasing during swing phase of the target limb and therefore did not cause a perturbation. These trials were also excluded from further analysis. The remaining 33 trials were sorted into early stance (ES), mid-stance (MS), and late stance (LS) categories based on their time of occurrence during stance. ES was defined as 0–33.3%, MS as 33.4– 66.7%, and LS as 66.8–100% of stance phase. Slip dis- tances, directions, and velocities were obtained from the motion of the sliding foot’s CoM. Distance was taken as the difference between the positions of the foot CoM at slip onset and at the following toe-off, while velocity was the maximum attained during the same period. Direc- tion was calculated relative to the subject’s bearing. The location of the next step following slip onset, which we will call the “recovery step”, was derived from the X and Y coordinates of the unperturbed foot’s CoM relative to those of the whole-body CoM. Due to missing whole- body CoM position data at the instant of recovery step touchdown, nine of the remaining 33 trials were not in- cluded in the recovery step calculations (Fig. 3b). temperature sufficient to sever the monofilament. Walking With and Without WASP g Wearer perceptions of their gait pattern were mixed, with 50% of responses indicating that a natural gait was maintained. Respondents generally felt safe from losing their balance while wearing the device (8.0 ± 1.8 {5–10}), but were less approving of its comfort (5.7 ± 1.7 {3–9}). Stride times while wearing WASP were not significantly different than without (0.656 ± 0.070 s with WASP vs. 0.625 ± 0.109 s without WASP; t (65) = 1.94, p = 0.056). However, the differences in gait speed (1.097 ± 0.055 statures/s with WASP vs. 1.138 ± 0.084 statures/s with- out WASP; t (434) = −7.09, p < 0.001), stride length (1.344 ± 0.151 m with WASP vs. 1.301 ± 0.180 m without WASP; t (434) = 3.37, p < 0.001), and stride width (0.164 ± 0.027 m with WASP vs. 0.152 ± 0.034 m without WASP; t (434) = 2.95, p = 0.003) were all significant (Fig. 5). Wearing WASP did not appear to substantially impact the kinematics of the lower extremities during over- ground walking, as indicated by low RMSE values (RMSE = 1.4026°, 3.2870°, and 3.4377°, respectively; Fig. 5) and very strong correlations between hip, knee, and ankle angles measured both with and without the devices (R = 0.9977, 0.9982, and 0.9383, respectively; Fig. 5). While wearing WASP, subjects on average exhib- ited an additional 0.3° of maximum hip extension and 1.7° of maximum hip flexion, as well as an additional Wearer perceptions of their gait pattern were mixed, with 50% of responses indicating that a natural gait was maintained. Respondents generally felt safe from losing their balance while wearing the device (8.0 ± 1.8 {5–10}), but were less approving of its comfort (5.7 ± 1.7 {3–9}). Stride times while wearing WASP were not significantly different than without (0.656 ± 0.070 s with WASP vs. 0.625 ± 0.109 s without WASP; t (65) = 1.94, p = 0.056). However, the differences in gait speed (1.097 ± 0.055 statures/s with WASP vs. 1.138 ± 0.084 statures/s with- out WASP; t (434) = −7.09, p < 0.001), stride length (1.344 ± 0.151 m with WASP vs. 1.301 ± 0.180 m without WASP; t (434) = 3.37, p < 0.001), and stride width (0.164 ± 0.027 m with WASP vs. 0.152 ± 0.034 m without WASP; t (434) = 2.95, p = 0.003) were all significant (Fig. 5). Data and Statistical Analysis The maximum temperature attained by the nichrome elem- ent (576.6 ± 29.7 {528.5–640.1}°C) consistently occurred after outsole release at one second post-triggering, as this was the instant that the mechanism’s relay ceased to supply power. The maximum temperature steadily dropped with repeated use. Available voltage to the sys- tem from the onboard 9-V battery decreased at a loga- rithmic rate through multiple uses, likely explaining the general downward trend in peak temperature. Functional Characteristics The average SCoF observed between the WASP outsole and tile floor surface was 0.54 ± 0.03 with a 75 lbs. load and 0.49 ± 0.03 under a 180 lbs. load. Friction is severely reduced following activation regardless of lubrication state, with dry and lubricated coefficients of 0.11 ± 0.01 and 0.08 ± 0.01 at 75 lbs., 0.11 ± 0.01 and 0.07 ± 0.004 at 180 lbs., and 0.13 ± 0.02 and 0.10 ± 0.02 at 240 lbs. (Fig. 4). Following reception of the trigger signal, the average (± SD {range}) delay until outsole release was 0.482 ± 0.181 {0.311–0.871} seconds. This delay was due to the time required for the nichrome wire to reach a Wearing WASP did not appear to substantially impact the kinematics of the lower extremities during over- ground walking, as indicated by low RMSE values (RMSE = 1.4026°, 3.2870°, and 3.4377°, respectively; Fig. 5) and very strong correlations between hip, knee, and ankle angles measured both with and without the devices (R = 0.9977, 0.9982, and 0.9383, respectively; Fig. 5). While wearing WASP, subjects on average exhib- ited an additional 0.3° of maximum hip extension and 1.7° of maximum hip flexion, as well as an additional SCoF across Simulated Weights and Interfaces Simulated Weights (lbs.) 75 180 240 Static Coeff. of Friction (µ) WASP Dry PTFE Lubricated PTFE 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Required for normal walking29 Fig. 4 Average SCoF coefficients (±1 SD) at 75, 180, and 240 lbs. SCoF across Simulated Weights and Interfaces Required for normal walking29 Fig. 4 Average SCoF coefficients (±1 SD) at 75, 180, and 240 lbs. Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Page 7 of 11 Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Page 7 of 11 Fig. 5 Sagittal plane lower extremity kinematics and spatiotemporal gait parameters observed during over-ground walking both with and without WASP 4.5° of knee flexion during loading and 3.0° during mid- swing. The ankle was the most effected, with a reduction of 4.8° in plantarflexion immediately after heel contact, of 0.5° in dorsiflexion at terminal stance, and of 4.6° in plantarflexion at toe-off. when triggered, thereby perturbing the base of support (BoS) of the wearer. Administered disturbances were highly variable, and appeared to be influenced at least in direction by onset phase. Functional Characteristics In its attached state, the device had a minimal effect on the wearers’ lower limb gait kinematics and caused small yet significant changes to a number of spatiotemporal parameters. Administered Perturbations p p p Also through our observations, we identified a number of improvements to be addressed in future iterations of wearable slip perturbation devices. First and perhaps the most important of these is the uncontrollable variability in outsole release, which renders the precise targeting of specific phases of gait by the experimenter difficult. This stems from two particular aspects of our device. The first aspect is the inconsistent delay between reception of the trigger command and outsole release. Because this release is caused by rapid melting of the monofilament, the delay is dependent on factors not easily controlled with our current mechanism. To address this issue, we developed an alternative release mechanism that uses a cam and follower system to detach the outsole after per- forming the experiments outlined previously. This mechanism still receives a wireless trigger signal from the experimenter, but instead of supplying power to a strand of nichrome wire, the Bluetooth-enabled relay de- livers electricity from a battery to a small DC motor. This motor is outfitted with a 1000:1 gear ratio trans- mission in order to produce a high degree of torque. The driveshaft of the motor connects to a drop cam, which depresses a spring-loaded pin that protrudes from the housing when in contact with the peak of the cam (see illustration in Fig. 7, Additional file 3). To attach the outsole, a loop of monofilament is threaded through None of the successfully administered perturbations met the criteria to be classified as a fall. Successfully inflicted slips were broadly distributed, occurring between 16 and 91% of stance phase (Fig. 6b). Slipping directions relative to the direction of progression fell between 10° and 178°, with angles generally growing as slips occurred later in stance (Fig. 6a). Slip distances ranged from a minimum of 1.6 cm to a maximum of 15.4 cm, and peak velocities from 4.5–163.2 cm/s. Recovery steps also covered a broad range of positions that varied in both the anterior- posterior and medial-lateral directions (Fig. 6c). In con- trast to the observed slipping angles, phase-distance, phase-velocity, and phase-recovery step relationships are not immediately clear. Participants reported that the perceived severity of the slips was low (2.8 ± 1.7 {1–6}), but were more moderate when asked if they seemed realistic (5.3 ± 1.5 {3–7}). Discussion Through the experiments described above, we assessed the functional characteristics of our novel slipping device and its utility to deliver unrestricted slipping perturba- tions in a laboratory setting. Our results demonstrate WASP’s ability to do this at any point during stance phase, which was a primary intention of our device. This is achieved through rapid reduction of friction underfoot Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Page 8 of 11 Page 8 of 11 Fig. 6 a Attributes of administered slips coded by slip phase. b The distribution of administered slips across stance phase. c Recovery step locations relative to the location of the whole-body CoM Fig. 6 a Attributes of administered slips coded by slip phase. b The distribution of administered slips across stance phas locations relative to the location of the whole-body CoM accurate and consistent to target specific gait phases or events repeatedly. Nevertheless, there will undoubtedly be errors between the intended and actual times of release. This potential for error can be remediated by developing an automatically released system. Such a system could rely on signals from footswitches or an inertial measurement unit to estimate gait phases and trigger outsole release. This would require the user to simply “arm” the device be- fore a trial to activate at a specified gait phase during the next step, after a number of strides, or after a predeter- mined length of time. The uncontrollable variability cre- ated by the two aspects above should not be confused with the desired “experimenter-controlled” variability in slip onset phase, which is determined primarily by the phase of stance at which the device is activated. If the goal was to deliver all slips at the same phase, a user would simply trigger the device at the same phase for every trial (for the present device, it should be triggered roughly 500 ms before the intended phase of release). Further, while the difference in supplied friction before and after trigger- ing was substantial, slip severity could be increased through greater reduction of traction underfoot. Increased the buckles of the nylon webbing straps and hooked to the protruding pin of the trigger mechanism. When acti- vated, the motor turns the cam, which allows the spring loaded pin to rapidly retract into the housing. Discussion This retrac- tion releases the monofilament loops and thereby the out- sole from the foot [see Additional file 3]. Reattaching the outsole requires the cam and pin to be reset to their “armed” positions by turning the motor, followed by hooking the monofilament back onto the protruding pin. Because outsole release does not rely on severing a loop of filament, there is no need to replace the filament after every trial. Using the same release delay testing method performed on the nichrome mechanism, we found that the cam and follower mechanism is both faster and more consistent (0.056 ± 0.02 s, Fig. 7). Based on this result, we plan to utilize the cam and follower release component in future studies using this wearable slip device, as improved control over the instant that friction is reduced will allow more targeted, consistent perturbations to be adminis- tered. Second, because the current form of WASP relies on an experimenter to manually release the outsole through a Bluetooth-connected device, one must be very Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Page 9 of 11 Page 9 of 11 Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation Fig. 7 A comparison of release delays following reception of the trigger command using the nichrome method employed in this study and an alternative cam and follower mechanism developed later. As reported, the mean ± 1 SD delay for the nichrome mechanism was 0.482 ± 0.181 s, while the cam and follower delay was 0.056 ± 0.02 s context plays in slip severity, specific reactive move- ments, and fall rates. For example, differences in slip at- tributes that follow slips initiated at distinct phases may be due to the natural course of kinematic and dynamic context over stance. In the case of ES slips, the anteriorly-directed shear forces may carry the slipping foot forward and away from the rest of the body [33]. Combined with the loss of shear ground reaction forces (GRFs), this creates an exaggerated moment arm that pitches the body backward. Recovering from this back- ward pitch is more challenging than respective forward rotations [34, 35]. MS and LS slip recoveries may benefit from less compromising body positions and passive dy- namics. For MS disturbances specifically, the affected foot appears to slide laterally from the CoM, likely caus- ing a momentary rotation of the body in the opposite direction. Discussion The unaffected foot at this time is in swing, and may be able to oppose the motion of the CoM by redirecting its trajectory and planting lateral to the CoM. LS slips might require even less intervention, as the generally posterior-directed slip may set the CoM in motion anteriorly toward the unperturbed foot that is already positioned ahead of the body. Each phase may offer a distinct set of initial conditions from which an in- dividual must find a unique, successful recovery strategy. Although our preliminary results on reactive movements are not sufficient to infer such effects of slip onset phase, they indicate differences that may be detectable in a fu- ture study using WASP. Fig. 7 A comparison of release delays following reception of the trigger command using the nichrome method employed in this study and an alternative cam and follower mechanism developed later. As reported, the mean ± 1 SD delay for the nichrome mechanism was 0.482 ± 0.181 s, while the cam and follower delay was 0.056 ± 0.02 s Fig. 7 A comparison of release delays following reception of the trigger command using the nichrome method employed in this study and an alternative cam and follower mechanism developed later. As reported, the mean ± 1 SD delay for the nichrome mechanism was 0.482 ± 0.181 s, while the cam and follower delay was 0.056 ± 0.02 s severity would increase the likelihood of eliciting falls, therefore providing greater challenges to the wearer’s bal- ance recovery abilities. The ability to modulate the avail- able friction after release would also be advantageous to actively change the severity of administered slips, analo- gous to controlling distance or velocity with treadmills or sliding platforms [13]. This feature in a wearable device could prove valuable to a progressive perturbation training program. Finally, from a clinical perspective, our device suffers from the need for a moveable overhead harness in order to be used safely, as well as a relatively large area for the wearer to walk through. These limitations are shared by all over-ground slip perturbation methods, and they may preclude settings that lack such space or equipment from using the device. Supplementary information pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12984-019-0602-0. pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12984-019-0602-0. Additional file 1. WASP Release Example Video. This high-speed video was recorded as part of the data set to characterize the release delay, as described in the Materials and Methods section. The WASP device is attached to a shod prosthetic foot as it would be to a subject, and activated through the reception of a trigger command issued by a Bluetooth-connected device. Upon receiving the command, the nichrome within the green housing heats up rapidly, eventually melting through the monofilament loop that the outsole is attached to. Note that the red LED visible in the video was not present during the human subject trials described within the manuscript, only during the benchtop analyses to indicate when the trigger command was received. A wearable device such as WASP enables the applica- tion of perturbation training to other locomotory modes and environments that have not been fully examined in the existing literature, yet describe a significant portion of the steps taken day-to-day. The vast majority of gait perturbation studies have focused on linear, forward gait across a level surface, yet walking in the community is far more complex. Turning comprises approximately 35– 45% of all steps taken [41], and a study by Crenshaw and colleagues indicated that 20% of reported falls in their sam- ple occurred while changing direction [9]. The required co- efficient of friction needed to safely execute a turn is 0.45 [42], more than twice the value for straight walking [33]. Slopes present a wide range of potential conditions that de- mand unique biomechanical adaptations to traverse. Walk- ing directly up or down an inclined surface generates significantly larger anterior-posterior shear GRFs [43], re- quiring greater friction as the magnitude of the incline in- creases [44]. These forces peak at different times during stance as well [43], which may lead to different “critical phases” where the risk of a slip is greatest. Crossing slopes in any other direction creates asymmetrical functional leg lengths [45], resulting in unique body positions that may in- fluence the repertoire of effective recovery strategies avail- able in the event of a slip. In addition, WASP allows for other mechanical consequences of the perturbation to be examined that are inhibited by the restricted nature of other techniques. Funding Funding The present work was supported by grants from the National Institutes of Health (P20GM109090, R15AG063106). Discussion Despite these limitations, we be- lieve the unconstrained and unpredictable nature of wear- able slipping devices may complement the knowledge already gained from existing methods by facilitating the study of unique slip features, which will be described in detail to follow. Successfully addressing our device’s draw- backs through alternative release mechanisms and mate- rials will streamline this research and allow for more targeted, consistent perturbations to be delivered. The wearable apparatus delivered slipping perturbations diverse in distance, direction, and velocity in response to a range of onset times. Likewise, diverse slips that occur when navigating the environment depend only on the available friction between the support surface and the out- sole as well as the distribution of forces acting over that interface [33], allowing an extremely complex and variable space of possible slip attributes. Compared to previous work, the slips provoked by WASP were slightly shorter and generally slower. In studies where these were con- trolled, displacements ranged from 1 cm up to 38 cm with velocities between 10 cm/s and 900 cm/s [12–14]. When not constrained, average displacements of 78 cm and 61 cm, as well as peak velocities of 200 cm/s and 184 cm/s, were observed in fallers and non-fallers, respectively [16]. These protocols only produced translations in a single plane [12–14, 16], however, imposing unnatural con- straints on the administered perturbations [26]. As sug- gested by Troy and Grabiner, greater experimental control is gained at the expense of realism of the task [26]. Indeed, context similarity seems to be an important factor to the success of task-specific perturbation training, as transfer of learned stabilization skills from sliding platform train- ing to an over-ground unconstrained slip was significantly greater compared to the same training done on a treadmill Our wearable device demonstrated the capability to produce slips with variable onset times. This capability enables the investigation of the role biomechanical Page 10 of 11 Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Page 10 of 11 [13]. A wearable perturbation device may offer even greater task specificity of lab-induced slips to those en- countered in the environment than both methods, there- fore future research should investigate the importance of task specificity by comparing improvements in stability gained from training with a wearable device to those from sliding platform and treadmill protocols. Acknowledgements h h ld l k The authors would like to thank Andrew Walski and Kyle Brozek for their contributions to device development and data collection. The authors would like to thank Andrew Walski and Kyle Brozek for their contributions to device development and data collection. Supplementary information One crucial example may be the freedom of foot orientation to change during a slip. Perturbed foot yaw may alter the area of the BoS and therefore the pos- sible locations of the CoP from which to generate counter- ing GRFs. To our knowledge, foot orientation during or after a slip has yet to be investigated. Additional file 2. WASP Example Subject Trial. This video shows the WASP device in action while the wearer is walking over-ground, demon- strating how it delivers a slip perturbation. This video was recorded solely for demonstration purposes and was not used for data collection in any way. Further, the individual in the video is an undergraduate member of our research team, and did not participate in the present study as a subject. Additional file 3. Example Cam and Follower Release. This video shows the cam and follower release mechanism developed after the experiments described in this article. This alternative mechanism was built to address the unreliable release delay that was a limitation of the nichrome, and will be used in future experiments with our wearable device. Additional file 3. Example Cam and Follower Release. This video shows the cam and follower release mechanism developed after the experiments described in this article. This alternative mechanism was built to address the unreliable release delay that was a limitation of the nichrome, and will be used in future experiments with our wearable device. Authors’ contributions CR and NH developed the device, designed the study, interpreted the analyzed data, and drafted and revised the manuscript. CR collected and analyzed the data. Both authors have read and approved the final manuscript. Availability of data and materials The datasets generated during the current study are available from the corresponding author upon reasonable request. Conclusions Our study indicates that wearable apparatuses like WASP are capable of instigating variable slipping pertur- bations during stance phase, in turn eliciting uncon- strained slip mechanics and recovery reactions. Slips encountered in nature are intrinsically complex due to the limited number of constraining factors present. By closely mimicking this complexity, future work using this device will investigate the possibility of developing a more comprehensive repertoire of reactive stabilization strategies that is effective against a broader range of slip- ping conditions and environments. Because slips caused by WASP are highly variable and unconstrained, the compensatory steps and foot orienta- tions in response may have to be equally diverse in order to successfully recover. Recovery steps, seemingly by be- ing placed in the direction a fall would otherwise occur [36], reconfigure the BoS to arrest the destabilizing an- gular momentum of the body through an opposing an- gular impulse [37] and maintain body weight support. Multiple studies have aimed to characterize reactive stepping ability [10, 38–40] and report improvements following a perturbation training protocol [12, 14]. It may be possible to supplement these advances through the use of more ecologically valid means, as a broader range of effective recovery steps could be practiced. References Phys Ther. 2014;94:1755–64. 12. Kurz I, Gimmon Y, Shapiro A, Debi R, Snir Y, Melzer I, et al. Unexpected perturbations training improves balance control and voluntary stepping times in older adults - a double blind randomized control trial. BMC Geriatr. 2016;16:58. 40. Maki BE, McIlroy WE. Control of rapid limb movements for balance recovery: age-related changes and implications for fall prevention. Age Ageing. 2006; 35:12–8. 13. Lee A, Bhatt T, Pai YC. Generalization of treadmill perturbation to overground slip during gait: effect of different perturbation distances on slip recovery. J Biomech. 2016;49:149–54. 41. Glaister BC, Bernatz GC, Klute GK, Orendurff MS. Video task analysis of turning during activities of daily living. Gait Posture. 2007;25:289–94. 42. Fino P, Lockhart TE. Required coefficient of friction during turning at self- selected slow, normal, and fast walking speeds. J Biomech. 2014;47:1395–400. 14. Patel P, Bhatt T. Adaptation to large-magnitude treadmill-based perturbations: improvements in reactive balance response. Phys Rep. 2015;3:1–10. 43. McIntosh AS, Beatty KT, Dwan LN, Vickers DR. Gait dynamics on an inclined walkway. J Biomech. 2006;39:2491–502. 15. Pai YC, Bhatt T, Yang F, Wang E. Perturbation training can reduce community-dwelling older adults’ annual fall risk: a randomized controlled trial. J Gerontol Ser A Biol Sci Med Sci. 2014;69:1586–94. 44. Redfern MS, DiPasquale J. Biomechanics of descending ramps. Gait Posture. 1997;6:119–25. 16. Sawers A, Pai YC, Bhatt T, Ting LH. Neuromuscular responses differ between slip-induced falls and recoveries in older adults. J Neurophysiol. 2017;117: 509–22. 45. Dixon PC, Pearsall DJ. Gait dynamics on a cross-slope walking surface. J Appl Biomech. 2010;26:17–25. 17. Yang F, Bhatt T, Pai YC. Role of stability and limb support in recovery against a fall following a novel slip induced in different daily activities. J Biomech. 2009;42:1903–8. References A systematic review of gait perturbation paradigms for improving reactive stepping responses and falls risk among healthy older adults. Eur Rev Aging Phys Act. 2017;14:3. 6. McCrum C, Gerards MH, Karamanidis K, Zijlstra W, Meijer K. A systematic review of gait perturbation paradigms for improving reactive stepping responses and falls risk among healthy older adults. Eur Rev Aging Phys Act. 2017;14:3. 32. Zeni JA, Richards JG, Higginson JS. Two simple methods for determining gait events during treadmill and overground walking using kinematic data. Gait Posture. 2008;27:710–4. 33. Redfern MS, Cham R, Gielo-Perczak K, Gronqvist R, Hirvonen M, Lanshammar H, et al. Biomechanics of slips. Ergonomics. 2001;44:1138–66. 7. Okubo Y, Schoene D, Lord SR. Step training improves reaction time, gait and balance and reduces falls in older people: a systematic review and meta-analysis. Br J Sports Med. 2017;51:586–93. 34. Mathiyakom W, McNitt-Gray JL, Wilcox R. Lower extremity control and dynamics during backward angular impulse generation in forward translating tasks. J Biomech. 2006;39:990–1000. 8. Berg WP, Alessio HM, Mills EM, Tong C. Circumstances and consequences of falls in independent community-dwelling older adults. Age Ageing. 1997;26: 261–8. 35. Winter DA, Prince F, Frank JS, Powell C, Zabjek KF. Unified theo 35. Winter DA, Prince F, Frank JS, Powell C, Zabjek KF. Unified theory regarding A/P and M/L balance in quiet stance. 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Immediate effects of perturbation treadmill training on gait and postural control in patients with Parkinson’s disease. Gait Posture. 2016;50:102–8. 39. Inness EL, Mansfield A, Lakhani B, Bayley M, McIlroy WE. Impaired reactive stepping among patients ready for discharge from inpatient stroke rehabilitation. Consent for publication Not applicable. 21. Bhatt T, Pai YC. Generalization of gait adaptation for fall prevention: from moveable platform to slippery floor. J Neurophysiol. 2009;101:948–57. 22. Bhatt T, Wang TY, Yang F, Pai YC. Adaptation and generalization to opposing perturbations in walking. Neuroscience. 2013;246:435–50. References 1. Kaufman KR, Wyatt MP, Sessoms PH, Grabiner MD. Task-specific f 1. Kaufman KR, Wyatt MP, Sessoms PH, Grabiner MD. Task-specific f 1. Kaufman KR, Wyatt MP, Sessoms PH, Grabiner MD. Task-specific fall ti t i i i ff ti f fi ht ith t tibi l t ti 1. Kaufman KR, Wyatt MP, Sessoms PH, Grabiner MD. 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Dijkstra BW, Horak FB, Kamsma YPT, Peterson DS. Older adults can improve compensatory stepping with repeated postural perturbations. Front Aging Neurosci. 2015;7:1–8. 3. Dijkstra BW, Horak FB, Kamsma YPT, Peterson DS. Older adults can improve compensatory stepping with repeated postural perturbations. Front Aging Neurosci. 2015;7:1–8. reality environment on slip-induced falls. Ann Biomed Eng. 2015;43:958 29. Engineering Toolbox. Emissivity coefficients materials. https://www. engineeringtoolbox.com/emissivity-coefficients-d_447.html. Accessed 10 March 2018. 4. Grabiner MD, Crenshaw JR, Hurt CP, Rosenblatt NJ, Troy KL. Exercise-based fall prevention: can you be a bit more specific? Exerc Sport Sci Rev. 2014;42:161–8. 30. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671–5. 5. Gerards MH, McCrum C, Mansfield A, Meijer K. Perturbation-based balance training for falls reduction among older adults: Current evidence and implications for clinical practice. Geriatr Gerontol. 2017;17:2294–303. 5. Gerards MH, McCrum C, Mansfield A, Meijer K. Perturbation-based balance training for falls reduction among older adults: Current evidence and implications for clinical practice. Geriatr Gerontol. 2017;17:2294–303. 31. Yang F, Pai YC. Automatic recognition of falls in gait-slip: a harness load cell based criterion. J Biomech. 2012;44:2243–9. based criterion. J Biomech. 2012;44:2243–9. 6. McCrum C, Gerards MH, Karamanidis K, Zijlstra W, Meijer K. Received: 22 May 2019 Accepted: 30 September 2019 25. Okubo Y, Brodie MA, Sturnieks DL, Hicks C, Carter H, Toson B, et al. Exposure to trips and slips with increasing unpredictability while walking can improve balance recovery responses with minimum predictive gait alterations. PLoS One. 2018;13:e0202913. Ethics approval and consent to participate h d d b h The present study was approved by the Institutional Review Board of the University of Nebraska Medical Center and adheres to all pertinent regulations. Written informed consent was obtained from all participants prior to beginning the study protocol. Page 11 of 11 Page 11 of 11 Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Rasmussen and Hunt Journal of NeuroEngineering and Rehabilitation (2019) 16:118 Consent for publication Not applicable. Competing interests 23. Wang TY, Bhatt T, Yang F, Pai YC. Generalization of motor adaptation to repeated-slip perturbation across tasks. Neuroscience. 2011;180:85–95. CR and NH have a provisional patent pending for the device described and used in the current study. 24. Epro G, Mierau A, McCrum C, Leyendecker M, Bruggemann GP, Karamanidis K. Retention of gait stability improvements over 1.5 years in older adults: effects of perturbation exposure and triceps surae neuromuscular exercise. J Neurophysiol. 2018;119:2229–40. Received: 22 May 2019 Accepted: 30 September 2019 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 18. Cham R, Redfern MS. Lower extremity corrective reactions to slip events. J Biomech. 2001;34:1439–45. 19. Troy KL, Donovan SJ, Grabiner MD. Theoretical contribution of the upper extremities to reducing trunk extension following a laboratory-induced slip. J Biomech. 2009;42:1339–44. 20. Brady RA, Pavol MJ, Owings TM, Grabiner MD. Foot displacement but not velocity predicts the outcome of a slip induced in young subjects while walking. J Biomech. 2000;33:803–8.
https://openalex.org/W3124618132	https://osf.io/836sm/download	English	null	The Narratives of Cyberspace Law (or, Learning from Casablanca)	null	2,017	public-domain	23,732	2. “The internet” being the collective term for the hardware and software technology that underlies interconnected computer networks and “cyberspace” being the collective term for human experiences of engaging with a computer-mediated, network environment. See Brett M. Frischmann, Reconciling Internet and Cyberspace, 35 LOY. U. CHI. L.J. (forthcoming 2004) (arguing that the two phenomena ought to be separated for legal analysis); Orin S. Kerr, The Problem of Perspective in Internet Law, 91 GEO. L.J. 357 (2003) (arguing that given legal problems involving online behavior can be analyzed from either perspective). In popular culture, these terms are often used interchangeably. As the technology of the internet becomes ever more invisible, where the “technology” leaves off and the “experience” begins is difficult to determine. I use the term “cyberspace” for this Article, but I do not draw a hard and fast distinction between the two terms. But cf. Reno v. ACLU, 521 U.S. 844, 851 (1997) (“Taken together, these tools [e-mail, newsgroups, the World Wide Web, etc.] constitute a unique medium—known to its users as ‘cyberspace’—located in no particular geographical location but available to anyone, anywhere in the world, with access to the internet.”). The Narratives of Cyberspace Law (or, Learning From Casablanca) Michael J. Madison∗ * Assistant Professor, University of Pittsburgh School of Law. Email: madison@law.pitt.edu. My thanks to Michael Froomkin, whose use of the phrase “papers, please” in a talk on privacy technologies unintentionally inspired this paper, and to George Taylor, Fred Yen, Martha Woodmansee and Aviam Soifer for thoughtful comments and criticism. 1. Crystallization of these fields is evident in the flourishing market for relevant law school casebooks. See, e.g., PATRICIA L. BELLIA ET AL., CYBERLAW: PROBLEMS OF POLICY AND JURISPRUDENCE IN THE INFORMATION AGE (2003); RAYMOND S. R. KU ET AL., CYBERSPACE LAW: CASES AND MATERIALS (2002); MARK A. LEMLEY ET AL., SOFTWARE AND INTERNET LAW (2d ed. 2003); PETER B. MAGGS, INTERNET AND COMPUTER LAW: CASES—COMMENTS—QUESTIONS (2001); LAWRENCE LESSIG ET AL., INTERNET LAW (forthcoming 2004). 3. See, e.g., Joseph H. Sommer, Against Cyberlaw, 15 BERKELEY TECH. L.J. 1145, 1150-51 (2000). Sommer nonetheless makes his argument in the form of “stories” about the internet and lega questions related to it. * Assistant Professor, University of Pittsburgh School of Law. Email: madison@law.pitt.edu. My thanks to Michael Froomkin, whose use of the phrase “papers, please” in a talk on privacy technologies unintentionally inspired this paper, and to George Taylor, Fred Yen, Martha Woodmansee and Aviam Soifer for thoughtful comments and criticism. 1. Crystallization of these fields is evident in the flourishing market for relevant law school casebooks. See, e.g., PATRICIA L. BELLIA ET AL., CYBERLAW: PROBLEMS OF POLICY AND JURISPRUDENCE IN THE INFORMATION AGE (2003); RAYMOND S. R. KU ET AL., CYBERSPACE LAW: CASES AND MATERIALS (2002); MARK A. LEMLEY ET AL., SOFTWARE AND INTERNET LAW (2d ed. 2003); PETER B. MAGGS, INTERNET AND COMPUTER LAW: CASES—COMMENTS—QUESTIONS (2001); LAWRENCE LESSIG ET AL., INTERNET LAW (forthcoming 2004). 249 * Assistant Professor, University of Pittsburgh School of Law. Email: madison@law.pitt.edu. My thanks to Michael Froomkin, whose use of the phrase “papers, please” in a talk on privacy technologies unintentionally inspired this paper, and to George Taylor, Fred Yen, Martha Woodmansee and Aviam Soifer for thoughtful comments and criticism. 1. Crystallization of these fields is evident in the flourishing market for relevant law school casebooks. See, e.g., PATRICIA L. BELLIA ET AL., CYBERLAW: PROBLEMS OF POLICY AND JURISPRUDENCE IN THE INFORMATION AGE (2003); RAYMOND S. R. KU ET AL., CYBERSPACE LAW: CASES AND MATERIALS (2002); MARK A. LEMLEY ET AL., SOFTWARE AND INTERNET LAW (2d ed. 2003); PETER B. MAGGS, INTERNET AND COMPUTER LAW: CASES—COMMENTS—QUESTIONS (2001); LAWRENCE LESSIG ET AL., INTERNET LAW (forthcoming 2004). 2. “The internet” being the collective term for the hardware and software technology that underlies interconnected computer networks and “cyberspace” being the collective term for human experiences of engaging with a computer-mediated, network environment. See Brett M. Frischmann, Reconciling Internet and Cyberspace, 35 LOY. U. CHI. L.J. (forthcoming 2004) (arguing that the two phenomena ought to be separated for legal analysis); Orin S. Kerr, The Problem of Perspective in Internet Law, 91 GEO. L.J. 357 (2003) (arguing that given legal problems involving online behavior can be analyzed from either perspective). In popular culture, these terms are often used interchangeably. As the technology of the internet becomes ever more invisible, where the “technology” leaves off and the “experience” begins is difficult to determine. I use the term “cyberspace” for this Article, but I do not draw a hard and fast distinction between the two terms. But cf. Reno v. ACLU, 521 U.S. 844, 851 (1997) (“Taken together, these tools [e-mail, newsgroups, the World Wide Web, etc.] constitute a unique medium—known to its users as ‘cyberspace’—located in no particular geographical location but available to anyone, anywhere in the world, with access to the internet.”). 3. See, e.g., Joseph H. Sommer, Against Cyberlaw, 15 BERKELEY TECH. L.J. 1145, 1150-51 (2000). Sommer nonetheless makes his argument in the form of “stories” about the internet and legal questions related to it. 4. See Philip E. Agre, Cyberspace as American Culture, 11 SCI. AS CULTURE 171, 174 (2002) (“The ideology of cyberspace . . . is spectacularly wrong; yet facts do not seem to refute it.”); Philip E. Agre, Yesterday’s Tomorrow, TIMES LITERARY SUPPLEMENT, July 3, 1998, at 3-4 (arguing that “cyberspace” is an artifact of the evolutionary interaction between network computing technology and the institutions and relationships into which it is being integrated). Information professionals have largely mastered this understanding. See, e.g., CHRISTINE L. BORGMAN, FROM GUTENBERG TO THE GLOBAL INFORMATION INFRASTRUCTURE: ACCESS TO INFORMATION IN THE NETWORKED WORLD (2000) (analyzing libraries as a set of information practices that may include networked technology). But their insights have penetrated popular consciousness—and law—only to a limited extent. See, e.g., Kerr, supra note 2, at 357 (developing the argument that legal analysis regarding computer networks might take an “internal” or “external” perspective regarding the experience of the network). g p p g g p ) 5. ACLU v. Reno, 929 F. Supp. 824, 836-37 (E.D. Pa. 1996), aff’d, 521 U.S. 844 (1997). Cf. Margaret Jane Radin, Online Standardization and the Integration of Text and Machine, 70 FORDHAM L. REV. 1125, 1139-45 (2002) (noting that the blurring of text and machine in the digital world leads to an erosion of distinctions between legal standards and technical standards); WILLIAM J. MITCHELL, CITY OF BITS: SPACE, PLACE, AND THE INFOBAHN 65 (1995) (describing online blurring of traditional distinctions between performer and audience). 6. See Dan Hunter, Cyberspace as Place and the Tragedy of the Digital Anticommons, 91 CAL. L. REV. 439 (2003); eBay, Inc. v. Bidder’s Edge, Inc., 100 F. Supp. 2d 1058, 1070 (N.D. Cal. 2000) (granting preliminary injunction against auction aggregator service that committed “trespass” to the plaintiff’s auction website, where interference with plaintiff’s property interest consisted of unauthorized access to plaintiff’s computer servers). , ( ) 11. See Matt Jackson, One Step Forward, Two Steps Back: An Historical Analysis of Copyright Liability, 20 CARDOZO ARTS & ENT. L.J. 367, 414 (2002); Larry E. Ribstein & Bruce H. Kobayashi, State Regulation of Electronic Commerce, 51 EMORY L.J. 1, 19-23 (2002). g f ( ) 12. See Tom W. Bell, Book Review, 28 J. MAR. L. & COM. 185, 186 (1997) (reviewing HENRY H. PERRITT, JR., LAW AND THE INFORMATION SUPERHIGHWAY (1996)) (comparing the internet to the ocean). 9. See Jane C. Ginsburg, Putting Cars on the “Information Superhighway”: Authors, Exploiters, and Copyright in Cyberspace, 95 COLUM. L. REV. 1466, 1466-68 (1995); Brookfield Communications, Inc. v. West Coast Entm’t Corp., 174 F.3d 1036, 1062-64 (9th Cir. 1999) (explaining doctrine of “initial interest confusion” on the internet by analogy to billboard confusion on an interstate highway). INTRODUCTION The number of legal and other social issues created or affected by computer networks and our use of computer technology appears overwhelmingly diverse. In law, these issues have been collected and analyzed as a “law of the internet” or “law of cyberspace.”1 Critics have objected that neither the artifact of the internet nor the experience we call cyberspace2 justifies unifying perspectives on such topics as copyright law, defamation, taxation, contract formation, unreasonable searches and personal jurisdiction.3 Despite the criticism, lawyers, legal scholars and even judges intuit that something having to do with modern computing technology must connect the changes now observed in each of these fields. Both popular and technical understanding refer to “the internet” and to “cyberspace” as y y ) 3. See, e.g., Joseph H. Sommer, Against Cyberlaw, 15 BERKELEY TECH. L.J. 1145, 1150-51 (2000). Sommer nonetheless makes his argument in the form of “stories” about the internet and legal questions related to it. 249 COLUMBIA JOURNAL OF LAW & THE ARTS [27:2 250 unitary phenomena, knowing full well that there is no such single thing beneath either label.4 Surely this understanding, and not simply the scholar’s instinct to abstract and generalize, has produced the persistence of debates over the right answer to the question: what is cyberspace? The range of answers to date includes the propositions that cyberspace (or the internet) is or is not analogous to one or more of a host of mostly physicalized constructs. Cyberspace is “a single body of knowledge.”5 Cyberspace is a place,6 a collection of places,7 a network,8 a highway9 and an information “commons.”10 It is a marketplace,11 an open space,12 7. See James Boyle, A Politics of Intellectual Property: Environmentalism for the Net?, 47 DUKE L.J. 87, 108-112 (1997); James Boyle, The Second Enclosure Movement and the Construction of the Public Domain, LAW & CONTEMP. PROBS., Winter/Spring 2003, at 33; Mark A. Lemley, Place and Cyberspace, 91 CAL. L. REV. 521 (2003); Lawrence Lessig, Reading the Constitution in Cyberspace, 45 EMORY L.J. 869, 883-95 (1996) (observing that the internet is capable of and is in fact being “zoned”). 8. See Yochai Benkler, From Consumers to Users: Shifting the Deeper Structures of Regulation Toward Sustainable Commons and User Access, 52 FED. COMM. L.J. 561, 562-63 (2000) (describing internet as a series of network layers); I. Trotter Hardy, Computer RAM “Copies”: A Hit or a Myth? 7. See James Boyle, A Politics of Intellectual Property: Environmentalism for the Net?, 47 DUKE L.J. 87, 108-112 (1997); James Boyle, The Second Enclosure Movement and the Construction of the Public Domain, LAW & CONTEMP. PROBS., Winter/Spring 2003, at 33; Mark A. Lemley, Place and Cyberspace, 91 CAL. L. REV. 521 (2003); Lawrence Lessig, Reading the Constitution in Cyberspace, 45 EMORY L.J. 869, 883-95 (1996) (observing that the internet is capable of and is in fact being “zoned”). 10. See LAWRENCE LESSIG, THE FUTURE OF IDEAS: THE FATE OF THE COMMONS IN A CONNECTED WORLD 23-26 (2001); Lawrence Lessig, Commons and Code, 9 FORDHAM INTELL. PROP. MEDIA & ENT. L.J. 405, 405-12 (1999). 8. See Yochai Benkler, From Consumers to Users: Shifting the Deeper Structures of Regulation Toward Sustainable Commons and User Access, 52 FED. COMM. L.J. 561, 562-63 (2000) (describing internet as a series of network layers); I. Trotter Hardy, Computer RAM “Copies”: A Hit or a Myth? Historical Perspectives on Caching as a Microcosm of Current Copyright Concerns, 22 U. DAYTON L. REV. 423, 436-37 (1997). INTRODUCTION Historical Perspectives on Caching as a Microcosm of Current Copyright Concerns, 22 U. DAYTON L. REV. 423, 436-37 (1997). 9. See Jane C. Ginsburg, Putting Cars on the “Information Superhighway”: Authors, Exploiters, and Copyright in Cyberspace, 95 COLUM. L. REV. 1466, 1466-68 (1995); Brookfield Communications, Inc. v. West Coast Entm’t Corp., 174 F.3d 1036, 1062-64 (9th Cir. 1999) (explaining doctrine of “initial interest confusion” on the internet by analogy to billboard confusion on an interstate highway). 10. See LAWRENCE LESSIG, THE FUTURE OF IDEAS: THE FATE OF THE COMMONS IN A CONNECTED WORLD 23-26 (2001); Lawrence Lessig, Commons and Code, 9 FORDHAM INTELL. PROP. MEDIA & ENT. L.J. 405, 405-12 (1999). 11. See Matt Jackson, One Step Forward, Two Steps Back: An Historical Analysis of Copyright Liability, 20 CARDOZO ARTS & ENT. L.J. 367, 414 (2002); Larry E. Ribstein & Bruce H. Kobayashi, State Regulation of Electronic Commerce, 51 EMORY L.J. 1, 19-23 (2002). g f , , ( ) 12. See Tom W. Bell, Book Review, 28 J. MAR. L. & COM. 185, 186 (1997) (reviewing HENRY H. PERRITT, JR., LAW AND THE INFORMATION SUPERHIGHWAY (1996)) (comparing the internet to the ocean). 2004] 251 THE NARRATIVES OF CYBERSPACE LAW a “frontier,”13 a virtual community (or set of communities)14 and a set of medieval status-based relationships.15 Cyberspace has been characterized as a giant photocopier,16 as a book17 and as a library.18 “Cyberspace” itself is a metaphor, of course;19 in part the exercise has been to tie one metaphor (place, for example) to another (cyberspace) in order to define an artifact (the internet). But cyberspace is not any one of these things, though the effort to find a worthy metaphorical counterpart to this strange new technology and experience is an appropriate one. The metaphorical inquiry is inevitable, and it is necessary: The alleged artifact itself almost defies stable description on technical terms alone.20 Neither the internet nor cyberspace is a “thing” capable of re-conceptualization merely in terms of another “thing.” The challenge is to conceptualize cyberspace for legal purposes, in a way that accommodates—if not resolves—the myriad of technologies, theories and practices to which cyberspace connects, and to do so in a way that is both comprehensible in contemporary terms and allows for evolution and adaptation in the future. I propose instead to characterize cyberspace (or, if one prefers, the internet) as a story, a narrative. 18. See Mainstream Loudoun v. Bd. of Trs. of Loudoun County Library, 2 F. Supp. 2d 783, 793- 94 (E.D. Va. 1998) (rejecting library characterization in favor of “internet-as-encyclopedia”). Cf. New York Times Co. v. Tasini, 533 U.S. 483, 502-04 (2001) (comparing databases to libraries). At this point, the metaphorical journey has come full circle to Borges. See JORGE LUIS BORGES, The Library of Babel, in LABYRINTHS: SELECTED STORIES & OTHER WRITINGS 51, 51 (Donald A. Yates & James E. Irby eds., 1964). In the present spirit, this collection of metaphors might be referred to as “the usual suspects” after Captain Renault’s famous line at the end of Casablanca: “Major Strasser has been shot. Round up the usual suspects.” CASABLANCA (Warner Brothers Pictures 1942). Early predictions that existing metaphorical frameworks would prove inadequate for cyberspace have proved clearly wrong. See David R. Johnson & Kevin A. Marks, Mapping Electronic Data Communications onto Existing Legal Metaphors: Should We Let Our Conscience (and Our Contracts) Be Our Guide?, 38 VILL. L. REV. 487, 514-15 (1993). 13. The canonical expression of this metaphor comes from Mitchell Kapor and John Perry Barlow, in their 1990 essay, Across the Electronic Frontier, at http://www.eff.org//Misc/Publications/ Mitch_Kapor/electronic_frontier.eff (July 10, 1990). 15. See Alfred C. Yen, Western Frontier or Feudal Society?: Metaphors and Perceptions of Cyberspace, 17 BERKELEY TECH. L.J. 1207, 1232-48 (2002). 17. See generally Ann Bartow, Electrifying Copyright Norms and Making Cyberspace More Like a Book, 48 VILL. L. REV. 13 (2003). 13. The canonical expression of this metaphor comes from Mitchell Kapor and John Perry Barlow, in their 1990 essay, Across the Electronic Frontier, at http://www.eff.org//Misc/Publications/ Mitch_Kapor/electronic_frontier.eff (July 10, 1990). 14. See HOWARD RHEINGOLD, THE VIRTUAL COMMUNITY: HOMESTEADING ON THE ELECTRONIC FRONTIER (1993). 16. See Religious Tech. Ctr. v. Netcom On-Line Communication Servs., Inc., 907 F. Supp. 1361 1369 (N.D. Cal. 1995). _ p _ ( y , ) 14. See HOWARD RHEINGOLD, THE VIRTUAL COMMUNITY: HOMESTEADING ON THE ELECTRONIC FRONTIER (1993). , ( ) 19. The novelist William Gibson famously characterized his fictional “cyberspace” as a “consensual hallucination.” WILLIAM GIBSON, NEUROMANCER 51 (1984). INTRODUCTION I do so both to echo its dynamic character and, more provocatively, to suggest that cyberspace has a plot, or perhaps multiple plots; that these plots have characters who interact with one another through some structure; that there are themes and motifs; that there are “good” characters, “bad” characters, winners and losers; and that eventually (though not necessarily simultaneously), 16. See Religious Tech. Ctr. v. Netcom On-Line Communication Servs., Inc., 907 F. Supp. 1361, 369 (N.D. Cal. 1995). 17. See generally Ann Bartow, Electrifying Copyright Norms and Making Cyberspace More Like a Book, 48 VILL. L. REV. 13 (2003). 18. See Mainstream Loudoun v. Bd. of Trs. of Loudoun County Library, 2 F. Supp. 2d 783, 793- 94 (E.D. Va. 1998) (rejecting library characterization in favor of “internet-as-encyclopedia”). Cf. New York Times Co. v. Tasini, 533 U.S. 483, 502-04 (2001) (comparing databases to libraries). At this point, the metaphorical journey has come full circle to Borges. See JORGE LUIS BORGES, The Library of Babel, in LABYRINTHS: SELECTED STORIES & OTHER WRITINGS 51, 51 (Donald A. Yates & James E. Irby eds., 1964). In the present spirit, this collection of metaphors might be referred to as “the usual suspects” after Captain Renault’s famous line at the end of Casablanca: “Major Strasser has been shot. Round up the usual suspects.” CASABLANCA (Warner Brothers Pictures 1942). Early predictions that existing metaphorical frameworks would prove inadequate for cyberspace have proved clearly wrong. See David R. Johnson & Kevin A. Marks, Mapping Electronic Data Communications onto Existing Legal Metaphors: Should We Let Our Conscience (and Our Contracts) Be Our Guide?, 38 VILL. L. REV. 487, 514-15 (1993). 19. The novelist William Gibson famously characterized his fictional “cyberspace” as a “consensual hallucination.” WILLIAM GIBSON, NEUROMANCER 51 (1984). 20. See Agre, Cyberspace as American Culture, supra note 4, at 182-83 (describing how computing theory has failed to evolve in step with the embedding of networked technology beyond computing devices themselves). COLUMBIA JOURNAL OF LAW & THE ARTS [27:2 252 these plots may wind to a conclusion and new plots may begin. In the best of these senses, cyberspace, for now, is a thoroughly conventional popular narrative melodrama. 24. In 1998, a large panel of critics and other film industry participants convened by the American Film Institute listed the 100 greatest films ever made. Citizen Kane, Orson Welles’s artistic tour de force of the moviemaker’s art, was ranked first. Casablanca, competently produced but with no artistic ambitions, finished second. The rankings are available at American Film Institute’s “AFI’s 100 Years . . . 100 Movies,” at http://www.afi.com/tv/movies.asp. 22. To be thoroughly contemporary, I could characterize cyberspace as a single computer game being played out on an unprecedented scale. But my argument would fail on the ground that it might be literally true. See, e.g., David Becker, “EverQuest” Spins Its Own Economy, at http://news.com.com/2100-1040-823260.html?tag=bplst (describing an international economy built by participants in online multi-character interactive computer game); F. Gregory Lastowka & Dan Hunter, The Laws of the Virtual Worlds, CAL. L. REV. (forthcoming) (describing legal problems emanating from massive multi-player online games). INTRODUCTION In an earlier era, I might have characterized it as a work of literary fiction.21 Today, connecting the suggestion to popular understanding means invoking a more contemporary literary form—the motion picture.22 If the narrative construct fits, others may search for their own celluloid analogs. The more stories and counterstories, the better.23 For now, and below, I argue that cyberspace is the electronic equivalent of the very best melodramatic, story-based narrative film ever made.24 Cyberspace is Casablanca.25 23. See Richard Delgado, Storytelling for Oppositionists and Others: A Plea for Narrative, 87 MICH. L. REV. 2411, 2439-40 (1989). I. FADE IN: CYBERSPACE-AS-NARRATIVE Enough with the metaphors, one might say,26 before I venture into stories and 21. Within literature, I am thoroughly anticipated by Jorge Luis Borges. See BORGES, supra note 18, at 51 (“The universe (which others call the Library) is composed of an indefinite and perhaps infinite number of hexagonal galleries, with vast air shafts between, surrounded by very low railings.”). The infinite scale of the Library permits it to accommodate all words ever written and still yet to be written, so that hope at the availability of all knowledge collapses into despair at the impossibility of cataloguing it. 25. Other legal phenomena “are” Casablanca as well, or at least Casablanca has proved a useful foil for legal analysis in other contexts. See, e.g., Aviam Soifer, Complacency and Constitutional Law, 42 OHIO ST. L.J. 383 (1981); Daniel J. Steinbock, Refuge and Resistance: Casablanca’s Lessons for Refugee Law, 7 GEO. IMMIGR. L.J. 649 (1993); cf. Richard A. Epstein, Let “The Fundamental Things Apply”: Necessary and Contingent Truths in Legal Scholarship, 115 HARV. L. REV. 1288, 1313 (2002) (invoking a line from “As Time Goes By,” performed by the piano player Sam (played by Dooley Wilson) in Casablanca); Neil Weinstock Netanel, Copyright Alienability Restrictions and the Enhancement of Author Autonomy: A Normative Evaluation, 24 RUTGERS L.J. 347 (1993): 25. Other legal phenomena “are” Casablanca as well, or at least Casablanca has proved a useful foil for legal analysis in other contexts. See, e.g., Aviam Soifer, Complacency and Constitutional Law, 42 OHIO ST. L.J. 383 (1981); Daniel J. Steinbock, Refuge and Resistance: Casablanca’s Lessons for Refugee Law, 7 GEO. IMMIGR. L.J. 649 (1993); cf. Richard A. Epstein, Let “The Fundamental Things Apply”: Necessary and Contingent Truths in Legal Scholarship, 115 HARV. L. REV. 1288, 1313 (2002) (invoking a line from “As Time Goes By,” performed by the piano player Sam (played by Dooley Wilson) in Casablanca); Neil Weinstock Netanel, Copyright Alienability Restrictions and the Enhancement of Author Autonomy: A Normative Evaluation, 24 RUTGERS L.J. 347 (1993): f y , ( ) [A] viewer of the film, Casablanca, would be free to see it as a metaphor for whatever she wishes and to express that viewpoint in critical commentary. 25. Other legal phenomena “are” Casablanca as well, or at least Casablanca has proved a useful foil for legal analysis in other contexts. See, e.g., Aviam Soifer, Complacency and Constitutional Law, 42 OHIO ST. L.J. 383 (1981); Daniel J. Steinbock, Refuge and Resistance: Casablanca’s Lessons for Refugee Law, 7 GEO. IMMIGR. L.J. 649 (1993); cf. Richard A. Epstein, Let “The Fundamental Things Apply”: Necessary and Contingent Truths in Legal Scholarship, 115 HARV. L. REV. 1288, 1313 (2002) (invoking a line from “As Time Goes By,” performed by the piano player Sam (played by Dooley Wilson) in Casablanca); Neil Weinstock Netanel, Copyright Alienability Restrictions and the Enhancement of Author Autonomy: A Normative Evaluation, 24 RUTGERS L.J. 347 (1993): I. FADE IN: CYBERSPACE-AS-NARRATIVE She also would be entitled to take inspiration from the film and to borrow from its basic themes, story and characters in creating her own work, so long as her work did not cross the fine line between independent expression and a modified version of the director’s and screenwriter’s expression. Id. at 408. [A] viewer of the film, Casablanca, would be free to see it as a metaphor for whatever she wishes and to express that viewpoint in critical commentary. She also would be entitled to take inspiration from the film and to borrow from its basic themes, story and characters in creating her own work, so long as her work did not cross the fine line between independent expression and a modified version of the director’s and screenwriter’s expression. Id. at 408. The enduring popularity of the film has made it an appropriate foil for recent political commentary. See Morgan Meis, Usual Suspect, THE AMERICAN PROSPECT, (June 20, 2003), at http:// www.prospect.org/webfeatures/2003/06/meis-m-06-20.html (arguing that suicide bombings in Casablanca recall humane themes of Casablanca). 26. See Hunter, supra note 6, at 514-16 (arguing for displacement of cyberspace-as-place THE NARRATIVES OF CYBERSPACE LAW 2004] 253 motion pictures, let alone one particular film. But metaphors and analogies are all but inevitable, not only for understanding computer technology, but for understanding and explaining any area of law. We need them both because we need to develop procedures and tools to guide interpretation and the creation of meaning in legal contexts generally27 and because they play a central role in the particular descriptive enterprise of developing, applying and extending precedent.28 p p p p g, pp y g g p Law might approach cyberspace as a sui generis phenomenon and deliberately avoid applying policy or precedent from other fields, reasoning instead from principles based on some “true” understanding of the internet itself. Thus, some courts and scholars have tried to address legal issues “in” cyberspace or “on” the internet by focusing on technical characteristics of the network or digital technology rather than on human experiences situated in it.29 This perspective includes arguments that cyberspace analysis should focus on the relevant technical “layer” of the internet involved in a particular policy or litigation problem.30 A different solution suggests deconstructing cyberspace into categories defined by theoretical abstractions. y p ( ) 35. See LESSIG, THE FUTURE OF IDEAS, supra note 10, at 103-41. I. FADE IN: CYBERSPACE-AS-NARRATIVE Cyberspace describes a problem of information theory,31 property rights theory,32 economics,33 governance and institutional legitimacy,34 innovation and creativity35 and privacy and security,36 among other things. metaphor); Jonathan H. Blavin & I. Glenn Cohen, Note, Gore, Gibson, and Goldsmith: The Evolution of Internet Metaphors in Law and Commentary, 16 HARV. J.L. & TECH. 265, 285 (2002) (arguing for abandonment of search for overarching metaphor for the internet and acceptance of localized metaphors); Timothy Wu, Application-Centered Internet Analysis, 85 VA. L. REV. 1163, 1163-66 (1999) (arguing against application of single analogy for all internet analysis). 27. See George H. Taylor, Critical Hermeneutics: The Intertwining of Explanation and Understanding as Exemplified in Legal Analysis, 76 CHI.-KENT L. REV. 1101, 1117 (2000) (“The relationship between understanding and explanation itself forms a hermeneutic circle: understanding is mediated by explanatory procedures, and explanation needs contextualization within interpretive understanding.”). 28. See generally Dan Hunter, Reason is Too Large: Analogy and Precedent in Law, 50 EMORY L.J. 1197, 1204-29 (2001) (describing cognitive bases for analogical models of legal reasoning). 29. See Frischmann, supra note 2; Marobie-FL, Inc. v. Nat’l Ass’n of Fire Equip. Distribs., 983 F. Supp. 1167, 1176 (N.D. Ill. 1997) (holding World Wide Web user liable for infringement of plaintiff’s right to distribute copyrighted work when defendant copied file containing plaintiff’s work to web server). The trend under contemporary copyright law to treat all electronic “copying” as infringing “reproduction” under the Copyright Act can be traced to MAI Sys. Corp. v. Peak Computer, Inc., 991 F.2d 511 (9th Cir. 1993). 30. See Wu, supra note 26, at 1194-1203 (using example of debates over scope to be accorded to private ordering “on the internet” to argue that internet analysis should focus on the characteristics of the software application in question); Yochai Benkler, Net Regulation: Taking Stock and Looking Forward, 71 U. COLO. L. REV. 1203, 1260 (2000) (urging recognition of the different layers of the internet— physical, logical and content—and how they relate to one another). 31. See Yochai Benkler, Free as the Air to Common Use: First Amendment Constraints on Enclosure of the Public Domain, 74 N.Y.U. L. REV. 354, 377-86 (1999). 32. See Trotter Hardy, Property (and Copyright) in Cyberspace, 1996 U. CHI. LEGAL F. 217, 236-58. 33. See Tom W. Bell, Fair Use vs. Fared Use: The Impact of Automated Rights Management on Copyright’s Fair Use Doctrine, 76 N.C. L. REV. 557, 579-600 (1998). py g ( ) 34. See A. Michael Froomkin, Habermas@Discourse.net: Toward a Critical Theory of Cyberspace, 116 HARV. L. REV. 749, 855-71 (2003). metaphor); Jonathan H. Blavin & I. Glenn Cohen, Note, Gore, Gibson, and Goldsmith: The Evolution of Internet Metaphors in Law and Commentary, 16 HARV. J.L. & TECH. 265, 285 (2002) (arguing for abandonment of search for overarching metaphor for the internet and acceptance of localized metaphors); Timothy Wu, Application-Centered Internet Analysis, 85 VA. L. REV. 1163, 1163-66 (1999) (arguing against application of single analogy for all internet analysis). 33. See Tom W. Bell, Fair Use vs. Fared Use: The Impact of Automated Rights Management on Copyright’s Fair Use Doctrine, 76 N.C. L. REV. 557, 579-600 (1998). 36. See Marc Rotenberg, Fair Information Practices and the Architecture of Privacy (What Larry Doesn’t Get), 2001 STAN. TECH. L. REV. 1, ¶¶51-100, at http://stlr.stanford.edu/STLR/Symposia/ Cyberspace/00_rotenberg_1/article.htm. 37. See Taylor, supra note 27. 38. In a relatively short time, the Supreme Court’s perspective on the internet has undergone a radical transformation. Compare Reno v. ACLU, 521 U.S. 844, 851 (1997) (analyzing federal law regulating access to online pornography from the premise that the internet is a unique and wholly new medium), with Ashcroft v. ACLU, 535 U.S. 564, 583 (2002) (confirming applicability of existing “community standards” obscenity jurisprudence to analysis of follow-on federal law regulating access to pornography). The “speaker centered” analysis of the internet obscenity cases contrasts with the “user centered” analysis applied in the Court’s first internet copyright case, New York Times Co. v. Tasini, 533 U.S. 483, 499 (2001) (finding that newspaper publishers infringed copyrights of freelance authors when publishers caused articles to be re-distributed in online computer databases). 41. See David Nimmer, Brains and Other Paraphernalia of the Digital Age, 10 HARV. J. L. & TECH. 1, 4-9 (1998) (discussing causes and effects of “despatialization” in cyberspace). p p 40. See Agre, Cyberspace as American Culture, supra note 4, at 183-85 (describing the conflation of computing technology and ordinary patterns of human activity); Radin, supra note 5, at 1143-44 (describing the conflation of text and technology). The evolution of both technology and practice from “wired” network communications to “wireless” communications, for data as well as voice, is further breaking down barriers between “online” and “offline” activity. For a perceptive critique of how wireless networks are changing fundamental assumptions about telecommunications regulation, see Yochai Benkler, Some Economics of Wireless Communications, 16 HARV. J.L. & TECH. 25, 40-47 (2002). p p 39. See Kerr, supra note 2; Hunter, supra note 6, at 472-99. 37. See Taylor, supra note 27. 36. See Marc Rotenberg, Fair Information Practices and the Architecture of Privacy (What Larry Doesn’t Get), 2001 STAN. TECH. L. REV. 1, ¶¶51-100, at http://stlr.stanford.edu/STLR/Symposia/ Cyberspace/00_rotenberg_1/article.htm. 37. See Taylor, supra note 27. 38. In a relatively short time, the Supreme Court’s perspective on the internet has undergone a radical transformation. Compare Reno v. ACLU, 521 U.S. 844, 851 (1997) (analyzing federal law regulating access to online pornography from the premise that the internet is a unique and wholly new medium), with Ashcroft v. ACLU, 535 U.S. 564, 583 (2002) (confirming applicability of existing “community standards” obscenity jurisprudence to analysis of follow-on federal law regulating access to pornography). The “speaker centered” analysis of the internet obscenity cases contrasts with the “user centered” analysis applied in the Court’s first internet copyright case, New York Times Co. v. Tasini, 533 U.S. 483, 499 (2001) (finding that newspaper publishers infringed copyrights of freelance authors when publishers caused articles to be re-distributed in online computer databases). 39. See Kerr, supra note 2; Hunter, supra note 6, at 472-99. 40. See Agre, Cyberspace as American Culture, supra note 4, at 183-85 (describing the conflation of computing technology and ordinary patterns of human activity); Radin, supra note 5, at 1143-44 (describing the conflation of text and technology). The evolution of both technology and practice from “wired” network communications to “wireless” communications, for data as well as voice, is further breaking down barriers between “online” and “offline” activity. For a perceptive critique of how wireless networks are changing fundamental assumptions about telecommunications regulation, see Yochai Benkler, Some Economics of Wireless Communications, 16 HARV. J.L. & TECH. 25, 40-47 (2002). 41. See David Nimmer, Brains and Other Paraphernalia of the Digital Age, 10 HARV. J. L. & TECH. 1, 4-9 (1998) (discussing causes and effects of “despatialization” in cyberspace). I. FADE IN: CYBERSPACE-AS-NARRATIVE py g ( ) 34. See A. Michael Froomkin, Habermas@Discourse.net: Toward a Critical Theory o Cyberspace, 116 HARV. L. REV. 749, 855-71 (2003). 254 [27:2 COLUMBIA JOURNAL OF LAW & THE ARTS None of these approaches is wrong even though each may teach different things. Cyberspace and the internet represent all of these things: metaphor, technology and theory, even when the lessons of each approach appear to contradict one another. What the technology describes, the theory explains; what the theory explains, the metaphor understands.37 What is missing is a meta-analysis, a way to connect the technical, the theoretical and the metaphorical in a way that captures the dynamism of the field. What we have today is a collection of metaphors, each with adherents and impact on the development of the law, applied with no sense that they might be related to one another.38 The metaphor may not be the only thing driving legal analysis, but the legal landscape is littered with conflicts produced in part by disagreements over perspective and characterization.39 Theoretical, technical and metaphorical approaches tend to assume the existence of an object—“cyberspace,” or “the internet,” whether those are one thing or two— that is both fixed and distinct. If that characterization continues to make sense remains to be seen. We think and act as if there is “an internet” and “cyberspace,” but the border between how people use the internet (or experience cyberspace), and the internet or cyberspace itself, from a technical standpoint, is increasingly blurry.40 And neither cyberspace nor the internet is static. If any one thing has characterized both over the last three decades, it is a state of constant, rapid change. Metaphors that rely on mapping characteristics from source phenomenon to target and theories that project their constructs onto objects of analysis have a difficult time analyzing a fast-moving target, large portions of which are being absorbed into the fabric of broader experience.41 Finding the “right” metaphor for cyberspace at a universal level may be an , p ; , p , 40. See Agre, Cyberspace as American Culture, supra note 4, at 183-85 (describing the conflation of computing technology and ordinary patterns of human activity); Radin, supra note 5, at 1143-44 (describing the conflation of text and technology). 42. See GIBSON, supra note 19. 43. The relationships among law, literature and narrative form a rich field of scholarly inquiry. Scholars urging critical examination of jurisprudence from literary, aesthetic and/or narrative perspectives include: MARTHA C. NUSSBAUM, POETIC JUSTICE: THE LITERARY IMAGINATION AND PUBLIC LIFE (1995); RICHARD WEISBERG, POETHICS AND OTHER STRATEGIES OF LAW AND LITERATURE (1992); ROBIN WEST, NARRATIVE, AUTHORITY, AND LAW (1993); Robert M. Cover, Foreword: Nomos and Narrative, 97 HARV. L. REV. 4 (1983). The role of literary perspectives in constructing a normative vision of law and legal practice is explored in the work of James Boyd White. See, e.g., JAMES BOYD WHITE, ACTS OF HOPE: CREATING AUTHORITY IN LITERATURE, LAW, AND POLITICS (1994); JAMES BOYD WHITE, JUSTICE AS TRANSLATION: AN ESSAY IN CULTURAL AND LEGAL CRITICISM (1990); THE LEGAL IMAGINATION: STUDIES IN THE NATURE OF LEGAL THOUGHT AND EXPRESSION (1973); JAMES BOYD WHITE, Reading Law and Reading Literature: Law as Language, in HERACLES’ BOW: ESSAYS ON THE RHETORIC AND POETICS OF THE LAW (1985). 42. See GIBSON, supra note 19. 43. The relationships among law, literature and narrative form a rich field of scholarly inquiry. Scholars urging critical examination of jurisprudence from literary, aesthetic and/or narrative perspectives include: MARTHA C. NUSSBAUM, POETIC JUSTICE: THE LITERARY IMAGINATION AND PUBLIC LIFE (1995); RICHARD WEISBERG, POETHICS AND OTHER STRATEGIES OF LAW AND LITERATURE (1992); ROBIN WEST, NARRATIVE, AUTHORITY, AND LAW (1993); Robert M. Cover, Foreword: Nomos and Narrative, 97 HARV. L. REV. 4 (1983). The role of literary perspectives in constructing a normative vision of law and legal practice is explored in the work of James Boyd White. See, e.g., JAMES BOYD WHITE, ACTS OF HOPE: CREATING AUTHORITY IN LITERATURE, LAW, AND POLITICS (1994); JAMES BOYD WHITE, JUSTICE AS TRANSLATION: AN ESSAY IN CULTURAL AND LEGAL CRITICISM (1990); THE LEGAL IMAGINATION: STUDIES IN THE NATURE OF LEGAL THOUGHT AND EXPRESSION (1973); JAMES BOYD WHITE, Reading Law and Reading Literature: Law as Language, in HERACLES’ BOW: ESSAYS ON THE RHETORIC AND POETICS OF THE LAW (1985). 44. See Cover, supra note 43, at 4-5. 45. See NUSSBAUM, supra note 43, at 3-12. 46. This is the classic Aristotelian perspective on narrative. [I]t is not the poet’s function to relate actual events, but the kinds of things that might occur and are possible in terms of probability or necessity. The difference between the historian and the poet is not that between using verse or prose; Herodotus’ work could be versified and would be just as much a kind of history in verse as in prose. No, the difference is this: that the one relates actual events, the other the kinds of things that might occur. ARISTOTLE, POETICS 51b, at 59 (Stephen Halliwell trans., 1995). I. FADE IN: CYBERSPACE-AS-NARRATIVE The evolution of both technology and practice from “wired” network communications to “wireless” communications, for data as well as voice, is further breaking down barriers between “online” and “offline” activity. For a perceptive critique of how wireless networks are changing fundamental assumptions about telecommunications regulation, see Yochai Benkler, Some Economics of Wireless Communications, 16 HARV. J.L. & TECH. 25, 40-47 (2002). THE NARRATIVES OF CYBERSPACE LAW 2004] 255 impossible task. Gibson’s characterization of “cyberspace” as a “consensual hallucination”42 suggested long before cyberspace became “real” in popular understanding that the technology that sustains this form of electronic interaction could be understood only with more than ordinary application of the metaphorical and imaginative arts. I argue, modestly, that cyberspace-as-story, and cyberspace- as-Casablanca as one story in particular, supplies a better and appropriate framework. I focus on narrative in two distinct, but related, senses. First, there is the modern academic sense of “narrative” as a social construct that integrates diverse phenomena into some meaningful, if necessarily incomplete, whole.43 This is partly Robert Cover’s nomos: No set of legal institutions or prescriptions exists apart from the narratives that locate it and give it meaning. For every constitution there is an epic, for each decalogue a scripture. Once understood in the context of the narratives that give it meaning, law becomes not merely a system of rules to be observed, but a world in which we live.44 It is also partly what Martha Nussbaum has described and commended as an act of “literary imagination.”45 The narrative permits us to both see the world as it is, by structuring apparently disparate phenomena, and perhaps to understand or imagine, as a writer might do, the world as it might be.46 Situating legal problems of cyberspace in the context of a narrative helps us to describe the character of the issues that we face and to derive prescriptions from the narrative structure for resolving those issues. 45. See NUSSBAUM, supra note 43, at 3-12. ARISTOTLE, POETICS 51b, at 59 (Stephen Halliwell trans., 1995). 44. See Cover, supra note 43, at 4-5. 46. This is the classic Aristotelian perspective on narrative. [I]t is not the poet’s function to relate actual events, but the kinds of things that might occur and are possible in terms of probability or necessity. The difference between the historian and the poet is not that between using verse or prose; Herodotus’ work could be versified and would be just as much a kind of history in verse as in prose. No, the difference is this: that the one relates actual events, the other the kinds of things that might occur. A P 51b 59 (S h H lli ll 1995) 46. This is the classic Aristotelian perspective on narrative. 42. See GIBSON, supra note 19. I. FADE IN: CYBERSPACE-AS-NARRATIVE It is also partly what Martha Nussbaum has described and commended as an act of “literary imagination.”45 The narrative permits us to both see the world as it is, by structuring apparently disparate phenomena, and perhaps to understand or imagine, as a writer might do, the world as it might be.46 Situating legal problems of cyberspace in the context of a narrative helps us to describe the character of the issues that we face and to derive prescriptions from the narrative structure for resolving those issues. Second, there is the related but both older and more popular sense of “narrative” as story, as a tale that has power and relevance because it has a beginning, a middle COLUMBIA JOURNAL OF LAW & THE ARTS [27:2 256 and an end. The cyberspatial metaphor is this Aristotelian narrative47 rather than Platonic essence (“what is the internet?”). Theoretical constructs, metaphors and technologies become characters, settings, themes and plots. In both senses, thinking about cyberspace as a narrative can help lawyers, judges, policy makers and jurors structure intersecting and overlapping arguments and interests about conflicts involving phenomena in cyberspace.48 Cyberspace is not a thing, in other words, nor is it a place or merely a collection of wires and bits, nor an instantiation of information theory. Cyberspace is a narrative. Cyberspace is a story.49 47. ARISTOTLE, supra note 46, 50b, at 55 (“[A] whole is that which has a beginning, a middle, and an end.”). Cf. PAUL RICOEUR, 1 TIME AND NARRATIVE 31-51 (Kathleen McLaughlin & David Pellauer trans., 1984) (providing an account of the centrality of narrative in Aristotelian thought to rhetoric and ethics, as well as poetics). Mark A. Lemley & Lawrence Lessig, The End of End-to-End: Preserving the Architecture of the internet in the Broadband Era, 48 UCLA L. REV. 925, 930-31 (2001). The e2e argument organizes the placement of functions within a network. It counsels that the ‘intelligence’ in a network should be located at the top of a layered system—at its ‘ends,’ where users put information and applications onto the network. The communications protocols themselves (the ‘pipes’ through which information flows) should be as simple and as general as possible. 50. This statement echoes the “end-to-end” (or “e2e”) design principle on which the technology o the internet is based. 48. See generally Nancy Pennington & Reid Hastie, A Cognitive Theory of Juror Decision Making: The Story Model, 13 CARDOZO L. REV. 519 (1991) (finding that jurors tend to structure evidence into stories in order to understand confusing and disconnected material). 49. Among social scientists, American Studies scholars and some legal scholars, cyberspace and computer technology have previously been characterized as generators of discourses and products (or features) of narratives. See, e.g., Philip E. Agre, The Next Internet Hero, TECHNOLOGY REVIEW, Nov./Dec. 1997, at 61 (“Every new technology is accompanied by a grand narrative.”); Keith Aoki, (Intellectual) Property and Sovereignty: Notes Toward a Cultural Geography of Authorship, 48 STAN. L. REV. 1293 (1996); CHRISTOPHER BRADFORD CHESHER, COMPUTERS AS INVOCATIONAL MEDIA (2000) (unpublished Ph.D. dissertation, Macquarie University) (on file with author); RICHARD COYNE, TECHNOROMANTICISM: DIGITAL NARRATIVE, HOLISM, AND THE ROMANCE OF THE REAL (1999); Brian F. Fitzgerald, Software as Discourse: The Power of Intellectual Property in Digital Architecture, 18 CARDOZO ARTS & ENT. L.J. 337 (2000); N. KATHERINE HAYLES, HOW WE BECAME POSTHUMAN: VIRTUAL BODIES IN CYBERNETICS, LITERATURE, AND INFORMATICS (1999); Rob Kling, Hopes and Horrors: Technological Utopianism and Anti-utopianism in Narratives of Computerization, in COMPUTERIZATION AND CONTROVERSY: VALUE CONFLICTS AND SOCIAL CHOICES, at 40 (Rob Kling ed., 2d ed. 1996); BRENDA LAUREL, COMPUTERS AS THEATRE (1991); DAVID E. NYE, NARRATIVES AND SPACES: TECHNOLOGY AND THE CONSTRUCTION OF AMERICAN CULTURE (1997); ANDREW ROSS, STRANGE WEATHER: CULTURE, SCIENCE AND TECHNOLOGY IN THE AGE OF LIMITS (1991); Vivian Sobchack, New Age Mutant Ninja Hackers: Reading Mondo 2000, 92 S. ATLANTIC Q. 569 (1993). 47. ARISTOTLE, supra note 46, 50b, at 55 (“[A] whole is that which has a beginning, a middle, and an end.”). Cf. PAUL RICOEUR, 1 TIME AND NARRATIVE 31-51 (Kathleen McLaughlin & David Pellauer trans., 1984) (providing an account of the centrality of narrative in Aristotelian thought to rhetoric and ethics, as well as poetics). 48. See generally Nancy Pennington & Reid Hastie, A Cognitive Theory of Juror Decision Making: The Story Model, 13 CARDOZO L. REV. 519 (1991) (finding that jurors tend to structure evidence into stories in order to understand confusing and disconnected material). 49. Among social scientists, American Studies scholars and some legal scholars, cyberspace and computer technology have previously been characterized as generators of discourses and products (or features) of narratives. See, e.g., Philip E. Agre, The Next Internet Hero, TECHNOLOGY REVIEW, Nov./Dec. 1997, at 61 (“Every new technology is accompanied by a grand narrative.”); Keith Aoki, (Intellectual) Property and Sovereignty: Notes Toward a Cultural Geography of Authorship, 48 STAN. L. REV. 1293 (1996); CHRISTOPHER BRADFORD CHESHER, COMPUTERS AS INVOCATIONAL MEDIA (2000) (unpublished Ph.D. dissertation, Macquarie University) (on file with author); RICHARD COYNE, TECHNOROMANTICISM: DIGITAL NARRATIVE, HOLISM, AND THE ROMANCE OF THE REAL (1999); Brian F. Fitzgerald, Software as Discourse: The Power of Intellectual Property in Digital Architecture, 18 CARDOZO ARTS & ENT. L.J. 337 (2000); N. KATHERINE HAYLES, HOW WE BECAME POSTHUMAN: VIRTUAL BODIES IN CYBERNETICS, LITERATURE, AND INFORMATICS (1999); Rob Kling, Hopes and Horrors: Technological Utopianism and Anti-utopianism in Narratives of Computerization, in COMPUTERIZATION AND CONTROVERSY: VALUE CONFLICTS AND SOCIAL CHOICES, at 40 (Rob Kling ed., 2d ed. 1996); BRENDA LAUREL, COMPUTERS AS THEATRE (1991); DAVID E. NYE, NARRATIVES AND SPACES: TECHNOLOGY AND THE CONSTRUCTION OF AMERICAN CULTURE (1997); ANDREW ROSS, STRANGE WEATHER: CULTURE, SCIENCE AND TECHNOLOGY IN THE AGE OF LIMITS (1991); Vivian Sobchack, New Age Mutant Ninja Hackers: Reading Mondo 2000, 92 S. ATLANTIC Q. 569 (1993). 50. This statement echoes the “end-to-end” (or “e2e”) design principle on which the technology of 51. See, e.g., J. DAVID BOLTER, WRITING SPACE: THE COMPUTER, HYPERTEXT, AND THE HISTORY OF WRITING 107-46 (1991); RICHARD A. LANHAM, THE ELECTRONIC WORD: DEMOCRACY, TECHNOLOGY, AND THE ARTS 124-25 (1993); GEORGE P. LANDOW, HYPERTEXT 2.0: THE CONVERGENCE OF CONTEMPORARY CRITICAL THEORY AND TECHNOLOGY 49-60, 178-92 (1997); JANET H. MURRAY, HAMLET ON THE HOLODECK: THE FUTURE OF NARRATIVE IN CYBERSPACE 65-94, 173-75 (1997); Robert Coover, The End of Books, N.Y. TIMES, June 21, 1992, (Book Review), at 1. Early essays investigating the implications of computer technology for the concept of “the book” are collected in THE FUTURE OF THE BOOK (Geoffrey Nunberg ed., 1996). The idea of “hypertext” is generally traced to Vannevar Bush, As We May Think, 176 THE ATLANTIC MONTHLY No. 1, at 101-08 (July 1945). II. DISSOLVE TO: CODE-AS-NARRATIVE Though this suggestion is obviously—but only partly—metaphorical itself, before proceeding further I should note and respond to the immediate objection that, in many respects, cyberspace is the very antithesis of a narrative, either in the sense that narrative consists of a constructed ordering of apparently disparate phenomena or in the sense that narrative serves as a conventional text. By design, cyberspace has no beginning, middle or end. By design, it has structure only at its most basic technical layers.50 Literary theorists who discovered “hypertext” online The e2e argument organizes the placement of functions within a network. It counsels that the ‘intelligence’ in a network should be located at the top of a layered system—at its ‘ends,’ where users put information and applications onto the network. The communications protocols themselves (the ‘pipes’ through which information flows) should be as simple and as general as possible THE NARRATIVES OF CYBERSPACE LAW 2004] 257 in the mid-1990s foresaw the end (or at least the compromising) of the traditional novel along with the end to the traditional physical bounded-ness of the book.51 A novel’s conventional narrative structure requires the linearity defined by the book. Online hypertext encourages, or at least permits, the reader to create a non-linear experience. Narrative theory, then, should have little application to hypertext or to cyberspace. This argument eventually made its way to law, both in the larger law of “cyberspace” and in the better-defined field of copyright law. Initial efforts to define a law of “cyberspace” focused on its boundless nature and on the fact that conventional state-based legal regulation could not be sustained.52 The lack of “bounded-ness” in cyberspace implies a corresponding shift in the kinds of stories that can be told about law.53 Copyright commentators have noted that the internet lacks the material characteristics that defined traditional copyright law. In particular, the lack of “book-ish”-ness of information created, stored and distributed on electronic networks has been a cause for concern on the ground that it appears to signify the passing of an effective doctrine of first sale,54 among other things. The question “What is a book?” has to be re-asked when text is presented in electronic form. So far, it appears, an electronic book is not “a book.”55 Copyright’s conventional stories do not apply. But the lack of boundaries in cyberspace may be more apparent than real. More recent literary criticism has grasped this. 55. See Random House LLC v. Rosetta Books LLC, 150 F. Supp. 2d 613 (S.D.N.Y. 2001) (denying a motion for preliminary injunction brought by a book publisher authorized to publish novel “in book form” against electronic “book” publisher distributing same novel in electronic form), aff’d, 283 F.3d 490 (2d Cir. 2002). 52. This position is most clearly associated with David R. Johnson & David Post, Law And Borders—The Rise of Law in Cyberspace, 48 STAN. L. REV. 1367 (1996). Johnson and Post’s article inspired a response (see Jack L. Goldsmith, Against Cyberanarchy, 65 U. CHI. L. REV. 1199 (1998) (contending that cyberspace presents an unexceptional range of jurisdictional and choice-of-law problems)) and a reply (see David G. Post, Against “Against Cyberanarchy,” 17 BERKELEY TECH. L.J. 1365 (2002) (maintaining unrepentant exceptionalism)). 54. See Bartow, supra note 17, at 110-18. 53. See Shulamit Almog, From Sterne and Borges to Lost Storytellers: Cyberspace, Narrative, and Law, 13 FORDHAM INTELL. PROP. MEDIA & ENT. L.J. 1 (2002). With its essentially dynamic character: The internet initiates and continuously induces important shifts in our storytelling practices and narrative cognizance. These shifts carry significant implications in the domain of law. They influence the way we practice law and the way we perceive it. They affect our comprehension of law and the range of anticipations, hopes, and emotions related to it. Id. at 3. 51. See, e.g., J. DAVID BOLTER, WRITING SPACE: THE COMPUTER, HYPERTEXT, AND THE HISTORY OF WRITING 107-46 (1991); RICHARD A. LANHAM, THE ELECTRONIC WORD: DEMOCRACY, TECHNOLOGY, AND THE ARTS 124-25 (1993); GEORGE P. LANDOW, HYPERTEXT 2.0: THE CONVERGENCE OF CONTEMPORARY CRITICAL THEORY AND TECHNOLOGY 49-60, 178-92 (1997); JANET H. MURRAY, HAMLET ON THE HOLODECK: THE FUTURE OF NARRATIVE IN CYBERSPACE 65-94, 173-75 (1997); Robert Coover, The End of Books, N.Y. TIMES, June 21, 1992, (Book Review), at 1. Early essays investigating the implications of computer technology for the concept of “the book” are collected in THE FUTURE OF THE BOOK (Geoffrey Nunberg ed., 1996). The idea of “hypertext” is generally traced to Vannevar Bush, As We May Think, 176 THE ATLANTIC MONTHLY No. 1, at 101-08 (July 1945). 52. This position is most clearly associated with David R. Johnson & David Post, Law And Borders—The Rise of Law in Cyberspace, 48 STAN. L. REV. 1367 (1996). Johnson and Post’s article inspired a response (see Jack L. Goldsmith, Against Cyberanarchy, 65 U. CHI. L. REV. 1199 (1998) (contending that cyberspace presents an unexceptional range of jurisdictional and choice-of-law problems)) and a reply (see David G. Post, Against “Against Cyberanarchy,” 17 BERKELEY TECH. L.J. 1365 (2002) (maintaining unrepentant exceptionalism)). 53. See Shulamit Almog, From Sterne and Borges to Lost Storytellers: Cyberspace, Narrative, and Law, 13 FORDHAM INTELL. PROP. MEDIA & ENT. L.J. 1 (2002). With its essentially dynamic character: The internet initiates and continuously induces important shifts in our storytelling practices and narrative cognizance. These shifts carry significant implications in the domain of law. They influence the way we practice law and the way we perceive it. They affect our comprehension of law and the range of anticipations, hopes, and emotions related to it. Id. at 3. 54. See Bartow, supra note 17, at 110-18. 55. See Random House LLC v. Rosetta Books LLC, 150 F. Supp. 2d 613 (S.D.N.Y. 2001) (denying a motion for preliminary injunction brought by a book publisher authorized to publish novel “in book form” against electronic “book” publisher distributing same novel in electronic form), aff’d, 283 F.3d 490 (2d Cir. 2002). II. DISSOLVE TO: CODE-AS-NARRATIVE Cyberspace is not necessarily defined by 52. This position is most clearly associated with David R. Johnson & David Post, Law And Borders—The Rise of Law in Cyberspace, 48 STAN. L. REV. 1367 (1996). Johnson and Post’s article inspired a response (see Jack L. Goldsmith, Against Cyberanarchy, 65 U. CHI. L. REV. 1199 (1998) (contending that cyberspace presents an unexceptional range of jurisdictional and choice-of-law problems)) and a reply (see David G. Post, Against “Against Cyberanarchy,” 17 BERKELEY TECH. L.J. 1365 (2002) (maintaining unrepentant exceptionalism)). g 53. See Shulamit Almog, From Sterne and Borges to Lost Storytellers: Cyberspace, Narrative, and Law, 13 FORDHAM INTELL. PROP. MEDIA & ENT. L.J. 1 (2002). With its essentially dynamic character: 53. See Shulamit Almog, From Sterne and Borges to Lost Storytellers: Cyberspace, Narrative, and Law, 13 FORDHAM INTELL. PROP. MEDIA & ENT. L.J. 1 (2002). With its essentially dynamic character: The internet initiates and continuously induces important shifts in our storytelling practices and narrative cognizance. These shifts carry significant implications in the domain of law. They influence the way we practice law and the way we perceive it. They affect our comprehension of law and the range of anticipations, hopes, and emotions related to it. Id at 3 55. See Random House LLC v. Rosetta Books LLC, 150 F. Supp. 2d 613 (S.D.N.Y. 2001) (denying a motion for preliminary injunction brought by a book publisher authorized to publish novel “in book form” against electronic “book” publisher distributing same novel in electronic form), aff’d, 283 F.3d 490 (2d Cir. 2002). COLUMBIA JOURNAL OF LAW & THE ARTS [27:2 258 its form and format, as a traditional book is, but by the production of the form itself. Cyberspace does not have an essence and instead is determined and bounded by the processes by which it is created and experienced. The boundaries of cyberspace are recursive, generated dynamically and constantly re-determined, rather than static. Boundaries do exist, if in a non-traditional sense.56 At its most basic technical layer, cyberspace is defined by software, embodied in the programs and protocols that enable different computers to transmit data between them. Protocols are sets of rules that define the conditions under which computer programs will operate; that is, they set limits on the behavior of computer systems. Computer programs do likewise, in that they set limits on the behavior of both computers and their human users. 56. JOSEPH TABBI, COGNITIVE FICTIONS xi (2002) (“A similar interdependence [between the individual and the environment], through which a bounded and stable structure is continually produced, might better describe what is actually going on within today’s changing media environment.”). This perspective identifies traditional text-based narratives as a historically contingent instantiation of a cognitive framework for the recognition of meaning in context. The narrative construct does not depend on a particular technology. See id. The same argument is evident in a recent short story by Richard Powers. See Richard Powers, Literary Devices, 6 ZOETROPE, No. 4 (Winter 2002), which echoes the hermeneutic perspective offered above. Also See Taylor, supra note 27, at 1123: 60. See, e.g., Paul Schiff Berman, The Globalization of Jurisdiction, 151 U. PA. L. REV. 311, 321- 22 (2002) (attempting to rethink jurisdictional questions in terms of dynamic community affiliations enabled by the internet). The idea that technology can create borders, and thus, meaning, is not new, at least in internet time. See generally Joel R. Reidenberg, Lex Informatica: The Formulation of Information Policy Rules Through Technology, 76 TEX. L. REV. 553 (1998); Joel R. Reidenberg, Governing Networks and Rule-Making in Cyberspace, 45 EMORY L.J. 911 (1996). And the unconventional idea of property (an artifact typically characterized as timeless) as narrative has been 58. HTML stands for Hyper Text Markup Language, the software protocol used to frame the basic appearance of pages on the World Wide Web. The model for law need not necessarily derive from the deterministic, nomological form of explanation commonly identified in the physical sciences but may find more appropriate analogue in the interrelation of regularity and exception found in biology. A sophisticated narrative approach—informed by both interpretive understanding and explanation—can be an appropriate, indeed arguably is the most appropriate, methodology in law. 57. Lawrence Lessig makes an extended argument in support of this point in LAWRENCE LESSIG CODE AND OTHER LAWS OF CYBERSPACE (1999). See also MITCHELL, supra note 5, at 111. 59. See Johnson & Post, supra note 52. 56. JOSEPH TABBI, COGNITIVE FICTIONS xi (2002) (“A similar interdependence [between the individual and the environment], through which a bounded and stable structure is continually produced, might better describe what is actually going on within today’s changing media environment.”). This perspective identifies traditional text-based narratives as a historically contingent instantiation of a cognitive framework for the recognition of meaning in context. The narrative construct does not depend on a particular technology. See id. The same argument is evident in a recent short story by Richard Powers. See Richard Powers, Literary Devices, 6 ZOETROPE, No. 4 (Winter 2002), which echoes the hermeneutic perspective offered above. Also See Taylor, supra note 27, at 1123: The model for law need not necessarily derive from the deterministic, nomological form of explanation commonly identified in the physical sciences but may find more appropriate analogue in the interrelation of regularity and exception found in biology. A sophisticated narrative approach—informed by both interpretive understanding and explanation—can be an appropriate, indeed arguably is the most appropriate, methodology in law. 57. Lawrence Lessig makes an extended argument in support of this point in LAWRENCE LESSIG, CODE AND OTHER LAWS OF CYBERSPACE (1999). See also MITCHELL, supra note 5, at 111. 58. HTML stands for Hyper Text Markup Language, the software protocol used to frame the basic appearance of pages on the World Wide Web. 59. See Johnson & Post, supra note 52. 60. See, e.g., Paul Schiff Berman, The Globalization of Jurisdiction, 151 U. PA. L. REV. 311, 321- 22 (2002) (attempting to rethink jurisdictional questions in terms of dynamic community affiliations enabled by the internet). The idea that technology can create borders, and thus, meaning, is not new, at least in internet time. See generally Joel R. Reidenberg, Lex Informatica: The Formulation of Information Policy Rules Through Technology, 76 TEX. L. REV. 553 (1998); Joel R. Reidenberg, Governing Networks and Rule-Making in Cyberspace, 45 EMORY L.J. 911 (1996). And the unconventional idea of property (an artifact typically characterized as timeless) as narrative has been 64. Any “cast of cyberspace” is inevitably incomplete. Some of the better known names include John Perry Barlow (cyberspace rhetoritician), Vinton Cerf and Bob Metcalfe (networking pioneers), the late Jon Postel (original keeper of the domain names registry), Marc Andreesen and Jim Clark (popularizers of the Netscape Navigator web browser) and Steve Case (founder of America Online). See Agre, The Next Instant Hero, supra note 49, at 61 (anticipating the emergence of the “public hacker” as the next hero of the grand narrative of the internet). 63. The myth that the internet was designed to survive a nuclear war has been largely debunked, but it remains the case that much of the research that produced the computer networks that evolved into today’s internet was funded through the Department of Defense during the Cold War. See M. MITCHELL WALDROP, THE DREAM MACHINE: J.C.R. LICKLIDER AND THE REVOLUTION THAT MADE COMPUTING PERSONAL 259-332 (2001) II. DISSOLVE TO: CODE-AS-NARRATIVE Those limits define the scope of permitted conduct. Code regulates.57 Even hypertext is not truly boundless. The manner in which a reader of hypertext can jump from link to link is determined by HTML58 codes that specify the existence of, and connections between, links. The reader is merely establishing one pattern from among a large number of patterns whose potential is embedded in the software. There are many beginnings and many ends, rather than one of each. All of these choices exist within the boundaries set by the technology. The reader or user chooses and, in doing so, selects the boundaries of his or her own experience. A comparable notion is present in legal scholarship, though described in different terms. From initial arguments that legal doctrine would be forced to accommodate the essential openness and indeterminacy of the internet,59 the legal literature now focuses more precisely on the ways in which cyberspace both creates and can help resolve boundary problems.60 Code regulates not only in a purely THE NARRATIVES OF CYBERSPACE LAW 2004] 259 behavioral sense, but also in a meaningful legal sense, by dynamically shaping the choices faced by both communities and individuals online. What I suggest, then, is that this emerging dynamic notion of boundaries and limits in cyberspace, from both literary and legal perspectives, can be borrowed to construct the foundations for a narrative, a story, of cyberspace. ( ) p p y ( ) 61. It might be argued that existing metaphorical efforts in cyberspace analysis (see supra notes 5 19 and accompanying text) constitute narratives of their own. explored in the context of property theory generally. See Carol M. Rose, Property as Storytelling: Perspectives from Game Theory, Narrative Theory, Feminist Theory, 2 YALE J.L. & HUMAN. 37, 38-39 (1990); Anupam Chander, The New, New Property, 81 TEX. L. REV. 715 (2003). p y g ) 62. See Philip N. Meyer, Making the Narrative Move: Observations Based Upon Reading Gerry Spence’s Closing Argument in the Estate of Karen Silkwood v. Kerr-McGee, Inc., 9 CLINICAL L. REV. 229, 244-46 (2002). On the use of literary devices in judicial opinions, see Patricia M. Wald, The Rhetoric of Results and the Results of Rhetoric: Judicial Writings, 62 U. CHI. L. REV. 1371, 1386-90 (1995). 65. See infra notes 128-36 and accompanying text. explored in the context of property theory generally. See Carol M. Rose, Property as Storytelling: Perspectives from Game Theory, Narrative Theory, Feminist Theory, 2 YALE J.L. & HUMAN. 37, 38-39 (1990); Anupam Chander, The New, New Property, 81 TEX. L. REV. 715 (2003). 61. It might be argued that existing metaphorical efforts in cyberspace analysis (see supra notes 5- 19 and accompanying text) constitute narratives of their own. 62. See Philip N. Meyer, Making the Narrative Move: Observations Based Upon Reading Gerry Spence’s Closing Argument in the Estate of Karen Silkwood v. Kerr-McGee, Inc., 9 CLINICAL L. REV. 229, 244-46 (2002). On the use of literary devices in judicial opinions, see Patricia M. Wald, The Rhetoric of Results and the Results of Rhetoric: Judicial Writings, 62 U. CHI. L. REV. 1371, 1386-90 (1995). 63. The myth that the internet was designed to survive a nuclear war has been largely debunked, but it remains the case that much of the research that produced the computer networks that evolved into today’s internet was funded through the Department of Defense during the Cold War. See M. MITCHELL WALDROP, THE DREAM MACHINE: J.C.R. LICKLIDER AND THE REVOLUTION THAT MADE COMPUTING PERSONAL 259-332 (2001) 64. Any “cast of cyberspace” is inevitably incomplete. Some of the better known names include John Perry Barlow (cyberspace rhetoritician), Vinton Cerf and Bob Metcalfe (networking pioneers), the late Jon Postel (original keeper of the domain names registry), Marc Andreesen and Jim Clark (popularizers of the Netscape Navigator web browser) and Steve Case (founder of America Online). See Agre, The Next Instant Hero, supra note 49, at 61 (anticipating the emergence of the “public hacker” as the next hero of the grand narrative of the internet). 65. See infra notes 128-36 and accompanying text. 67. Efforts to cast internet policy debates as arguments over use of the “commons” (see supra notes 6-10) draw on property theories that echo the forms of Greek drama. See Carol Rose, The Comedy of the Commons: Custom, Commerce, and Inherently Public Property, 53 U. CHI. L. REV. 711 (1986); Michael A. Heller, The Tragedy of the Anticommons: Property in the Transition from Marx to Markets, 111 HARV. L. REV. 621 (1998). 70. See NUSSBAUM, supra note 43, at 6. For examples of legal scholarship framed by film, see Shubha Ghosh, The Merits of Ownership; Or, How I Learned to Stop Worrying and Love Intellectual Property, 15 HARV. J.L. & TECH. 453 (2002) (Dr. Strangelove); Ann Althouse, Invoking Rashomon, WIS. L REV. 503 (2000) (Rashomon); Steven L. Winter, Bull Durham and the Uses of Theory, 42 STAN. L. REV. 639 (1990) (Bull Durham); Robert B. Porter-Odawi, Two Kinds of Indians, Two Kinds of Indian Nation Sovereignty: A Surreply to Professor LaVelle, 11 KANSAS J. L. & PUB. POL’Y 629 (2002) (The Matrix). Cf. Aviam Soifer, Beyond Mirrors: Lawrence Friedman’s Moving Pictures, 22 LAW & SOC’Y REV. 995 (1988) (analogizing Professor Friedman to a one-man Warner Brothers motion picture studio); Jeffrey L. Harrison & Amy R. Mashburn, Jean-Luc Godard and Critical Legal Studies (Because We Need the Eggs), 87 MICH. L. REV. 1924 (1989) (comparing Critical Legal Studies to French New Wave cinema). Cf. United States v. Syufy Enterprises, 903 F.2d 659 (9th Cir. 1990) (Kozinski, J.) (opinion analyzing antitrust claims against motion picture theater operator that is rife with references to motion pictures); The Syufy Rosetta Stone, 1992 BYU. L. REV. 457 (decoding Judge Kozinski’s opinion). 72. Following Bogart’s death, a theater in Harvard Square began screening Casablanca, building a cult following for the film that eventually spread across the country. See Old Bogart Films Packing Them In, N.Y. TIMES, Jan. 28, 1965, at 19; About the Brattle Theatre, at http://www.brattlefilm.org/ timeline.html. The film’s place in popular culture was likely secured by 1971 with the release of PLAY IT AGAIN, SAM (Paramount Pictures 1972), starring Woody Allen as a neurotic single male haunted by the need to model the worldly cynicism of Bogart’s character in Casablanca. 66. See infra notes 92-94 and accompanying text. III. CUT TO: LEARNING FROM CASABLANCA Narrative theory might stop at this point and encourage scholars and other critics to examine cyberspace, its texts and other forms, for explicit and implicit narratives. There is not a single story of cyberspace, but multiple dynamically- structured stories of cyberspace.61 The best of them, the most useful stories for conceptualizing this phenomenon, emerge organically from critical understandings of the experience of cyberspace. Yet, while narrative at such an abstract level might be valuable critically, it is of relatively little use practically to the sitting judge or the practicing lawyer. The judge and the lawyer may value story-telling more for its role in advocacy than for its help in conceptualization. They care less for the philosophical or psychological justifications for narrative and more for its effectiveness. A good story turns the abstract and the confusing into the relatively structured and concrete.62 Pragmatically (and frankly, somewhat speculatively) speaking, I suggest not just that cyberspace is a story, but that it is some particular story. To press my argument to this level, I ask: if cyberspace is a story, then what story could it be? It is clearly a great one. It is timeless, yet of its time,63 both popular and elite, with international and domestic dimensions, compelling characters,64 multiple plots,65 cynicism and romance,66 comedy and drama67 and COLUMBIA JOURNAL OF LAW & THE ARTS [27:2 260 original68 and derivative69 elements alike. A classic novel might do here, but in contemporary culture, motion pictures provide the legal system’s preferred currency.70 No motion picture fits this bill better than Casablanca. Casablanca was released in late 1942 and was named Best Picture in 1943 by the Academy of Motion Picture Arts and Sciences.71 Its rise to iconic status began shortly after the premature death of its star, Humphrey Bogart, in 1957.72 Since then, it has remained one of a handful of films universally regarded as “classics.”73 67. Efforts to cast internet policy debates as arguments over use of the “commons” (see supra notes 6-10) draw on property theories that echo the forms of Greek drama. See Carol Rose, The Comedy of the Commons: Custom, Commerce, and Inherently Public Property, 53 U. CHI. L. REV. 711 (1986); Michael A. Heller, The Tragedy of the Anticommons: Property in the Transition from Marx to Markets, 111 HARV. L. REV. 621 (1998). 68. See Lawrence Lessig, The Law of the Horse: What Cyberlaw Might Teach, 113 HARV. 73. The film itself has been the subject of some legal analysis. Warner Brothers, which produced the motion picture, twice attempted to adapt the same material into a “prequel” television series. In its second attempt, the studio was sued by the authors of the original play. See Burnett v. Warner Bros. Pictures, 493 N.Y.S.2d 326 (App. Div. 1985) (affirming order granting summary judgment to the defendants on the ground that the plaintiffs had assigned to the defendants all rights in the work). Casablanca is the basis of an apocryphal copyright law legend involving the Marx Brothers. Groucho et al. were threatened by a humorless Warner Brothers executive over the rights to the name “Casablanca” while preparing the film, A Night in Casablanca. With characteristic wit, Groucho threatened a countersuit based on the Warners’ misuse of “Brothers,” and the matter was dropped. See SIVA VAIDHYANATHAN, COPYRIGHTS & COPYWRONGS: THE RISE OF INTELLECTUAL PROPERTY AND HOW IT III. CUT TO: LEARNING FROM CASABLANCA L REV. 501, 514-34 (1999). 69. See Frank H. Easterbrook, Cyberspace and the Law of the Horse, 1996 U. CHI. LEGAL F. 207, 08 69. See Frank H. Easterbrook, Cyberspace and the Law of the Horse, 1996 U. CHI. LEGAL F. 207 207-08. 70. See NUSSBAUM, supra note 43, at 6. For examples of legal scholarship framed by film, see Shubha Ghosh, The Merits of Ownership; Or, How I Learned to Stop Worrying and Love Intellectual Property, 15 HARV. J.L. & TECH. 453 (2002) (Dr. Strangelove); Ann Althouse, Invoking Rashomon, WIS. L REV. 503 (2000) (Rashomon); Steven L. Winter, Bull Durham and the Uses of Theory, 42 STAN. L. REV. 639 (1990) (Bull Durham); Robert B. Porter-Odawi, Two Kinds of Indians, Two Kinds of Indian Nation Sovereignty: A Surreply to Professor LaVelle, 11 KANSAS J. L. & PUB. POL’Y 629 (2002) (The Matrix). Cf. Aviam Soifer, Beyond Mirrors: Lawrence Friedman’s Moving Pictures, 22 LAW & SOC’Y REV. 995 (1988) (analogizing Professor Friedman to a one-man Warner Brothers motion picture studio); Jeffrey L. Harrison & Amy R. Mashburn, Jean-Luc Godard and Critical Legal Studies (Because We Need the Eggs), 87 MICH. L. REV. 1924 (1989) (comparing Critical Legal Studies to French New Wave cinema). Cf. United States v. Syufy Enterprises, 903 F.2d 659 (9th Cir. 1990) (Kozinski, J.) (opinion analyzing antitrust claims against motion picture theater operator that is rife with references to motion pictures); The Syufy Rosetta Stone, 1992 BYU. L. REV. 457 (decoding Judge Kozinski’s opinion). 71. See 75th Annual Academy Awards, at http://www.oscar.com/legacy/pastwin/picture6.html. The making and subsequent history of Casablanca are documented in ALJEAN HARMETZ, ROUND UP THE USUAL SUSPECTS: THE MAKING OF CASABLANCA—BOGART, BERGMAN, AND WORLD WAR II (1992); JEFF SPIEGEL, THE CASABLANCA COMPANION (1992); William Donnelly, Love and Death in Casablanca, in PERSISTENCE OF VISION: A COLLECTION OF FILM CRITICISM (Joseph McBride ed., 1968); CHARLES FRANCISCO, YOU MUST REMEMBER THIS (1980); CASABLANCA (Richard J. Anobile ed., 1974). 73. The film itself has been the subject of some legal analysis. Warner Brothers, which produced the motion picture, twice attempted to adapt the same material into a “prequel” television series. In its second attempt, the studio was sued by the authors of the original play. See Burnett v. Warner Bros. Pictures, 493 N.Y.S.2d 326 (App. Div. III. CUT TO: LEARNING FROM CASABLANCA 1985) (affirming order granting summary judgment to the defendants on the ground that the plaintiffs had assigned to the defendants all rights in the work). Casablanca is the basis of an apocryphal copyright law legend involving the Marx Brothers. Groucho et al. were threatened by a humorless Warner Brothers executive over the rights to the name “Casablanca” while preparing the film, A Night in Casablanca. With characteristic wit, Groucho threatened a countersuit based on the Warners’ misuse of “Brothers,” and the matter was dropped. See SIVA VAIDHYANATHAN, COPYRIGHTS & COPYWRONGS: THE RISE OF INTELLECTUAL PROPERTY AND HOW IT 2004] 261 THE NARRATIVES OF CYBERSPACE LAW Casablanca is pure character, plot and theme, a fact that makes it far better for my purpose than any film highly regarded more for cinematic techniques than for pure emotional appeal. More than fifty years have passed since its initial release, but no contemporary popular or critical audience can fail to understand Casablanca.74 p y p p The plot of Casablanca appears to be a simple one. An expatriate American, Rick Blaine (played by Bogart), is marooned in French Morocco during the early stages of World War II for reasons that are left obscure.75 While managing his nightclub (“Rick’s Café Americain”) and respecting the interests of the local French prefect (played by Claude Rains), Rick encounters an old flame recently arrived in Casablanca, Ilsa Lund (a Swede, played by Ingrid Bergman).76 Ilsa is accompanied by her Czech husband, Victor Laszlo (played by Paul Henreid), who happens to be the most famous and notorious Resistance fighter in Europe and who happens to have arrived in Casablanca one short step ahead of his Nazi pursuers. The Nazis (personified by Major Strasser, played by Conrad Veidt) track Laszlo to Rick’s, eventually forcing Rick to decide whether his love for Ilsa means reclaiming her for his own or letting her stay with husband Laszlo as Laszlo tries to escape to Lisbon and then to America. By the end of the movie, Rick appears to have decided to join Ilsa on board the Lisbon plane. At the last moment, Laszlo reappears, putting Rick to the film’s climactic choice.77 Of course, the love story between Rick and Ilsa is not the story of Casablanca at all. The movie opens with the murder of two German couriers carrying “letters of transit” that entitle their bearer to unquestioned passage out of Casablanca. THREATENS CREATIVITY 1-2 (2001) (citing Groucho Marx letter of 1944, published in MARX, THE GROUCHO LETTERS: LETTERS TO AND FROM GROUCHO MARX (1967)). Groucho’s famous reply appears to have been more publicity stunt than genuine outrage. The original concept for A Night in Casablanca was a parody of the original Casablanca, and Warner Brothers may have had legitimate grounds to object to more than mere misuse of the title. See A Night in Casablanca (2000), at http://www.snopes.com/movies/films/anightin.htm. 74. See Umberto Eco, Casablanca: Cult Movies and Intertextual Collage, 47 SUBSTANCE No. 2, at 3-12 (1985), reprinted in UMBERTO ECO, TRAVELS IN HYPER REALITY: ESSAYS 197-211 (1986). THREATENS CREATIVITY 1-2 (2001) (citing Groucho Marx letter of 1944, published in MARX, THE GROUCHO LETTERS: LETTERS TO AND FROM GROUCHO MARX (1967)). Groucho’s famous reply appears to have been more publicity stunt than genuine outrage. The original concept for A Night in Casablanca was a parody of the original Casablanca, and Warner Brothers may have had legitimate grounds to object to more than mere misuse of the title. See A Night in Casablanca (2000), at http://www.snopes.com/movies/films/anightin.htm. 74. See Umberto Eco, Casablanca: Cult Movies and Intertextual Collage, 47 SUBSTANCE No. 2, at 3-12 (1985), reprinted in UMBERTO ECO, TRAVELS IN HYPER REALITY: ESSAYS 197-211 (1986). 75. Rick was in Paris when the German army arrived to occupy that city. He escaped to Casablanca. Why he chose Casablanca is part of the man’s mystery: (Renault) I have often speculated on why you don’t return to America. Did you abscond with the church funds? Did you run off with a Senator’s wife? I like to think that you killed a man. It’s the romantic in me. (Rick) It was a combination of all three. (Renault) Then what in heaven’s name brought you to Casablanca? (Rick) My health. I came to Casablanca for the waters. (Renault) Waters? What waters? We’re in the desert. (Rick) I was misinformed. HOWARD KOCH, CASABLANCA: SCRIPT AND LEGEND 59 (3rd ed. 1973). 76. Rick, speaking to no one in particular, considers the likelihood that he would ever see Ilsa again: “Of all the gin joints in all the towns in all the world, she walks into mine!” Id. at 94. 77. On the tarmac, Rick tells Ilsa that she has to go with her husband: (Rick) Inside of us we both know you belong with Victor. You’re part of his work, the thing that keeps him going. If that plane leaves the ground and you’re not with him, you’ll regret it . . . . Maybe not today, maybe not tomorrow, but soon, and for the rest of your life. (Ilsa) But what about us? (Rick) We’ll always have Paris. We didn’t have it, we’d lost it until you came to Casablanca. We got it back last night. (Ilsa) And I said I would never leave you . . . . (Rick) And you never will. Id. at 175. 75. Rick was in Paris when the German army arrived to occupy that city. He escaped to Casablanca. Why he chose Casablanca is part of the man’s mystery: (Renault) I have often speculated on why you don’t return to America. Did you abscond with the church funds? Did you run off with a Senator’s wife? I like to think that you killed a man. It’s the romantic in me. (Rick) It was a combination of all three. (Renault) Then what in heaven’s name brought you to Casablanca? (Rick) My health. I came to Casablanca for the waters. (Renault) Waters? What waters? We’re in the desert. (Rick) I was misinformed. HOWARD KOCH, CASABLANCA: SCRIPT AND LEGEND 59 (3rd ed. 1973). 76. Rick, speaking to no one in particular, considers the likelihood that he would ever see Ilsa again: “Of all the gin joints in all the towns in all the world, she walks into mine!” Id. at 94. you never will. Id. at 175. III. CUT TO: LEARNING FROM CASABLANCA The prefect, Captain Renault, correctly traces both the letters and the murderer to 74. See Umberto Eco, Casablanca: Cult Movies and Intertextual Collage, 47 SUBSTANCE No. 2 at 3-12 (1985), reprinted in UMBERTO ECO, TRAVELS IN HYPER REALITY: ESSAYS 197-211 (1986). 75. Rick was in Paris when the German army arrived to occupy that city. He escaped to Casablanca. Why he chose Casablanca is part of the man’s mystery: (Renault) I have often speculated on why you don’t return to America. Did you abscond with the church funds? Did you run off with a Senator’s wife? I like to think that you killed a man. It’s the romantic in me. (Rick) It was a combination of all three. (Renault) Then what in heaven’s name brought you to Casablanca? (Rick) My health. I came to Casablanca for the waters. (Renault) Waters? What waters? We’re in the desert. (Rick) I was misinformed. HOWARD KOCH, CASABLANCA: SCRIPT AND LEGEND 59 (3rd ed. 1973). 76. Rick, speaking to no one in particular, considers the likelihood that he would ever see Ilsa again: “Of all the gin joints in all the towns in all the world, she walks into mine!” Id. at 94. g g j 77. On the tarmac, Rick tells Ilsa that she has to go with her husband: (Rick) Inside of us we both know you belong with Victor. You’re part of his work, the thing that keeps him going. If that plane leaves the ground and you’re not with him, you’ll regret it . . . . Maybe not today, maybe not tomorrow, but soon, and for the rest of your life. (Ilsa) But what about us? (Rick) We’ll always have Paris. We didn’t have it, we’d lost it until you came to Casablanca. We got it back last night. (Ilsa) And I said I would never leave you . . . . (Rick) And you never will. Id at 175 COLUMBIA JOURNAL OF LAW & THE ARTS [27:2 262 Rick’s, the center of the social and political networks that characterize the city. Quietly, Rick accepts the letters for safekeeping from his friend, broker to the local underworld and the presumed murderer Ugarte (played by Peter Lorre), but Rick refuses to shield Ugarte from arrest by Renault. 78. See KEN MOGG ET AL., THE ALFRED HITCHCOCK STORY 93 (1999). 79. Ugarte, running from French police inside the nightclub, encounters Rick: (Ugarte) Rick! Rick, help me! (Rick) Don’t be a fool. You can’t get away. (Ugarte) Rick hide me. Do something! You must help me, Rick. Do something! Rick! Rick! (Ugarte is wrestled away.) . . . . (Customer) When they come to get me, Rick, I hope you’ll be more of a help. (Rick) I stick my neck out for nobody. 81. Renault witnesses Rick shoot the German Strasser. French gendarmes runs up to Renault waiting for instructions: “(Renault) Major Strasser’s been shot. (Pause) Round up the usual suspects.” Id. at 181. KOCH, supra note 75, at 67-68. p 80. Renault gives the order to close Rick’s in response to a German request: (Renault) Everybody is to leave here immediately! This café is closed until further notice! Clear the room at once! (Rick) How can you close me up? On what grounds? (Renault) I am shocked, shocked to find that gambling is going on in here! (Croupier) You’re [sic] winnings, sir. (Renault) Oh. Thank you, very much. Everybody out at once! Id. at 145. 78. See KEN MOGG ET AL., THE ALFRED HITCHCOCK STORY 93 (1999). 79. Ugarte, running from French police inside the nightclub, encounters Rick: (Ugarte) Rick! Rick, help me! (Rick) Don’t be a fool. You can’t get away. (Ugarte) Rick hide me. Do something! You must help me, Rick. Do something! Rick! Rick! (Ugarte is wrestled away.) . . . . (Customer) When they come to get me, Rick, I hope you’ll be more of a help. (Rick) I stick my neck out for nobody. KOCH, supra note 75, at 67-68. 80. Renault gives the order to close Rick’s in response to a German request: (Renault) Everybody is to leave here immediately! This café is closed until further notice! Clear the room at once! (Rick) How can you close me up? On what grounds? (Renault) I am shocked, shocked to find that gambling is going on in here! (Croupier) You’re [sic] winnings, sir. (Renault) Oh. Thank you, very much. Everybody out at once! Id. at 145. 81. Renault witnesses Rick shoot the German Strasser. French gendarmes runs up to Renault waiting for instructions: “(Renault) Major Strasser’s been shot. (Pause) Round up the usual suspects. Id. at 181. 80. Renault gives the order to close Rick s in response to a German request: (Renault) Everybody is to leave here immediately! This café is closed until further notice! Clear the room at once! (Rick) How can you close me up? On what grounds? (Renault) I am shocked, shocked to find that gambling is going on in here! (Croupier) You’re [sic] winnings, sir. (Renault) Oh. Thank you, very much. Everybody out at once! 80. Renault gives the order to close Rick’s in response to a German request: 83. According to the script, “[T]he facades of the Moorish buildings [give way] to a narrow, twisting street crowded with the polyglot life of a native quarter. The intense desert sun holds the scene in a torpid tranquility. Activity is unhurried.” See KOCH, supra note 75, at 37, 182. I have to resist the temptation to metaphorically characterize cyberspace as Casablanca, the fictional place. Cyberspace-as- a-fictional-film-environment has been explored in STUART BIEGEL, BEYOND OUR CONTROL? CONFRONTING THE LIMITS OF OUR LEGAL SYSTEM IN THE AGE OF CYBERSPACE 12-18 (2001) (comparing cyberspace to the “idea of the West” as exemplified in Shane and other Western films), and HIGH NOON ON THE ELECTRONIC FRONTIER: CONCEPTUAL ISSUES IN CYBERSPACE (Peter Ludlow ed., 1996). ) 84. See HARMETZ, supra note 71, at 227-38 (describing the writing and shooting of the climactic airport scene). Eco describes the result as the film’s “glorious incoherence.” Eco, supra note 74, at 4. III. CUT TO: LEARNING FROM CASABLANCA The letters serve as what Alfred Hitchcock would later call the movie’s “macguffin,” the plot device around which all other elements of the story revolve but that means relatively little in itself.78 Ilsa and Laszlo desperately need the letters in order to escape from Casablanca. The Nazis desperately need the letters in order to prevent their escape. Captain Renault wants the letters in order to keep Laszlo in Casablanca, please the Nazis and keep his position. Rick professes neutrality in the face of their various schemes. The fate of the letters remains unclear until the very end of the film, when the Lisbon plane is about to depart and Rick must choose whether to use the letters for himself and Ilsa or to offer them to Ilsa and Laszlo. But the melodrama of the letters of transit is no more the real story of Casablanca than is its love story. Rick is an American who fought on the Loyalist (losing) side in the Spanish Civil War. He professes neutrality in the fight against the Nazis,79 but when the Nazis show up in Casablanca (and in Rick’s Café) in pursuit of Laszlo, his cynicism is challenged. Whether to reclaim Ilsa or to let her go and whether to keep the letters of transit or to give them to Ilsa and Laszlo are political questions. The film was produced in 1942, at a point when the German Army controlled much of North Africa and the outcome of the war was hardly assured. Casablanca is political propaganda. Is Rick an American fighting for truth and justice both at home and abroad, or will his cynicism and isolationism allow him to permit the Nazis to arrest Laszlo and crush the idealism that he represents? The corrupt Renault faces a similar choice. Early in the film Renault cooperates with the Nazis in investigating the murder of the German couriers by arresting Ugarte and closing Rick’s.80 Renault’s friendship helps to eventually persuade Rick to conspire in the murder of the Nazi Strasser81 that allows Laszlo and Ilsa, ultimately, to board the Lisbon plane together. Rick and Renault close the film by walking off together into the mist of the night, making plans to join the 81. Renault witnesses Rick shoot the German Strasser. French gendarmes runs up to Renault, waiting for instructions: “(Renault) Major Strasser’s been shot. (Pause) Round up the usual suspects.” Id. at 181. 85. Eco, supra note 74, at 6. 86. The musical theme of Casablanca is the popular tune, “As Time Goes By.” The lyrics include the lines: “It’s still the same old story, a fight for love and glory, a case of do or die.” See Herman Hupfeld, As Time Goes By (1931), available at http://www.cyberblanca.com/lyrics.html. 88. See Easterbrook, supra note 69. THE NARRATIVES OF CYBERSPACE LAW French Resistance.82 89. See Lessig, supra note 7. 87. Renault’s reference to rounding up “the usual suspects” both opens the film (in his conversation with Major Strasser, who wants to know what Renault is doing to investigate the murder of the couriers) and closes it (in his instructions to the gendarmes, who are meant to investigate the murder of Strasser himself). In each case, both the characters and the audience understand that the phrase alludes to nothing more than an empty gesture of authority. 82. The fog of the night envelops the airport runway: “(Rick, walking off with Renault) Louis, I think this is the beginning of a beautiful friendship.” Id. at 31. III. CUT TO: LEARNING FROM CASABLANCA 2004] 263 p ) 85. Eco, supra note 74, at 6. 82. The fog of the night envelops the airport runway: “(Rick, walking off with Renault) Louis, I think this is the beginning of a beautiful friendship.” Id. at 31. 83. According to the script, “[T]he facades of the Moorish buildings [give way] to a narrow, twisting street crowded with the polyglot life of a native quarter. The intense desert sun holds the scene in a torpid tranquility. Activity is unhurried.” See KOCH, supra note 75, at 37, 182. I have to resist the temptation to metaphorically characterize cyberspace as Casablanca, the fictional place. Cyberspace-as- a-fictional-film-environment has been explored in STUART BIEGEL, BEYOND OUR CONTROL? CONFRONTING THE LIMITS OF OUR LEGAL SYSTEM IN THE AGE OF CYBERSPACE 12-18 (2001) (comparing cyberspace to the “idea of the West” as exemplified in Shane and other Western films), and HIGH NOON ON THE ELECTRONIC FRONTIER: CONCEPTUAL ISSUES IN CYBERSPACE (Peter Ludlow ed., 1996). 84. See HARMETZ, supra note 71, at 227-38 (describing the writing and shooting of the climactic airport scene). Eco describes the result as the film’s “glorious incoherence.” Eco, supra note 74, at 4. 85. Eco, supra note 74, at 6. 86. The musical theme of Casablanca is the popular tune, “As Time Goes By.” The lyrics include the lines: “It’s still the same old story, a fight for love and glory, a case of do or die.” See Herman Hupfeld, As Time Goes By (1931), available at http://www.cyberblanca.com/lyrics.html. 87. Renault’s reference to rounding up “the usual suspects” both opens the film (in his conversation with Major Strasser, who wants to know what Renault is doing to investigate the murder of the couriers) and closes it (in his instructions to the gendarmes, who are meant to investigate the murder of Strasser himself). In each case, both the characters and the audience understand that the phrase alludes to nothing more than an empty gesture of authority. 88. See Easterbrook, supra note 69. 89. See Lessig, supra note 7. , p , 86. The musical theme of Casablanca is the popular tune, “As Time Goes By.” The lyrics include the lines: “It’s still the same old story, a fight for love and glory, a case of do or die.” See Herman Hupfeld, As Time Goes By (1931), available at http://www.cyberblanca.com/lyrics.html. 87. Renault’s reference to rounding up “the usual suspects” both opens the film (in his conversation with Major Strasser, who wants to know what Renault is doing to investigate the murder of the couriers) and closes it (in his instructions to the gendarmes, who are meant to investigate the murder of Strasser himself). In each case, both the characters and the audience understand that the phrase alludes to nothing more than an empty gesture of authority. 88 See Easterbrook supra note 69 90. As the film writer Aljean Harmetz writes: There are better movies than Casablanca, but no other movie better demonstrates America’s mythological vision of itself—tough on the outside and moral within, capable of sacrifice and romance without sacrificing the individualism that conquered a continent, sticking its neck out for everybody when circumstances demand heroism. No other movie has so reflected the moment when it was made—the early days of World War II—and the psychological needs of audiences decades later. IV. DISSOLVE TO: THE NARRATIVES OF CYBERSPACE LAW How then to map cyberspace to Casablanca, or Casablanca to cyberspace? It might or might not do as a matter of film criticism to see Casablanca as an eerie, early example of cyberspatial boundlessness.91 The numerous of doctrinal and policy problems involving the internet overlap with one another to a degree that it can be difficult to keep their plot lines and major interests distinct, even assuming that distinctions of this sort are feasible and desirable. But both interests and their development can be roughly organized along the same three plot lines that characterize the film. This is not mere entertainment. There is a method, which is both descriptive (showing how different interests in fact play multiple parts) and predictive (offering one, hopeful version of ongoing developments). French Resistance.82 Any one of these stories alone would make Casablanca an ordinary film. Indeed, the thing to know about Casablanca is that the film, piece by piece, is almost entirely unoriginal. Even the film’s description of its Moroccan setting is little more than a collage of Hollywood stereotypes.83 Yet the multiplicity of plots, the banality of the setting and the almost chaotic manner in which the film was written and shot clearly create something that is greater than the sum of its parts.84 In the words of the critic Umberto Eco: Forced to improvise a plot, the authors whipped up a little of everything, and everything they put in came from a repertoire that had stood the test of time. When only a few stock formulas are used, the result is simply kitsch. But when the repertoire of formulas is used wholesale, the result is an architecture like Gaudi’s Holy Family Church [Sagrada Familia]—the same vertigo, the same stroke of brilliance.85 In this sense—an entirely unoriginal collection of experiences that has a comprehensively transformative effect—Casablanca is the perfect story for (and of) cyberspace. Casablanca is a classic because it is still the same old story,86 told in a starkly original way.87 It acknowledges the critics who argue that there is nothing new here, just the same old themes.88 It accommodates the critics who argue that there is a connectedness to these older phenomena online that combines old themes in new ways.89 And its endurance over the last 60 years suggests that its power derives not merely from its origins but at least equally from the meaning projected onto it by generations of fans. Casablanca, as Eco implies, derives its 264 COLUMBIA JOURNAL OF LAW & THE ARTS [27:2 brilliance from its reception and interpretation as much as from its production.90 brilliance from its reception and interpretation as much as from its production.90 HARMETZ, supra note 71, at 6. 91. Eco evokes this timeless, placeless sense of the film: Casablanca has succeeded in becoming a cult movie because it is not one movie. It is “the movies.” And this is the reason it works, in spite of any aesthetic theory. For it stages the powers of Narrativity in its natural state, before art intervenes to tame it. Eco, supra note 74, at 10. 92. Rick despairs at Ilsa’s appearance, see supra note 77, but he cannot resist the pull of their relationship: (Rick, to Sam, the piano player) You know what I want to hear. (Sam) No, I don’t. (Rick) You played it for her and you can play it for me. (Sam) Well, I don’t think I can remember it. (Rick) If she can stand it, I can. Play it! (Sam) Yes, boss. (Sam starts to play “As Time Goes By.”). KOCH, supra note 75, at 94-95. HARMETZ, supra note 71, at 6. 91. Eco evokes this timeless, placeless sense of the film: Casablanca has succeeded in becoming a cult movie because it is not one movie. It is “the movies.” And this is the reason it works, in spite of any aesthetic theory. For it stages the powers of Narrativity in its natural state, before art intervenes to tame it. p y y p y ( ) ( ) If she can stand it, I can. Play it! (Sam) Yes, boss. (Sam starts to play “As Time Goes By.”). KOCH, supra note 75, at 94-95. Eco, supra note 74, at 10. (Rick, to Sam, the piano player) You know what I want to hear. (Sam) No, I don’t. (Rick) You played it for her and you can play it for me. (Sam) Well, I don’t think I can remember it. (Rick) p 92. Rick despairs at Ilsa’s appearance, see supra note 77, but he cannot resist the pull of their relationship: 97. See, e.g., Mark Cooper, Antitrust as Consumer Protection in the New Economy, 52 HASTINGS L.J. 813 (2001) (on behalf of the Consumer Federation of America, analyzing antitrust claims in United States v. Microsoft Corp., 87 F. Supp. 2d 30, 44 (D.D.C. 2000), as forms of consumer protection). Application of trademark law principles to internet commerce also could be considered a form of consumer protection, though actual application of trademark law online has tended to focus more on the interests of trademark holders than on the interests of consumers. See Mark A. Lemley, The Modern Lanham Act and the Death of Common Sense, 108 YALE L.J. 1687, 1701-03 (1999). A. A CYBERSPACE LOVE STORY Rick loves Ilsa. Ilsa loves Rick. In a flashback sequence in Casablanca, we learn that they had a love affair in pre-war Paris that was cut short unexpectedly as the Germans arrived, leaving Rick bitter and alone. Ilsa later appears with her husband, Victor Laszlo, in Rick’s Café, suggesting the rekindling of the romance92 that cannot flourish so long as Laszlo is with her and needs her. When the opportunity arises for one pair of lovers to leave Casablanca on the Lisbon plane, who will go? The romantic pair—Rick and Ilsa? Or the pragmatic pair—Laszlo and Ilsa? The story of cyberspace has been an equivalent tale of romance and pragmatism. When the internet first burst upon popular consciousness in the early 1990s, pop philosophers of the network proclaimed that it was definitively different and THE NARRATIVES OF CYBERSPACE LAW 2004] 265 special, uncolored by crass commercial interests and destined to remain ever so.93 This internet was to be free of corporate interests and commercial demands. It would be a platform for pure play, self-expression, innovation and creativity. To the extent that “real world” interests began to intrude, those interests were to be dealt with at a distance. The internet was special and different and demanded distinct treatment.94 Even today, the romantic vision of the internet persists, among other places, in appeals for preserving the “end to end” network architecture of the internet as supporting a “commons” for creativity and innovation and for ending the “digital divide” between internet-haves and internet-have nots, among other places. The internet is special. The internet is not, of course, all Rick and Ilsa. It is also Ilsa and Laszlo, representing the familiar, the pragmatic, even the commercial and the corporate. The internet could not remain insulated from the demands of the outside world for long. As the German army intruded on Rick’s romance with Ilsa, the business community intruded on the independent internet,95 building business models and demanding the regulation of domain name space, copyright and trademark use. Business was not the only demanding third party. Use of the internet by individuals and consumers created expectations regarding form and content that were not easily satisfied by a “pure” internet vision. f , , ( ) 98. See, e.g., A. Michael Froomkin, Wrong Turn in Cyberspace: Using ICANN to Route Around the APA and the Constitution, 50 DUKE L.J. 17, 105-65 (2000) (arguing that the U.S. Department of Commerce supported the creation of the Internet Corporation for Assigned Names and Numbers (ICANN) in order to address concerns over American control of the internet, but in doing so avoided having to confront legitimate questions about democracy and government control). 96. See Mark A. Lemley, The Law and Economics of Internet Norms, 73 CHI.-KENT L. REV 1257, 1281 (1998) (noting eventual failure of the On-Line Service Provider (OSP) “enclave” model as more and more subscribers turned to AOL for its Internet Service Provider (ISP) facilities). 95. The internet was opened to commercial traffic in 1995 when the National Science Foundation stopped supplying backbone services. As a result, the NSF’s Acceptable Use Policy, which forbade commercial traffic on its facilities, ceased to play a significant role in influencing internet activity. See Michael Kende, The Digital Handshake: Connecting Internet Backbones, 11 COMMLAW CONSPECTUS 45, 47-48 (2003); Jay P. Kesan & Rajiv C. Shah, Fool Us Once Shame on You—Fool Us Twice Shame on Us: What We Can Learn from the Privatization of the Internet Backbone Network and the Domain Name System, 79 WASH. U. L.Q. 89, 111-130 (2001). 93. See John Perry Barlow, A Declaration of the Independence of Cyberspace (February 8, 1996), at http://www.eff.org/~barlow/Declaration-Final.html. p g 94. See Johnson & Post, supra note 52, at 1370-76. 93. See John Perry Barlow, A Declaration of the Independence of Cyberspace (February 8, 1996) at http://www.eff.org/~barlow/Declaration-Final.html. (Annina) We come from Bulgaria. Oh, things are very bad there, M’sieur. A devil has the people by the throat. So, Jan and I, we, we do not want our children to grow up in such a country. (Rick) So you decided to go to America. (Annina) Yes, but we have not much money, and traveling is so expensive and difficult. It was much more than we thought to get here. And then Captain Renault sees us and he is so kind. He wants to help us. (Rick) Yes, I’ll bet. (Annina) He tells me he can give us an exit visa, but we have no money. (Rick) Does he know that? (Annina) Oh, yes. (Rick) And he is still willing to give you a visa? (Annina) Yes, M’sieur. (Rick) And you want to know . . . . (Annina) Will he keep his word? (Rick) He always has. COLUMBIA JOURNAL OF LAW & THE ARTS COLUMBIA JOURNAL OF LAW & THE ARTS With the forces of romantic openness confronting the forces of both business and consumer-driven pragmatism, how does this tale end? We cannot know yet, though we can discern some of the relevant players. Government, at international, national, and local levels, has an important (if sometimes self-interested) role to play, just as Renault’s corrupt involvement in Rick’s affairs was essential to resolution of the love affair in Laszlo’s favor. The government plays both sides of this game, however: Ilsa leaves with Laszlo, but Renault himself walks off to join the French Resistance in the end with Rick. Perhaps one should not write off government regulation of the internet altogether. There are also the intermediary interests, those who have no stake in the ultimate resolution of the story but who wish only to profit from transactions along the way. Ugarte, who traffics in the letters of transit, and Ferrari (played by Sidney Greenstreet), a competitor of Rick who buys Rick’s Café, represent intermediary institutions of all sorts, both commercial (internet service providers and online service providers) and non- commercial (the Internet Corporation for Assigned Names and Numbers, the World Intellectual Property Organization). Strasser, the German major, stands in, not for a player or plot device, but for a theme. He and the Germans are the antithesis of romance.99 Strasser has no interest in the assignment of Ilsa’s affections. He wants to both imprison Laszlo and close Rick’s Café, to exercise complete and perfect control over Casablanca. The romantic vision of the internet holds it as a perfectly open space. Strasser’s vision of the internet is one of perfect “end to end”100 control. KOCH, supra note 75, at 134-35. Rick frustrates Renault’s plan by fixing a round of roulette, enabling the Bulgarians to purchase their exit visas for cash and confirming Renault’s suspicions about the nobility beneath Rick’s cynical veneer. 100. Cf. Lemley & Lessig, supra note 50. 99. The literary irony in this stereotype is inescapable. The Romantic Movement, celebrating the creative imagination of the individual, originated in German (and French) literature. 101. One of the melodramatic subplots of Casablanca involves a young refugee couple from Bulgaria, trying to escape to America. The wife is prepared to sacrifice her honor to Renault in exchange for permission to leave Casablanca. She comes to Rick to find out whether Renault can be trusted: 99. The literary irony in this stereotype is inescapable. The Romantic Movement, celebrating the creative imagination of the individual, originated in German (and French) literature. 100. Cf. Lemley & Lessig, supra note 50. 101. One of the melodramatic subplots of Casablanca involves a young refugee couple from Bulgaria, trying to escape to America. The wife is prepared to sacrifice her honor to Renault in exchange for permission to leave Casablanca. She comes to Rick to find out whether Renault can be trusted: (Annina) We come from Bulgaria. Oh, things are very bad there, M’sieur. A devil has the people by the throat. So, Jan and I, we, we do not want our children to grow up in such a country. (Rick) So you decided to go to America. (Annina) Yes, but we have not much money, and traveling is so expensive and difficult. It was much more than we thought to get here. And then Captain Renault sees us and he is so kind. He wants to help us. (Rick) Yes, I’ll bet. (Annina) He tells me he can give us an exit visa, but we have no money. (Rick) Does he know that? (Annina) Oh, yes. (Rick) And he is still willing to give you a visa? (Annina) Yes, M’sieur. (Rick) And you want to know . . . . (Annina) Will he keep his word? (Rick) He always has. KOCH, supra note 75, at 134-35. Rick frustrates Renault’s plan by fixing a round of roulette, enabling the Bulgarians to purchase their exit visas for cash and confirming Renault’s suspicions about the nobility beneath Rick’s cynical veneer. A. A CYBERSPACE LOVE STORY The astonishing early growth of America Online was an accommodation of consumer as well as business interests, an enormous private enclave of “content” and regulation directly at odds with the original philosophy of the open internet.96 Consumer interests also brought demands for consumer protection,97 for relief from unwanted email (“spam”) and even for democracy and accountability in internet governance.98 [27:2 266 B. A CYBERSPACE MELODRAMA Will the Nazis capture Laszlo? Will Captain Renault help the Nazis, or will he remain loyal to his friend Rick? Will Rick help the Nazis, or will he remain loyal to his former love Ilsa? What will happen to the letters of transit? Will the Bulgarians get out of Casablanca?101 Melodrama is the popular dramatic form of THE NARRATIVES OF CYBERSPACE LAW 2004] 267 good and evil, winners and losers, heroes and villains. The initial conflict, the climax and the resolution count as much as, if not more than, any apparent lesson taught by the story. The audience pays to participate vicariously in the chase, the hunt, the betrayal and redemption. y p These are the questions posed by business and commerce on the internet, and they are also the questions of the distribution of entitlements online generally. The demise of transaction costs in the electronic age is now a truism.102 As transaction costs decrease, it matters less where initial entitlements are allocated and more that market participants can negotiate over their eventual distribution. We watch the evolution of the law of electronic contracting and online “access” as a multi- generational negotiation over the quantum of disclosure required of a firm before a contracting partner is bound by offered terms and conditions.103 The answer may not matter if firms and individuals can easily and effectively avoid being bound in any event. Expansive (or limited) intellectual property rights on the internet are of interest in this context primarily as the background for negotiation over purchasing those rights, whether it is firms that purchase them from consumers (in the form of more expansive self-help) or consumers who purchase them from firms (in the form of increased licensing and other fees, or the exercise of rights of fair use). There is essentially a fixed amount of wealth at stake. What we watch are the battles for wins and losses.104 The same may be said of arguments over internet taxation,105 jurisdiction,106 spam107 and even privacy, publicity and defamation.108 Entitlements regarding all of these things are the internet’s letters of transit. What matters, then, are not the characters themselves and the roles they play, but the rise and fall of transaction costs. We watch the narrative to see how they develop and to see how they can be overcome. 103. See Specht v. Netscape Communications Corp., 306 F.3d 17, 28-32 (2nd Cir. 2002) (holding online agreement to arbitrate invalid because firm failed sufficiently to disclose terms of agreement to individuals downloading software). 102. See I. Trotter Hardy, The Proper Legal Regime for “Cyberspace,” 55 U. PITT. L. REV. 993, 1033-36 (1994). 102. See I. Trotter Hardy, The Proper Legal Regime for “Cyberspace,” 55 U. PITT. L. REV. 993, 1033-36 (1994). 103. See Specht v. Netscape Communications Corp., 306 F.3d 17, 28-32 (2nd Cir. 2002) (holding online agreement to arbitrate invalid because firm failed sufficiently to disclose terms of agreement to individuals downloading software). 104. See supra note 86 (“As Time Goes By”). 105. See infra note 124 (Rick letting Renault win at roulette). 106. See supra note 76 (“Of all the gin joints in all the towns in all the world, she walks into mine!”). 107. The melodrama of Casablanca is set in motion by the arrival of unwanted mail—the letters of transit. 108. Rick is in Casablanca, hiding. Laszlo comes to Casablanca, flaunting his identity. Eventually, Rick is forced first to disclose his true identity and then to disappear (again), providing an interesting commentary on changing approaches to values of identity, reputation, and free speech. 105. See infra note 124 (Rick letting Renault win at roulette). 104. See supra note 86 (“As Time Goes By”). 108. Rick is in Casablanca, hiding. Laszlo comes to Casablanca, flaunting his identity. Eventually, Rick is forced first to disclose his true identity and then to disappear (again), providing an interesting commentary on changing approaches to values of identity, reputation, and free speech. 109. Some take this view of injunctions against file sharing services in the contest for copyright law entitlements. See Alfred C. Yen, A Preliminary Economic Analysis of Napster: Internet Technology, Copyright Liability, and the Possibility of Coasean Bargaining, 26 U. DAYTON L. REV. 247, 263-76 (2001). Similar arguments could be made with respect to regulation of online gambling and online pornography. B. A CYBERSPACE MELODRAMA Ugarte murders two German couriers and delivers the letters to Rick for safekeeping, intending to sell them to Laszlo. Ugarte is a facilitator and a highly cynical one. Renault, knowing that the sale will take place at Rick’s and wanting to keep Laszlo in Casablanca, makes a bet with Rick that Laszlo will not use the letters to escape. Having thus set the wheels of transaction costs in motion, Renault then arrests Ugarte, putting an end to the planned sale. Melodramatically, this merely sets the stage for a more involved contest for the letters. Metaphorically, the corrupt Renault has imposed a 108. Rick is in Casablanca, hiding. Laszlo comes to Casablanca, flaunting his identity Eventually, Rick is forced first to disclose his true identity and then to disappear (again), providing an interesting commentary on changing approaches to values of identity, reputation, and free speech. COLUMBIA JOURNAL OF LAW & THE ARTS [27:2 268 transaction cost barrier that Rick (or some competitor) is challenged to overcome. (One could characterize this as government intermeddling, attempting to raise the cost of some activity by forbidding it.)109 With the letters hidden (by Rick) inside Sam’s piano, Strasser and the Germans make a play for the letters, soliciting the French to turn Rick’s nightclub upside down.110 This is a different sort of government intermeddling, perhaps an act of creative destruction of the market altogether.111 In either case, the transaction cost barrier to market control is too high even for them.112 Rick feints with the letters, using his relationship with Ilsa to secure safe passage for the lovers to the airport with Renault, only to double- cross Renault and deliver the letters to Laszlo in the end (A deal will be struck; the only question in the online world is the price). Rick ends the drama by solving the transaction cost difficulty, killing Strasser and sending Ilsa to Lisbon with Laszlo.113 Renault, recognizing that he has been played, yields to both the story and to the market paradigm. He has lost his bet with Rick. His departure with Rick for the free French garrison is either an exit from the market (al)together, or a bet that in the next market, he will get his money back. 112. Again, this view of the Germans plays against type: “(Ferrari) (Rick’s competitor in the nightclub business, played by Sidney Greenstreet) (speaking to Laszlo) We might as well be frank, M’sieur. It will take a miracle to get you out of Casablanca. And the Germans have outlawed miracles.” KOCH, supra note 75, at 124. 110. The Germans rely on the French, one transaction cost layered on top of another: “(Renault Well, I told Strasser he wouldn’t find the letters here. But I told my men to be especially destructive You know how that impresses Germans.” KOCH, supra note 75, at 125. 113. Putting Ilsa and Laszlo on the plane to Lisbon is, in this odd sense, an efficient result. Had Rick boarded and left Laszlo behind, Laszlo, soon to be imprisoned, would represent a deadweight loss. 114. Its political bent is summed up in the following exchange: “(Ferrari) (responding to Rick’s rejection of Ferrari’s offer to go into business together on the black market) My dear Rick, when will you realize that, in this world today, isolationism is no longer a practical policy?” KOCH, supra note 75, at 51. p p 111. See JOSEPH A. SCHUMPETER, CAPITALISM, SOCIALISM AND DEMOCRACY 83-84 (3d ed 1950) (describing innovation as part of a process of “creative destruction” of a market leader’s position). 109. Some take this view of injunctions against file sharing services in the contest for copyright law entitlements. See Alfred C. Yen, A Preliminary Economic Analysis of Napster: Internet Technology, Copyright Liability, and the Possibility of Coasean Bargaining, 26 U. DAYTON L. REV. 247, 263-76 (2001). Similar arguments could be made with respect to regulation of online gambling and online pornography. 110. The Germans rely on the French, one transaction cost layered on top of another: “(Renault) Well, I told Strasser he wouldn’t find the letters here. But I told my men to be especially destructive. You know how that impresses Germans.” KOCH, supra note 75, at 125. 111. See JOSEPH A. SCHUMPETER, CAPITALISM, SOCIALISM AND DEMOCRACY 83-84 (3d ed. 1950) (describing innovation as part of a process of “creative destruction” of a market leader’s position). 112. Again, this view of the Germans plays against type: “(Ferrari) (Rick’s competitor in the nightclub business, played by Sidney Greenstreet) (speaking to Laszlo) We might as well be frank, M’sieur. It will take a miracle to get you out of Casablanca. And the Germans have outlawed miracles.” KOCH, supra note 75, at 124. 113. Putting Ilsa and Laszlo on the plane to Lisbon is, in this odd sense, an efficient result. Had Rick boarded and left Laszlo behind, Laszlo, soon to be imprisoned, would represent a deadweight loss. 114. Its political bent is summed up in the following exchange: “(Ferrari) (responding to Rick’s rejection of Ferrari’s offer to go into business together on the black market) My dear Rick, when will you realize that, in this world today, isolationism is no longer a practical policy?” KOCH, supra note 75, at 51. 117. See Graeme B. Dinwoodie, A New Copyright Order: Why National Courts Should Create Global Norms, 149 U. PA. L. REV. 469, 477-89 (2000); Joel R. Reidenberg, Yahoo and Democracy on the Internet, 42 JURIMETRICS J. 261, 275-78 (2002) (arguing that assertion of jurisdiction by French court over American internet firm can help promote democratic values); Berman, supra note 60, at 314- 317. Casablanca makes a strong case for American leadership in matters electronic: “(Strasser) You give him [Rick] credit for too much cleverness. My impression was that he’s just another blundering American. (Renault) But we mustn’t underestimate American blundering. I was with them when they blundered into Berlin in 1918.” KOCH, supra note 75, at 104. 122. It would stretch credulity to suggest that open source systems development is a sort o internet Resistance. But cf. Yochai Benkler, Coase’s Penguin, or, Linux and the Nature of the Firm, 112 YALE L.J. 369 (2002) (exploring peer production structures as alternatives to hierarchical firms). Bu 115. See BRUCE A. LEHMAN, WORKING GROUP ON INTELLECTUAL PROP. RIGHTS, INTELLECTUAL PROPERTY AND THE NATIONAL INFORMATION INFRASTRUCTURE (Info. Infrastructure Task Force, 1995) (colloquially known as the “White Paper”), available at http://www.uspto.gov/web/offices/ com/doc/ipnii; Pamela Samuelson, The U.S. Digital Agenda at WIPO, 37 VA. J. INT’L L. 369, 379-80 (1997) (arguing that the White Paper formed the basis for an agenda regarding intellectual property rights on the internet that the United States pushed internationally). 121. Such as cases in the United States and the European Union to enforce antitrust laws agains Microsoft. See United States v. Microsoft Corp., 253 F.3d 34 (D.C. Cir. 2001). C. THE POLITICS OF CYBERSPACE The melodramatic metaphor is extended perhaps too far, as melodrama tends to be. As in the film itself, it must be tempered not only by the romantic plot line, but by the political theme as well. Will Rick recover his noble American spirit and join the fight for freedom? Will he join with Laszlo, who rouses the patrons of Rick’s Café with an impromptu version of La Marseillaise (symbolizing a free France) in response to the Nazis’ Die Wacht am Rhein? Will he sacrifice his love of Ilsa for the greater good? The film is hardly subtle on this point,114 but there are several themes at work, both in film and on the internet: the role of individual responsibility in the context of an oppressively ordered corporate or statist culture; THE NARRATIVES OF CYBERSPACE LAW 2004] 269 the role of government and governance in the international order; and the role of both individual and group liberty. In the movie, Rick’s role evolves along with his feelings for Ilsa and the barriers that both Germans and French put in his way. On the internet, we are far from a resolution of these issues, but one can see the outlines of analogous conflicts. The Rick of the early part of the film is the United States in the early days of the internet, selfishly framing internet policy issues solely on its own terms.115 Or perhaps, Rick is the modern iconoclast internet hacker, with no need for traditional government or regulation.116 We see the emergence of both public and private governance mechanisms on the internet. The public is seen as national governments and public international organizations are now taking a more active role in defining the scope of internet regulation, and the United States is effectively required to participate (if reluctantly at times) in international regimes.117 Private governance systems are becoming more robust in the sense that formal private organizations have stepped up to address certain interests,118 in the sense that institutional interests operate effectively as governance structures119 and in the sense that private standard-setting bodies120 continue to exert a powerful influence on the underlying technical architecture of the internet. Overreaching at every level is at least theoretically cabined by national and supranational enforcement of unfair competition law121 and by norms and customs of online behavior and systems development.122 Rick is redeemed at the end of the film, giving up Ilsa and 115. 116. Questioned informally by the Nazis, Rick confirms his “hacker” status: “(Strasser) What is your nationality? (Rick) I’m a drunkard. (Laughter) (Renault) That makes Rick a citizen of the world.” KOCH, supra note 75, at 69. C. THE POLITICS OF CYBERSPACE See BRUCE A. LEHMAN, WORKING GROUP ON INTELLECTUAL PROP. RIGHTS, INTELLECTUAL PROPERTY AND THE NATIONAL INFORMATION INFRASTRUCTURE (Info. Infrastructure Task Force, 1995) (colloquially known as the “White Paper”), available at http://www.uspto.gov/web/offices/ com/doc/ipnii; Pamela Samuelson, The U.S. Digital Agenda at WIPO, 37 VA. J. INT’L L. 369, 379-80 (1997) (arguing that the White Paper formed the basis for an agenda regarding intellectual property rights on the internet that the United States pushed internationally). 116. Questioned informally by the Nazis, Rick confirms his “hacker” status: “(Strasser) What is your nationality? (Rick) I’m a drunkard. (Laughter) (Renault) That makes Rick a citizen of the world.” KOCH, supra note 75, at 69. 116. Questioned informally by the Nazis, Rick confirms his “hacker” status: “(Strasser) What is your nationality? (Rick) I’m a drunkard. (Laughter) (Renault) That makes Rick a citizen of the world.” KOCH, supra note 75, at 69. 117. See Graeme B. Dinwoodie, A New Copyright Order: Why National Courts Should Create Global Norms, 149 U. PA. L. REV. 469, 477-89 (2000); Joel R. Reidenberg, Yahoo and Democracy on the Internet, 42 JURIMETRICS J. 261, 275-78 (2002) (arguing that assertion of jurisdiction by French court over American internet firm can help promote democratic values); Berman, supra note 60, at 314- 317. Casablanca makes a strong case for American leadership in matters electronic: “(Strasser) You give him [Rick] credit for too much cleverness. My impression was that he’s just another blundering American. (Renault) But we mustn’t underestimate American blundering. I was with them when they blundered into Berlin in 1918.” KOCH, supra note 75, at 104. 118. The leading example here would be ICANN, the Internet Corporation for Assigned Names and Numbers. 119. Examples here would be Internet Service Providers (ISPs) and On-line Service Providers (OSPs). 120. Such as the World Wide Web Consortium (W3C) and the Internet Engineering Task Force (IETF). 121. Such as cases in the United States and the European Union to enforce antitrust laws against Microsoft. See United States v. Microsoft Corp., 253 F.3d 34 (D.C. Cir. 2001). p ( ) 122. It would stretch credulity to suggest that open source systems development is a sort of internet Resistance. But cf. Yochai Benkler, Coase’s Penguin, or, Linux and the Nature of the Firm, 112 YALE L.J. 369 (2002) (exploring peer production structures as alternatives to hierarchical firms). both on the screen and in the world, norm-based governance requires trust: “Ugarte (asking Rick to hold the letters of transit): You know, Rick, I have many friends in Casablanca, but somehow, just because you despise me you are the only one I trust.” KOCH, supra note 75, at 64. As we know, Ugarte’s trust is misplaced, but Rick recovers his own sense of trust by the end of the film. Cf. Benkler, Coase’s Penguin, supra at 400-23 (describing networked communities for distributed production, such as the open source software community, as norm-based alternatives to the hierarchy of the firm). 124. Rick’s success depends on Renault’s cooperation: (Renault) Rick there are many exit visas sold in this café, but we know that you’ve never sold one. That is the reason we permit you to remain open. (Rick) I thought it was because we let you win at roulette. (Renault) Er, that is another reason.” Id. at 62. 126. See Raymond Shih Ku, The Creative Destruction of Copyright: Napster and the New Economics of Digital Technology, 69 U. CHI. L. REV. 263, 300-22 (2002) (arguing that the economics of digital works of authorship suggest dramatic contraction of the scope of copyright protection for such works). ) 127. The internet can be “zoned.” Whether it should be “zoned” is another matter. See supra note 7 and accompanying text (discussing use of “zoning” and other real place analogies to analyze cyberspace law problems). 125. See Eugene Volokh, Cheap Speech and What It Will Do, 104 YALE L.J. 1805, 1833-43 (1995). KOCH, supra note 75, at 175. C. THE POLITICS OF CYBERSPACE But COLUMBIA JOURNAL OF LAW & THE ARTS [27:2 270 venturing off with Renault for the nearby free French garrison,123 implying metaphorically that individual liberty (Rick himself) and government regulation (Renault) can be reconciled and the public interest vindicated. Conflicts between liberty and community online are manageable once both sides recognize the greater good to be had from their cooperation. Individual liberty cannot be had without government;124 government cannot succeed without at least the symbolic consent of the governed. Strasser and the Germans become large rent-seeking institutions seeking to wrest governance of the resource from both the individual and represented public. Perhaps this version is unduly optimistic, since the internet is far too young to have witnessed anything approaching the kind of climax that Casablanca implies, let alone a resolution. A different use of the political plot line of Casablanca contrasts cyberspace as the domain of the perfectly autonomous individual with cyberspace as the foundation for, and embodiment of, novel forms of community. The individuated cyberspace supplies the conditions for political empowerment and “cheap speech” by citizens with little need for access to traditional publishing intermediaries.125 It allows individuals to wrest control of cultural products—both physical control and cultural control—from their corporate producers.126 In theory, it allows the privatization of behavior that in the physical world would have significant negative external effects.127 From a commercial perspective, cyberspace and its technologies enable “mass customization,” the holy grail of the price-discriminating merchant who wants to sell to every potential customer for no less than that customer is willing to pay. Rick is all of these things: sole master of his political, cultural and commercial 123. Rick’s parting words to Ilsa are classic film dialogue: (Rick) But I’ve got a job to do too. Where I’m going you can’t follow. What I’ve got to do you can’t be any part of. Ilsa, I’m no good at being noble, but it doesn’t take much to see that the problems of three little people don’t amount to a hill of beans in this crazy world. Someday you’ll understand that. Not now. Here’s looking at you, kid. 125. See Eugene Volokh, Cheap Speech and What It Will Do, 104 YALE L.J. 1805, 1833-43 (1995). 126. See Raymond Shih Ku, The Creative Destruction of Copyright: Napster and the New Economics of Digital Technology, 69 U. CHI. L. REV. 123. Rick’s parting words to Ilsa are classic film dialogue: (Rick) But I’ve got a job to do too. Where I’m going you can’t follow. What I’ve got to do you can’t be any part of. Ilsa, I’m no good at being noble, but it doesn’t take much to see that the problems of three little people don’t amount to a hill of beans in this crazy world. Someday you’ll understand that. Not now. Here’s looking at you, kid. C. THE POLITICS OF CYBERSPACE 263, 300-22 (2002) (arguing that the economics of digital works of authorship suggest dramatic contraction of the scope of copyright protection for such works). 127. The internet can be “zoned.” Whether it should be “zoned” is another matter. See supra note 7 and accompanying text (discussing use of “zoning” and other real place analogies to analyze cyberspace law problems). 2004] THE NARRATIVES OF CYBERSPACE LAW 271 worlds, opening himself and his Café to all but a few, selectively closing off some because of history (Ilsa) or politics (a German banker who wishes to gamble in the back room). But individualism comes at a cost. The community inevitably makes demands on the individual, and the individual ultimately recognizes the value of the community. Free speech may cause harm. Control of the means (and ends) of cultural production creates responsibility for re-creating the cultural environment. Openness and security must be balanced. Privatization, even of “personal” activity, cannot isolate the individual forever. The Germans arrive. Their singing Die Wacht am Rhein in a display of German nationalism in the Café triggers a reaction (La Marseillaise, sung by the rest of the Café patrons), sanctioned by Rick. Accepting Ilsa back into his life, even briefly, leads Rick directly back to the community that he escaped earlier in his life. The ability to “customize” his commercial activity produces benefits, in that Rick can rig his roulette wheel to allow a refugee couple to win the cash they need to buy their way out of Casablanca (though to do so, he needs information about one member of the couple that many people would regard as private). It also imposes costs, since Rick can stay open only by allowing Renault access to the same illegality. I leave to the critic or the lawyer the task of compiling a useful synthesis of these interwoven stories. I have substituted as much of my imagination for theirs as may be fair to do. Whatever the outcome of the various plots in cyberspace, and whatever their overall synthesis turns out to be, Casablanca makes clear that no one story can be resolved without interweaving it with the others. Cyberspace is not merely romance or commerce. It is both, with a significant dose of political philosophy thrown in. 128. 123 S. Ct. 2297 (2003). 129. See American Library Ass’n, Inc. v. U.S., 201 F. Supp. 2d 401, 409-411 (E.D. Pa. 2002). V. DISSOLVE TO: USING THE NARRATIVES A comprehensive and useful synthesis of the multiple plots and themes of cyberspace may be beyond the scope of my imagination, but a more limited synthesis is accessible, and it serves as an illustration not only of how this narrative approach may be useful in law, but also of how it is already (at least implicitly) part of the relevant legal discourse. I take as my reference the recent case of United States v. American Library Association, in which the Supreme Court considered the constitutionality of part of the federal Children’s Internet Protection Act.128 The relevant portions of the statute conditioned local libraries’ receipt of federal funds to support internet access on those libraries’ installation of computer software that blocked patron access to obscene or pornographic images on websites. A district court enjoined enforcement of the statute, before it took effect, on the ground that libraries’ use of such filtering software would necessarily violate the First Amendment as a content-based restriction on access to a public forum.129 Accordingly, conditioning receipt of federal internet access funds on use of the software violated Congress’s authority under the Spending Clause. The decision [27:2 272 COLUMBIA JOURNAL OF LAW & THE ARTS was appealed directly to the Supreme Court. The Court reversed by a vote of 6-3. The Court majority comprised three opinions in all. Two dissents were filed. The details of the justices’ doctrinal analyses aside, most interesting for my purposes is that each of the opinions, save one,130 relied heavily on metaphorical and mostly narrative constructions of libraries, the internet, and behaviors within them. Chief Justice Rehnquist, writing for a plurality consisting of himself and Justices O’Connor, Scalia and Thomas, rejected the premise that access to the internet via a public library constitutes any sort of “public forum”: A public library does not acquire internet terminals in order to create a public forum for Web publishers to express themselves, any more than it collects books in order to provide a public forum for the authors of books to speak. It provides internet access . . . for the same reasons it offers other library resources: to facilitate research, learning, and recreational pursuits by furnishing materials of requisite and appropriate quality. . . . As Congress recognized, ‘[t]he internet is simply another method for making information available in a school or library.’ S. Rep. No. 106-141, p. 7 (1999). 130. Justice Kennedy’s concurrence suggested that an as-applied challenge to the statute, rather than a facial challenge, might better illustrate the extent to which library filtering interfered with legitimate adult interests in access to Constitutionally-protected material. See American Library, 123 S. Ct. at 2309-10 (Kennedy, J., concurring). 131. Id. at 2305. 132. Id. at 2311 (Breyer, J., concurring). 133. Id. at 2315 (Stevens, J., dissenting): Until a blocked site or group of sites is unblocked, a patron is unlikely to know what is being hidden and therefore whether there is any point in asking for the filter to be removed. It is as though the statute required a significant part of every library’s reading materials to be kept in unmarked, locked rooms or cabinets, which could be opened only in response to specific requests. Some curious readers would in time obtain access to the hidden materials, but many would not. Inevitably, the interest of the authors of those works in reaching the widest possible audience would be abridged. 130. Justice Kennedy’s concurrence suggested that an as-applied challenge to the statute, rather than a facial challenge, might better illustrate the extent to which library filtering interfered with legitimate adult interests in access to Constitutionally-protected material. See American Library, 123 S. Ct. at 2309-10 (Kennedy, J., concurring). 131. Id. at 2305. V. DISSOLVE TO: USING THE NARRATIVES It is ‘no more than a technological extension of the book stack.’131 Justice Breyer’s concurring opinion similarly characterized the library’s provision of internet access in narrative terms, though the opinion emphasized not the patron’s process of “research, learning, and recreation pursuits” but instead the librarian’s process of constructing the collection. His opinion viewed furnishing filtered internet access to websites as an extension of “the discretion necessary to create, maintain, or select a library’s ‘collection’ (broadly defined to include all the information the library makes available).”132 Justice Stevens, dissenting, took yet a third view of the narrative, explaining the steps that an adult patron would have to follow in order to request “unblocking” of a filtered site and concluding that the burden represented by those steps amounted to a significant (and unconstitutional) prior restraint on access to protected speech.133 The multiplicity of stories made possible by the intersection of libraries and cyberspace was most fully and interestingly explored, however, in the dissenting opinion of Justice Souter joined by Justice Ginsburg. Each of the three themes and Id. THE NARRATIVES OF CYBERSPACE LAW 2004] 273 plots derived above for Casablanca and cyberspace are brought to bear on his conclusion that the statute was properly enjoined as unconstitutional. The dissent first considered the possibility that a library’s decision to block or unblock a particular website is the legal equivalent of the library’s decision to purchase a particular book. This is the melodrama of the book, which the opinion revealed as a Hitchcockian “macguffin,” important not in itself but because of what it reveals about other interests: “Whereas traditional scarcity of money and space require a library to make choices about what to acquire . . . blocking is the subject of a choice made after the money for internet access has been spent or committed.”134 The dissent’s next step, from dismissing the “scarcity” rationale to characterizing internet filtering as “censorship,” was to move from melodrama to romance. The opinion invoked the historic role of American public libraries and “freedom from censorship” as a defining characteristic of libraries’ institutional mission: “Institutional history of public libraries in America discloses an evolution toward a general rule, now firmly rooted, that any adult entitled to use the library has access to any of its holdings.”135 Failure to extend that principle to cyberspace would effectively, but impermissibly, export public censorship. V. DISSOLVE TO: USING THE NARRATIVES One thread remained, which is the political story, and the opinion used it to conclude its argument: “Quite simply, we can smell a rat when a library blocks material already in its control, just as we do when a library removes books from its shelves for reasons having nothing to do with wear and tear, obsolescence, or lack of demand. Content-based blocking and removal tell us something that mere absence from the shelves does not.”136 Though not ultimately persuasive to a majority of the Court, the opinion demonstrates the utility for legal analysis of a complex interweaving of narratives, of cyberspace and, in this case, of libraries and literature. 134. Id. at 2321 (Souter, J., dissenting). 135. Id. at 2322 (Souter, J., dissenting). 136. Id. at 2324 (Souter, J., dissenting). 137. See supra notes 21-60 and accompanying text. 134. Id. at 2321 (Souter, J., dissenting). 135. Id. at 2322 (Souter, J., dissenting). 136. Id. at 2324 (Souter, J., dissenting). 137. See supra notes 21-60 and accompanying text. VI. CUT TO: CONCLUSION In laying out my case for narrative understandings of cyberspace law, I have consciously integrated, to some degree, two distinct perspectives. On the one hand, and in arguing above that the very structure of cyberspace supplies the boundedness that permits its construction as a narrative, I offer an essentially internal, structural perspective. Cyberspace consists of an inherent but essentially novel story, defined dynamically by the limits imposed by computer software and hardware.137 Cyberspace, at this level, is inherently both technical and metaphorical. Cyberspace is the metaphor that describes the many technologies that comprise “the internet,” and as a metaphor it is better understood as a narrative than as a “thing” in how it describes connections between and relationships among individuals, entities, concepts and technologies. On the other hand, and in offering the stories, characters and themes of Casablanca as a valuable example of a COLUMBIA JOURNAL OF LAW & THE ARTS 274 [27:2 narratival reading of cyberspace, I offer the external perspective of the reader, creating and interpreting “cyberspace” by mediating online experience via the patterns supplied by the classic plots, characters and themes combined in the film.138 My argument is certainly open to critique on the ground that I have offered both views and not distinguished or chosen clearly between them. g y I confess that I have not done so, but I suggest that such a clean distinction is not, in the end, feasible. The interpretive exercise that constitutes the “external” understanding of cyberspace-as-narrative is essential to the construct that supplies an “internal” understanding of cyberspace-as-narrative. The second perspective defines the conditions that make different features of the second perspective relevant; in effect, we construct the story of cyberspace via the stories through which we understand cyberspace. Here is the recursive character of cyberspace to which I referred above.139 The romantic story of cyberspace, the search for “openness” at different technical levels of the internet, leads to legal rules promoting (or discouraging) openness and ultimately to technologies that reinforce that openness. The melodrama of cyberspace, the search for markets implicit in the technology of the internet, leads to rules reinforcing (or disrupting) markets and eventually to technologies that do likewise. The political story of cyberspace, the conflict between public and private interests embedded in the technology, leads to rules that reify (or reconstruct) those interests, and then to technologies that confirm the rules. 138. See supra notes 61-127 and accompanying text. 139. See supra note 56 and accompanying text. See Richard J. Ross, Communications Revolutions and the Legal Culture: An Elusive Relationship, 27 LAW & SOC. INQUIRY 638, 648-49 (2002) (discussing Nazareth A.M. Pantaloni, III, Legal Databases, Legal Epistemology, and the Legal Order, 86 LAW LIB. J. 679 (1994)). 140. See MITCHELL, supra note 5. 141. See Eco, supra note 74, at 11: When all the archetypes burst in shamelessly, we reach Homeric depths. Two cliches make us laugh, but a hundred cliches move us because we sense dimly that the cliches are talking among themselves and celebrating a reunion. Just as extreme pain meets sensual pleasure, and extreme perversion borders on mystical energy, so does extreme banality allow us to catch a glimpse of the sublime. Nobody would have been able to achieve such a result intentionally. Nature has spoken here in place of men. 142. Not only is Casablanca a classic of Western film, it is also a classic of a now (mostly) outmoded cultural ideology that celebrates the reserved, but heroic, lone white male as the master (author) of his fate. See Robin West, The Literary Lawyer, 27 PAC. L.J. 1187, 1196-98, 1206-11 (1996) (noting the critique that the literary canon is exclusionary, elitist and “textual”). Then again, maybe little of that matters, at least here, and at least with this source. See Peter Jaszi, Who Cares Who Wrote “Shakespeare”?, 37 AM. U. L. REV. 617, 622-24 (1988) (noting that the value of Shakespeare for contemporary society lies in the openness of the texts to interpretation). VI. CUT TO: CONCLUSION The narrative metaphor is itself recursive. At the end of Casablanca, Rick Blaine himself walks away from Casablanca to join the Resistance, to become, in effect, a Victor Laszlo to replace the Laszlo now flying to America. The unproduced sequel to Casablanca is a version of Casablanca itself. The apparent implausibility of adopting the Casablanca narrative is thus more than merely rebutted by the equal implausibility of some, if not all, of narrative’s metaphorical predecessors. Narrative is clearly superior, in at least two key respects. In the end cyberspace, like any great and enduring story, owes its resonance to the ongoing emotional and intellectual investments of its interpreters as much as to the efforts of its creators. An enduring metaphor of cyberspace needs to account, as I have done indirectly, for the blurring of object and subject, audience and participant, and player and viewer, which characterizes the medium.140 A metaphor of place adopts the static distinctions of “place,” distinctions between the place and the person and between one place and another. A great story invites new reconstructions. It welcomes that blurring, and it welcomes change. The liberty I have taken in characterizing “cyberspace” as Casablanca is a liberty granted by both the story and the medium. My deconstructing and reconstructing different plots and attributes is an implicit argument that this is a permissible use of the film, that Casablanca belongs as THE NARRATIVES OF CYBERSPACE LAW 2004] 275 much to me as it does to Bogart, or to its director, Michael Curtiz. Rick and Ilsa matter not. They are archetypes, in which generations of film-goers have recognized universal experience.141 Cyberspace, for its part, “belongs” in a sense to me, as well as to all others who build it and use it. That point should be emphasized and extended. Cyberspace is defined not only by millions of individual experiences, but also by the experiences of social networks and of institutions. Wireless networks are “part” of the internet and, therefore, of cyberspace not merely because protocols permit the interchange of appropriate digitized information. They are part of cyberspace, because some significant population of users has elected to integrate wireless networking technology into their lives. How cyberspace is defined for regulatory purposes depends as much on these understandings as it depends on the understandings of computer technologists. Each depends on the other. VI. CUT TO: CONCLUSION Other films, other works, and other stories may, in the end, serve as well as or better than Casablanca in accounting for law in cyberspace.142 Any other story of cyberspace needs to have the flexibility to accommodate the many demands of its subject and its overlapping themes and plots. My use of Casablanca itself shows that cyberspace is no simple story, and there are few stories with massive popular appeal that capture the multiplicity represented in both this film and in cyberspace. But the moral of the story (of this Article) is this: the computers of the internet are out there, but we construct cyberspace. For that reason, and celluloid aside, the notion of cyberspace-as-narrative deserves an extended stay in the pantheon of metaphorical conceptions of cyberspace. Physicalized metaphors are appealing for now, and they are embedded in our language. They may not endure as technology and experience evolve. Even if they do endure, however, they may prove more limiting than illuminating. That risk exists with any metaphor. A narrative framework, at least, contains in itself a design for adaptation and evolution. It may eventually provide a better understanding of how to approach the legal and policy questions posed by this new technology.
https://openalex.org/W4243783942	https://www.research-collection.ethz.ch/bitstream/20.500.11850/578204/3/1-s2.0-S0165489622000749-main.pdf	English	null	Risk Allocation and Financial Intermediation	Social Science Research Network	2,021	cc-by	8,480	ETH Library ETH Library 1. Introduction reduced set of nonspecific securities, while the idiosyncratic risk components are insured through financial intermediation. Reach- ing Pareto optimality does not rely on a Law of Large Numbers approximation thanks to an original characterization of idiosyn- cratic and aggregate risks. The role played by financial intermedi- ation is complementary to the role played by security derivatives or dynamic trading. While frameworks without financial inter- mediation require a set of securities that distinguishes between all the states of nature, the present framework only requires a set of securities that distinguishes between states of nature with different aggregate endowments. In other words, in the present framework the securities rely on much less information by only making public aggregate endowments in the economy. Moreover, the additional insurance contracts between agents and financial intermediaries are also parsimonious as each contract depends on the state of the agent insured and on aggregate endowments, but not on the states of other agents (i.e., not on the true state of nature). In a real world economy exposed to risks, it is well known that the classic framework of Arrow (1964) and Debreu (1959) requires an unrealistic number of specific contingent markets or securities to reach Pareto optimality. Indeed, the Arrow–Debreu framework implies one security per state of nature, which is huge in practice. Moreover, the Arrow–Debreu securities require a large quantity of information since their implementation publicly reveals the true state of nature, in other words the state of each agent in the economy. These issues have led to the development of a branch of literature that attempts to improve the realism of these securities while maintaining Pareto optimality. This lit- erature has focused on how a reduced set of securities may be completed by security derivatives and how trading at different points in time rather than only once may reduce the need for securities (e.g., see Duffie, 2010 for an overview). However, these frameworks are demanding in terms of securities features since the reduced set of securities still needs to distinguish between all the states of nature. The present paper takes a different path by studying the role that can be played by financial intermediaries (i.e., entities acting between two parties). A simple example highlights the main results. a r t i c l e i n f o Article history: Received 10 January 2022 Received in revised form 19 July 2022 Accepted 25 September 2022 Available online 5 October 2022 The classic Arrow–Debreu framework requires a very large number of specific securities to reach Pareto optimality. The present paper shows that financial intermediation can play an important role in maintaining a more parsimonious market framework while still obtaining Pareto optimality. In the framework developed, the aggregate risk components of individual risks are exchanged through a highly reduced set of nonspecific securities, while the idiosyncratic risk components are insured through financial intermediation. Reaching Pareto optimality does not rest on a Law of Large Numbers approximation. The role of financial intermediation is complementary to the role of security derivatives and dynamic trading. Keywords: Risk allocation General equilibrium Securities Insurance Financial intermediation Market completeness © 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). E-mail address: agoussebaile@ethz.ch. Risk allocation and financial intermediation Journal Article Author(s): Goussebaile, Arnaud Publication date: 2022-11 Permanent link: https://doi.org/10.3929/ethz-b-000578204 Rights / license: Creative Commons Attribution 4.0 International Originally published in: Mathematical Social Sciences 120, https://doi.org/10.1016/j.mathsocsci.2022.09.004 This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. Rights / license: Originally published in: g y p Mathematical Social Sciences 120, https://doi.org/10.1016/j.mathsocsci.2022.09.004 This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. Mathematical Social Sciences 120 (2022) 78–84 p // g/ /j 0165-4896/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). E-mail address: agoussebaile@ethz.ch. ing. Financial intermediation thus plays a complementary role to security markets. https://doi.org/10.1016/j.mathsocsci.2022.09.004 0165-4896/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0 https://doi.org/10.1016/j.mathsocsci.2022.09.004 0165-4896/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Risk allocation and financial intermediation Arnaud Goussebaïle ETH Zürich, Scheuchzerstrasse 7, 8092 Zürich, Switzerland ://doi.org/10.1016/j.mathsocsci.2022.09.004 -4896/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativ 2. Setting and terminology While insurance contracts depend on individual states and the number of iden- tical agents affected in the framework of the latter two papers, they depend on individual states and aggregate endowments in my framework. More specifically, they simply constitute an indemnity equal to the realized individual loss minus the average individual loss in the realized aggregate endowment state. All in all, the present framework is more parsimonious than the one of Cass et al. (1996) and Malinvaud (1973). Definition 2. The extensive social state z = 1, . . . , Z is the subset of Ωcharacterized by {ω ∈Ω, ˜e = e(z)}. The set of extensive social states forms a partition of Ω. Since it is a refinement of the set of individual states of any agent i, I can define for any i the function si(.) : {1, . . . , Z} →{1, . . . , Si} which associates to each extensive social state the corresponding individual state of agent i. The number of extensive social states depends on the number of agents, the number of individual states for each agent, and the risk dependence between agents, which is not specified in the model. In particular, the number of extensive social states in the economy reaches Z = ∏N i=1 Si (e.g., Z = SN with identical Si) if each combination of individual states has a strictly positive probability of occurring. I denote π(z) = ∑ {ω∈Ω,˜e=e(z)} πΩ(ω) the strictly positive probability of obtaining the extensive social state z, which implies ∑Z z=1 π(z) = 1. Different states of nature corresponding to the same extensive social state may be considered as the same state of nature since there is no way to distinguish between them. Thus, an extensive social state may be assimilated to a state of nature. The paper also provides an insight into market incomplete- ness and financial intermediation that is complementary to other branches of the literature. A large body of literature has studied the ways in which market incompleteness for idiosyncratic risks affects the economy at a macroeconomic level (e.g., see Heath- cote et al., 2009 for a literature review). In the present paper, I also assume market incompleteness for idiosyncratic risks. Rather than focusing on its consequences, I analyze how markets may be completed in a simple way by financial intermediaries who are able to observe these risks. 2. Setting and terminology I consider a risky static exchange economy with C commodi- ties (c = 1, . . . , C) and N heterogeneous agents (i = 1, . . . , N). The set of states of nature is denoted Ωand the probability measure on Ωis denoted πΩ. I assume that all the agents agree on the probability measure. Individual state. Each agent i = 1, . . . , N is assumed to have an uncertain individual commodity endowment, denoted ˜ei = (˜ei,1, . . . , ˜ei,C),1 with outcomes in the space of individual commodity endowments RC +. I assume that ˜ei has a finite number, denoted Si, of outcomes, denoted (ei(1), . . . , ei(Si)). Definition 1. The individual state si = 1, . . . , Si of agent i is the subset of Ωcharacterized by {ω ∈Ω, ˜ei = ei(si)}. The set of individual states of agent i forms a partition of Ω. Extensive social state. An extensive social endowment is de- fined by a complete specification of the individual commodity endowments obtained by all the agents in the economy. Thus, ˜e = ( ˜e1, . . . , ˜eN) is the uncertain extensive social endowment with outcomes in the space of extensive social endowments RC·N + . Given that ˜ei has a finite number of outcomes, ˜e has a finite number, denoted Z, of outcomes, denoted (e(1), . . . , e(Z)). q The paper builds on Cass et al. (1996) and Malinvaud (1973), who analyze the role of insurance contracts in completing markets. These authors consider insurance contracts between identical agents for identical risks, while I model financial inter- mediaries competing to supply insurance contracts to heteroge- neous agents for heterogeneous risks. My main result shows that financial intermediaries can play a much larger role in the optimal allocation of risks than the insurance contracts considered in Cass et al. (1996) and Malinvaud (1973). In these papers, contingent markets are still required to reallocate all the idiosyncratic risks that are either heterogeneous or borne by heterogeneous agents. In the present framework, this is not the case because financial intermediation eliminates any idiosyncratic risks (regardless of the heterogeneity across risks and across agents). Moreover, insurance contracts are no more complex in my framework than those in Cass et al. (1996) and Malinvaud (1973). 1 Vectors are denoted in bold in this paper. In mathematical formulas, the product of two vectors corresponds to the scalar product. A. Goussebaïle Mathematical Social Sciences 120 (2022) 78–84 In the general setting, I consider a static exchange economy characterized by multiple commodities and heterogeneous risk- averse agents facing individual endowment risks. I introduce an original dichotomy of individual endowment risks: in contrast with standard definitions, my definitions of the idiosyncratic and aggregate risk components of individual risks do not rely on the Law of Large Numbers. An aggregate endowment state is defined as a set of all the states of nature featuring identical aggregate endowments (such as states 1 and 2 in the example). The idiosyncratic risks are the parts of individual risks within each aggregate endowment state, while the aggregate risks are the parts of individual risks across aggregate endowment states. I assume the existence of one contingent market per aggre- gate endowment state. Although agents can exchange aggregate risks through these markets, they cannot hedge idiosyncratic risks. In addition, I consider financial intermediaries competing to supply insurance contracts for idiosyncratic risks. Assuming that insurance contracts cannot be sold below actuarially fair prices, I show that at least one Pareto optimal equilibrium exists and any equilibrium has the following features. Financial inter- mediaries supply insurance contracts at actuarially fair prices and agents purchase full coverage for their idiosyncratic risks. Financial intermediaries do not default in any states of nature thanks to the original risk dichotomy that does not rest on the Law of Large Numbers. In addition to purchasing full cover- age for their idiosyncratic risks, agents exchange aggregate risks and commodities through contingent markets. Finally, the com- modity consumption levels are identical to those in a classic Arrow–Debreu equilibrium. market incompleteness for idiosyncratic risks can be seen as the consequence of transaction costs, which prevent agents from contracting with each other on these risks. Rather than focusing on how transaction costs lead to the emergence of the financial intermediaries who insure these risks, I analyze to what extent the latter complete the markets when they incur transaction costs small enough to be ignored. The paper is organized as follows. Section 2 presents the setting and some terminology. Section 3 studies the decentralized economy with equilibrium allocations. Section 4 concludes. 1. Introduction In a static econ- omy with multiple agents and multiple states of nature, assume that agent A is endowed with WA in state 1 and WA −L in state 2, while agent B is endowed with WB −L in state 1 and WB in state 2. Assume also that all other agents have the same endowment in states 1 and 2. These two states are thus very similar and there might be no security that gives two strictly different pay-offs in these two states. In this case, agents A and B cannot hedge their risk across states 1 and 2, even with security derivatives, and dynamic trading is irrelevant in this static economy. A financial intermediary may insure these risks and eliminate them by pool- ing. Financial intermediation thus plays a complementary role to security markets. The paper shows that financial intermediation can play an im- portant role in maintaining a parsimonious market framework. In an economy where heterogeneous agents are exposed to endow- ment risks, I demonstrate that Pareto optimality is reached with a simple market framework featuring financial intermediaries and a dichotomy of individual endowment risks. The aggregate risk components of individual risks are exchanged through a highly 2. Setting and terminology t 1 2 3 4 5 6 7 E 0 1 2 3 4 5 6 Ft 1 2,3 4,5,9 6,7,10,11 8,12,13 14,15 16 Gt {1} {2,3} {4},{5,9} {6,7},{10,11} {8,12},{13} {14,15} {16} Π 1 16 2 16 3 16 4 16 3 16 2 16 1 16 e1 0 1/2 1/3 1/2 2/3 1/2 1 e2 0 1/2 1/3 1/2 2/3 1/2 1 e3 0 0 2/3 1 4/3 2 2 e4 0 0 2/3 1 4/3 2 2 Definition 3. The aggregate endowment state t = 1, . . . , T is the subset of Ωcharacterized by {ω ∈Ω, ˜E = E(t)}. Definition 3. The aggregate endowment state t = 1, . . . , T is the subset of Ωcharacterized by {ω ∈Ω, ˜E = E(t)}. denote by Gt the set of collective states included in the aggregate endowment state t. Example. To highlight the differences between extensive so- cial states, aggregate endowment states, and collective states, I consider an economy with C = 1 commodity and N = 4 agents. Agents 1 and 2 are identical and face 2 individual states with an endowment in {0, 1}. Agents 3 and 4 are identical and face 2 individual states with an endowment in {0, 2}. I assume that the economy has Z = 16 states of nature or extensive social states, each one with a 1/16 probability of occurring (Table 1), which corresponds to independence between individual endow- ment risks and a 1/2 probability of being in each individual state, for each agent. Extensive social states can be arranged into T = 7 aggregate endowment states (Table 2). On the other hand, following the approach of Cass et al. (1996) and Malinvaud (1973), extensive social states can be arranged into T ′ = 10 collective states (fourth line in Table 2). The set of aggregate endowment states forms a partition of Ω. Since the set of extensive social states is a refinement of the set of aggregate endowment states, I can define the func- tion t(.) : {1, . . . , Z} →{1, . . . , T} which associates to each extensive social state the corresponding aggregate endowment state. The number of aggregate endowment states also depends on the number of agents, the number of individual states for each agent, and the risk dependence between agents. 2. Setting and terminology I denote the probability of obtaining the aggregate endowment state t by Π(t) and the conditional probability of obtaining the extensive social state z, given the occurrence of aggregate endowment state t, by π(z\|t). I denote the set of extensive social states included in the aggregate endowment state t by Ft, i.e. Ft = {z ∈ {1, . . . , Z}, ∑N i=1 ei(si(z)) = E(t)}. These notations imply that ∑T t=1 #Ft = Z, Π(t) = ∑ z∈Ft π(z), ∑T t=1 Π(t) = 1, and π(z\|t) = π(z)/Π(t) for any z ∈Ft, and π(z\|t) = 0 for any z /∈Ft. When ei(t) denotes the average endowment of agent i in the aggregate endowment state t, then ei(t) = ∑ z∈Ft π(z\|t)ei(si(z)). Moreover, ∑N i=1 ei(si(z)) = E(t) for all z ∈Ft and ∑N i=1 ei(t) = E(t). Risk dichotomy. An individual endowment risk can be de- composed into an idiosyncratic risk component and an aggregate risk component. The idiosyncratic risk is defined as the part of the risk within each aggregate endowment state. The aggregate risk is defined as the remaining part of the risk. Mathematically, the different parts of the individual risk of agent i are such that: Literature. My setting is related to the ones of Cass et al. (1996) and Malinvaud (1973). They also assume that all the agents agree on the probability measure. Definition 1 is a formal definition of an ‘‘individual state’’, which corresponds to the non- formal definition of an ‘‘individual state’’ in Cass et al. (1996) and Malinvaud (1973). Definition 2 is a formal definition of an ‘‘extensive social state’’, which corresponds to the non-formal definition of an ‘‘extensive social state’’ in Cass et al. (1996) and the non-formal definition of a ‘‘social state’’ in Malinvaud (1973). Besides, the latter two papers build a partition of Ωthat differs from the set of aggregate endowment states. Indeed, they build subsets of Ωcalled ‘‘collective states’’ or ‘‘aggregate social states’’ (which I refer to as ‘‘collective states’’ in the rest of the paper to distinguish it from ‘‘aggregate endowment states’’), in which states of nature are similar to individual endowment switches between identical agents. The set of collective states is a refine- ment of the set of aggregate endowment states, since the states of nature that are similar to individual endowment switches between identical agents necessarily have the same aggregate endowments. 2. Setting and terminology Another large body of literature has studied the role of financial intermediation in the presence of information asymmetries or transaction costs (e.g., see Gorton and Winton, 2003 for a literature review). In the present paper, Aggregate endowment state. An aggregate endowment is defined by the sum of the individual commodity endowments of all the agents in the economy. Thus, ˜E = ∑N i=1 ˜ei is the uncertain aggregate endowment with outcomes in the space of aggregate endowments RC +. Given that ˜ei has a finite number of outcomes, ˜E has a finite number, denoted T, of outcomes, denoted (E(1), . . . , E(T)). 79 Mathematical Social Sciences 120 (2022) 78–84 A. Goussebaïle Table 1 The table shows the probability of occurrence π and the endowment ei of each agent i for each extensive social state z. z 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 π 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 e1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 e2 0 0 1 1 0 0 1 1 0 0 1 1 0 0 1 1 e3 0 0 0 0 2 2 2 2 0 0 0 0 2 2 2 2 e4 0 0 0 0 0 0 0 0 2 2 2 2 2 2 2 2 Table 2 The table shows the aggregate endowment E, the set Ft of extensive social states, the set Gt of collective states, the probability of occurrence Π, and the average endowment ei of each agent i for each aggregate endowment state t. t 1 2 3 4 5 6 7 E 0 1 2 3 4 5 6 Ft 1 2,3 4,5,9 6,7,10,11 8,12,13 14,15 16 Gt {1} {2,3} {4},{5,9} {6,7},{10,11} {8,12},{13} {14,15} {16} Π 1 16 2 16 3 16 4 16 3 16 2 16 1 16 e1 0 1/2 1/3 1/2 2/3 1/2 1 e2 0 1/2 1/3 1/2 2/3 1/2 1 e3 0 0 2/3 1 4/3 2 2 e4 0 0 2/3 1 4/3 2 2 Definition 3. The aggregate endowment state t = 1, . . . 2. Setting and terminology , T is the subset of Ωcharacterized by {ω ∈Ω, ˜E = E(t)}. denote by Gt the set of collective states included in the aggreg endowment state t. Table 1 The table shows the probability of occurrence π and the endowment ei of each agent i for each extensive social state z. z 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 π 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 1 16 e1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 e2 0 0 1 1 0 0 1 1 0 0 1 1 0 0 1 1 e3 0 0 0 0 2 2 2 2 0 0 0 0 2 2 2 2 e4 0 0 0 0 0 0 0 0 2 2 2 2 2 2 2 2 Table 2 The table shows the aggregate endowment E, the set Ft of extensive social states, the set Gt of collective states, the probability of occurrence Π, and the average endowment ei of each agent i for each aggregate endowment state t. t 1 2 3 4 5 6 7 E 0 1 2 3 4 5 6 Ft 1 2,3 4,5,9 6,7,10,11 8,12,13 14,15 16 Gt {1} {2,3} {4},{5,9} {6,7},{10,11} {8,12},{13} {14,15} {16} Π 1 16 2 16 3 16 4 16 3 16 2 16 1 16 e1 0 1/2 1/3 1/2 2/3 1/2 1 e2 0 1/2 1/3 1/2 2/3 1/2 1 e3 0 0 2/3 1 4/3 2 2 e4 0 0 2/3 1 4/3 2 2 Definition 3. The aggregate endowment state t = 1, . . . , T is h b f Ω h i d b { Ω˜E E( )} denote by Gt the set of collective states included in the aggregate d Table 2 The table shows the aggregate endowment E, the set Ft of extensive social states, the set Gt of collective states, the probability of occurrence Π, and the average endowment ei of each agent i for each aggregate endowment state t. 3. Decentralized economy In the classic Arrow–Debreu framework, agents can hedge all their individual risks with the C · Z contingent markets. In the framework of Cass et al. (1996) and Malinvaud (1973), in- surance contracts between identical agents for identical risks are considered to deal with the individual risks within each collective state, which cannot be covered with the C ·T ′ contingent markets. In the present framework, I assume the existence of M compet- ing, profit-maximizer financial intermediaries (k = 1, . . . , M) (i.e., M is large enough to have perfect competition, meaning that financial intermediaries are price-takers). I make the following assumptions for these financial intermediaries. They agree on the probability measure πΩ. They observe individual risks and supply insurance contracts for idiosyncratic risks with no transaction costs. They do not participate in contingent markets and do not default in any state of nature. (5) Market equilibrium. An allocation (xi(t), δi, ϵki, p(t), λi) with t ∈ {1, . . . , T}, i ∈ {1, . . . , N}, and k ∈ {1, . . . , M} is an equilibrium if it simultaneously satisfies the utility maximization problem (2) for each agent i ∈{1, . . . , N}, the profit maximization problem (3) for each financial intermediary k ∈{1, . . . , M}, the contingent market’s clearing condition (4) for each aggregate endowment state t ∈{1, . . . , T}, and the insurance market’s clearing condition (5) for each agent i ∈{1, . . . , N}.3 For the following propositions, I assume that aggregate endowments are strictly positive for any aggregate endowment state (i.e., E(t) ≥0 for any t ∈{1, . . . , T}). Proposition 1 (Equilibrium Existence). An equilibrium with δi = 1, ϵki = 1/M, and λi = 0 for any i ∈{1, . . . , N} and k ∈ {1, . . . , M} exists. In this equilibrium the allocation is Pareto optimal and identical to an equilibrium allocation of the classic Arrow– Debreu framework in terms of contingent commodity consumptions. Insurance markets. 2 The contingent indemnity ei(t(z)) −ei(si(z)) may be either positive or negative. Note that, by defining emax i as a certain endowment vector such that emax i −ei(si(z)) ≥0 for any z, it is possible to see the contingent indemnity as a positive contingent indemnity emax i −ei(si(z)) minus an ex post positive premium emax i −ei(t(z)) contingent on the aggregate endowment state. 3. Decentralized economy I assume the existence of a reduced number of competitive contingent markets and financial intermediaries competing to supply insurance contracts for idiosyncratic risks. I analyze the equilibrium in this decentralized economy. max xi,δi Z ∑ z=1 π(z)vi ( ei(si(z)) + xi(t(z)) + ( ei(t(z)) −ei(si(z))) δi ) s.t. T ∑ t=1 p(t)xi(t) + λiδi ≤0. (2) (2) s.t. ∑ t=1 p(t)xi(t) + λiδi ≤0. Contingent markets. I assume the existence of C · T com- petitive contingent markets, one per commodity and aggregate endowment state. In the classic Arrow–Debreu framework, there are C · Z competitive contingent markets, one market per com- modity and extensive social state. In the framework of Cass et al. (1996) and Malinvaud (1973), there are C · T ′ competitive contingent markets, one market per commodity and collective state. The number of contingent markets in the latter framework is necessarily larger than in the present framework since the number of collective states is larger than the number of aggregate endowment states. In the present framework, purchasing one commodity unit contingent on the aggregate endowment state t gives one unit in all the extensive social states z corresponding to t (i.e., z ∈Ft), in exchange for an ex ante payment corresponding to the contingent commodity unit price. The price vector for the C commodities contingent on the aggregate endowment state t is denoted by p(t). The quantity of commodities contingent on the aggregate endowment state t purchased by agent i is denoted by xi(t) (which might be either positive or negative, representing a purchase or a sale, respectively). Each financial intermediary k chooses the fraction ϵki of insur- ance supplied to each agent i in order to maximize profit under contingent solvency constraints: max ϵki N ∑ i=1 λiϵki s.t. N ∑ i=1 ( ei(t(z)) −ei(si(z))) ϵki ≤0, ∀z ∈{1, . . . , Z}. (3) (3) Market clearing. The clearing condition for the commodity market contingent on the aggregate endowment state t is: N ∑ i=1 xi(t) = 0. (4) N ∑ i=1 xi(t) = 0. (4) The clearing condition for the insurance market associated to agent i is: δi = M ∑ k=1 ϵki. (5) δi = M ∑ k=1 ϵki. Financial intermediaries. To deal with idiosyncratic risks, which cannot be covered with the C ·T contingent markets, I con- sider financial intermediaries supplying insurance contracts for these risks. 3 At any equilibrium, p ≥ 0, because agents are never satiated with commodities. On the other hand, it is not clear that λi ≥0, as agents become satiated with risk coverage. 2. Setting and terminology Thus, the number of collective states, denoted T ′, is larger than the number of aggregate endowment states T. I also ei(si(z)) = ei(si(z)) −ei(t(z))    idiosyncratic risk + ei(t(z)) − T ∑ t=1 Π(t)ei(t)    aggregate risk + T ∑ t=1 Π(t)ei(t). (1) ei(si(z)) = ei(si(z)) −ei(t(z))    idiosyncratic risk + ei(t(z)) − T ∑ t=1 Π(t)ei(t)    aggregate risk + T ∑ t=1 Π(t)ei(t). (1) idiosyncratic risk idiosyncratic risk (1) aggregate risk Preferences. I assume that agents have preferences satisfying the von Neumann–Morgenstern axioms. I denote by ∑Z z=1 π(z)vi (.) the expected utility function of agent i, where vi : RC + →R is the utility index function of agent i. For instance, if there are no exchanges between agents, the consumption plan of agent i is ei(si(z)) in the extensive social state z and her expected utility is ∑Z z=1 π(z)vi(ei(si(z))). The utility index function vi is assumed to be twice continuously differentiable. Agents are assumed to be strictly non-satiated (i.e., vi strictly increasing in each argument) and strictly risk averse (i.e., vi strictly concave with a negative definite Hessian matrix). 80 A. Goussebaïle Mathematical Social Sciences 120 (2022) 78–84 and the purchased fraction δi of insurance in order to maximize expected utility under budget constraint: and the purchased fraction δi of insurance in order to maximize expected utility under budget constraint: 4. Conclusion Parsimony of the framework. In the present paper, Pareto optimality is obtained with C ·T markets contingent on aggregate endowments plus N insurance markets in which financial inter- mediaries compete to supply insurance contracts for idiosyncratic risks to the N agents. In the classic Arrow–Debreu framework, Pareto optimality is obtained with C · Z markets contingent on the extensive social states. In the framework of Cass et al. (1996) and Malinvaud (1973), Pareto optimality is obtained with C · T ′ markets contingent on the collective states plus insurance con- tracts between identical agents for identical risks. Note that in each case, the C ·X contingent markets could be replaced by X se- curities plus C markets. The framework developed in the present paper is more parsimonious than previous frameworks developed in the literature. The Arrow–Debreu framework or other frame- works with security derivatives are demanding in terms of the number of securities required or in terms of their features, since they make public the true extensive social state (i.e., the state of each agent in the economy). The framework of Cass et al. (1996) and Malinvaud (1973) can also be demanding since it makes public the true collective state, which is identical to the true extensive social state when agents are fully heterogeneous for instance. The framework developed in the present paper is less demanding. The contingent markets or securities require less information as they only make public aggregate endowments in the economy. Moreover, an insurance contract between an agent and a financial intermediary depends on the state of the agent and on aggregate endowments, but not on the states of other agents (i.e., not on the true extensive social state). More specifically, it simply constitutes a null premium ex ante and an indemnity ex post equal to the realized individual loss minus the average individual loss in the realized aggregate endowment state (which might be positive or negative). The reallocation of risks across agents in an economy can provide significant welfare gains. However, this reallocation may require a highly demanding market framework in terms of the number of contingent markets and their specificity. The present paper shows that financial intermediation can play an impor- tant role in maintaining a parsimonious market framework. 3. Decentralized economy In this context, there is no difference between the framework of Arrow (1964) and Debreu (1959) and the one of Cass et al. (1996) and Malinvaud (1973). In both of these cases, the number of contingent markets is 100 and there are no complementary insurance contracts. To be effective, the 100 markets require to make public the realized extensive social state, in other words the loss incurred specifically by each agent in the economy. By contrast, in the present framework the number of contingent markets is 1 and the number of insurance markets is 10 (each one associated to a specific agent). Given that the expected loss for one agent is 10, the full insurance contract is such that the agent pays a null premium ex ante and receives an indemnity ex post equal to her loss minus 10, which does not depend on the loss incurred by any other specific agent in the economy. insurance markets are also balanced. Finally, with λi = 0, δi = 1, and ϵki = 1/M, the equilibrium problem is simplified and is similar to a standard Arrow–Debreu economy with T extensive social states and T contingent markets. It is well known that an equilibrium allocation for such a problem exists and that it is Pareto optimal (Chapter 17 of Mas-Colell et al., 1995), which concludes the proof. Proposition 2 (Equilibrium Uniqueness/Multiplicity). Assume that insurance contracts cannot be sold below actuarially fair prices (i.e., λi ≥0 for any i ∈{1, . . . , N}). In any equilibrium, δi = 1 and λi = 0 for any i ∈{1, . . . , N}, and the allocation is identical to an equilibrium allocation of the classic Arrow–Debreu framework in terms of contingent commodity consumptions. Proof. If insurance contracts cannot be sold below actuarially fair prices (i.e., λi ≥0 for any i ∈{1, . . . , N}), the price of each insurance contract will necessarily be null (i.e., λi = 0 for any i ∈{1, . . . , N}). Otherwise, the profit maximization problem (3) would lead financial intermediaries to sell an infinite quantity of insurance contracts and the insurance markets’ clearing condi- tions (5) would not be satisfied. 3. Decentralized economy A full insurance contract for the idiosyn- cratic risks of agent i comprises a contingent indemnity (ei(t(z))− ei(si(z))) in any extensive social state z in exchange for an ex ante premium λi.2 Note that an insurance contract for an agent does not depend on the individual states of other agents but on her individual state si and on the aggregate endowment state. The fraction of insurance supplied by financial intermediary k to agent i is denoted ϵki. The fraction of insurance purchased by agent i from the whole population of financial intermediaries is denoted δi. Proof. The formal proof of Proposition 1 is detailed in Appendix. When λi = 0, agents maximize their utility by purchasing full insurance δi = 1 for idiosyncratic risks, since it is supplied at an actuarially fair price. The commodities of any agent after covering idiosyncratic risks are thus identical across extensive social states corresponding to the same aggregate endowment state, which corresponds to the mutuality principle proposed in Borch (1962). Moreover, when λi = 0, financial intermediaries maximize their profit regardless of their insurance supply ϵki. When ϵki = 1/M, the solvency constraints of financial intermedi- aries are met thanks to maximal diversification. There is no need for a Law of Large Numbers approximation. When ϵki = 1/M, Choices. Each agent i chooses the purchased quantity xi(t) of commodities contingent on the aggregate endowment state t 81 Mathematical Social Sciences 120 (2022) 78–84 A. Goussebaïle a risk of loss. In any state of nature, the aggregate loss is 100, which means that the number of aggregate endowment states is T = 1. In the different states of nature, the loss incurred by one of the agents is assumed to be either 19, 28, 37, 46, 55, 64, 73, 82, 91, or 100, while all other agents incur the same individual loss equal to either 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0, respectively. The former agent (i.e., the one incurring the larger loss) can be any of the 10 agents, which means that the number of extensive social states is Z = 100. Since the agents are fully heterogeneous, it also means that the number of collective states is T ′ = 100. Each extensive social state is assumed to have a probability 0.01 of occurrence. 4. Conclusion In an economy where heterogeneous agents are exposed to en- dowment risks, I demonstrate that Pareto optimality is reached with a simple market framework featuring financial interme- diaries and a dichotomy of individual risks. The idiosyncratic risk components of individual risks are insured through finan- cial intermediation, while the aggregate risk components are exchanged through a highly reduced set of nonspecific securities. The set of securities only needs to distinguish between states of nature with different aggregate endowments. Conversely, stan- dard frameworks without financial intermediation rely on a set of securities that must distinguish between all the states of nature. This paper thus emphasizes that the role played by financial intermediation is complementary to the role played by security markets, including derivatives or dynamic trading. 3. Decentralized economy With null prices for insurance contracts, the first order condition of the utility maximization problem (2) relative to δi implies that agents purchase full insur- ance for idiosyncratic risks (i.e., δi = 1 for any i ∈{1, . . . , N}). The market equilibrium problem then simplifies to a standard Arrow– Debreu economy with T extensive social states and T contingent markets, which concludes the proof. Appendix. Proof of Proposition 1 (18) (18) T ∑ t=1 Π(t)∇vi ( xi(t) + ei(t)) · (∑ z∈Ft π(z\|t)( ei(t(z)) −ei(si(z)))) = 0, T ∑ t=1 Π In a Pareto optimal allocation, the consumption plan ePO(z) and the vector ρ(z)/π(z) are identical across extensive social states z corresponding to the same aggregate endowment state t. This is the classic mutuality principle presented by Borch (1962), which is proved as follows. Problem (16) implies that the weighted sum of utility of all the agents in each extensive social state has to be maximized under its own extensive social state feasibility constraint. As all the extensive social states z corresponding to the same aggregate endowment state t have the same feasibility constraint, they have the same consumption plan ePO(z), which also implies with (17) that they have the same vector ρ(z)/π(z). With χi = ψi and ρ(z) = π(z)p(t(z))/Π(t(z)), (13) and (15) correspond respectively to (17) and (18) for any i and z, which means that the equilibrium allocation(s) with the features defined in the first part of the proof is (are) Pareto optimal. In a Pareto optimal allocation, the consumption plan ePO(z) and the vector ρ(z)/π(z) are identical across extensive social states z corresponding to the same aggregate endowment state t. This is the classic mutuality principle presented by Borch (1962), which is proved as follows. Problem (16) implies that the weighted sum of utility of all the agents in each extensive social state has to be maximized under its own extensive social state feasibility constraint. As all the extensive social states z corresponding to the same aggregate endowment state t have the same feasibility constraint, they have the same consumption plan ePO(z), which also implies with (17) that they have the same vector ρ(z)/π(z). With χi = ψi and ρ(z) = π(z)p(t(z))/Π(t(z)), (13) and (15) correspond respectively to (17) and (18) for any i and z, which means that the equilibrium allocation(s) with the features defined in the first part of the proof is (are) Pareto optimal. (10) which is verified, given the definition of ei(t(z)). which is verified, given the definition of ei(t(z)). To finish the first part of the proof, the final step is to show that p(t), xi(t), and φi exist with t ∈{1, . . . , T} and i ∈{1, . . . , N} such that (4), (7), and (8) are verified. Appendix. Proof of Proposition 1 Appendix. Proof of Proposition 1 et al. (1995), an equilibrium allocation exists for this problem, given that agents are strictly non-satiated and strictly risk averse, and that aggregate endowments are strictly positive. This con- cludes the proof that at least one equilibrium allocation exists with the features defined above. First part of the proof. I show that an equilibrium allocation exists such that δi = 1, ϵki = 1/M, and λi = 0 for any i ∈ {1, . . . , N} and k ∈{1, . . . , M}. Suppose an allocation with these features. When λi = 0, problem (2) of agent i simplifies to: Note that the equilibrium allocation(s) with the features de- fined above has (have) commodity allocations that, for any agent, are identical across extensive social states z corresponding to the same aggregate endowment state t and such that (11) and (12) are satisfied. Moreover, x and p solve (11) and (12) if and only if x, p, and ψ solve: max xi,δi Z ∑ z=1 π(z)vi ( ei(si(z)) + xi(t(z)) + ( ei(t(z)) −ei(si(z))) δi ) s.t. T ∑ t=1 p(t)xi(t) ≤0. (6 max xi δi Z ∑ π(z)vi ( ei(si(z)) + xi(t(z)) + ( ei(t(z)) −ei(si(z))) δi ) (6) s.t. ∑ t=1 p(t)xi(t) ≤0. s.t. ∑ 1 p(t)xi(t) ≤0. ψi∇vi ( ei(t) + xi(t)) = p(t) Π(t), ∀i ∈{1, . . . , N}, ∀t ∈{1, . . . , T}, I denote ei(si(z)) + xi(t(z)) + (ei(t(z)) −ei(si(z)))δi by yi(z). By denoting the Lagrangian multiplier of the constraint in (6) by 1/φi, the first order condition of (6) relative to xi(t) and the constraint in (6) can be respectively written as: (13) T ∑ t=1 p(t)xi(t) = 0, ∀i ∈{1, . . . , N}, (14) N ∑ i=1 xi(t) = 0, ∀t ∈{1, . . . , T}. (15) (14) φi ∑ z∈Ft π(z)∇vi(yi(z)) = p(t), (7) T ∑ t=1 p(t)xi(t) = 0, (8) (7) (7) z∈Ft T ∑ t=1 p(t)xi(t) = 0, (8) (15) T ∑ t=1 p(t)xi(t) = 0, (8) T ∑ t=1 p(t)xi(t) = 0, (8) Second part of the proof. I now show that the equilibrium allocation(s) with the features defined in the first part of the proof is (are) Pareto optimal. Appendix. Proof of Proposition 1 A feasible allocation ePO is Pareto optimal if a positive χi exists for each agent i such that the allocation is the solution of the following problem: in which ∇vi(.) is the vector of the C commodity derivatives of vi(.). Moreover, the first order condition of (6) relative to δi is: Z ∑ z=1 π(z)∇vi(yi(z)) · ( ei(t(z)) −ei(si(z))) = 0, (9) (9) max ePO N ∑ i=1 χi Z ∑ z=1 π(z)vi(ePO i (z)) s.t. N ∑ i=1 ePO i (z) ≤E(t(z)), ∀z ∈{1, . . . , Z}. (16) in which the dot represents the scalar product of the vectors. An allocation with λi = 0 then satisfies the conditions for being an equilibrium allocation if and only if the allocation and φi simultaneously satisfy (3), (4), (5), (7), (8), and (9). (16) With δi = 1, ϵki = 1/M, and λi = 0, (3), (5), and (9) are verified. When λi = 0, problem (3) of financial intermediary k is verified as soon as the solvency constraints are met, which is the case when ϵki = 1/M, since ∑N i=1 ei(t) = E(t) and ∑N i=1 ei(si(z)) = E(t) for z ∈Ft. When δi = 1 and ϵki = 1/M, the insurance market clearing condition (5) is verified. The value δi = 1 gives yi(z) = xi(t(z)) + ei(t(z)), which is identical across z ∈Ft. Then, (9) can be written as: The derivation of problem (16) indicates that a feasible allocation ePO is Pareto optimal if and only if a positive χi exists for each agent i and a positive vector ρ(z) of dimension C exists for each extensive social state z such that: The derivation of problem (16) indicates that a feasible allocation ePO is Pareto optimal if and only if a positive χi exists for each agent i and a positive vector ρ(z) of dimension C exists for each extensive social state z such that: χi∇vi(ePO i (z)) = ρ(z) π(z), ∀i ∈{1, . . . , N}, ∀z ∈{1, . . . , Z}, (17) (17) N ∑ i=1 ePO i (z) = E(t(z)), ∀z ∈{1, . . . , Z}. Acknowledgments I would like to thank two anonymous referees, Antoine Bom- mier, Jean-Marc Bourgeon, Pierre Boyer, Arthur Charpentier, Georges Dionne, Alexis Direr, Darrell Duffie, François Le Grand, Denis Gromb, Daniel Heyen, Dirk Krueger, Alexis Louaas, Jean- Baptiste Michau, Erwann Michel-Kerjan, Alexander Muermann, and Pierre Picard for their comments and suggestions. The re- search described in this paper did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Example. To highlight the differences in terms of parsimony between the present framework and the one of Cass et al. (1996) and Malinvaud (1973), I consider an economy with C = 1 commodity and N = 10 agents. Agents are fully heterogeneous in terms of risk aversion or initial wealth. They are exposed to 82 A. Goussebaïle Mathematical Social Sciences 120 (2022) 78–84 Appendix. Proof of Proposition 1 Thus, (ei(t(z)) + xi(t(z)), π(z\|t)p(t(z)), ψi) and (ei(si(z)) + xAD i (z), pAD(z), ψAD i ) solve the same equations, which implies that com- modity consumptions ei(t(z)) + xi(t(z)) and ei(si(z)) + xAD i (z) are identical. With χi = ψAD i and ρ(z) = pAD(z), (21) and (23) correspond respectively to (17) and (18) for any i and z, which means that the equilibrium allocation(s) of the Arrow–Debreu framework is (are) Pareto optimal. Finally, the third part of the proof is con- cluded as follows. Replacing (ei(t(z)) + xi(t(z)), π(z\|t)p(t(z)), ψi) by (ei(si(z)) + xAD i (z), pAD(z), ψAD i ) in (13) gives (21). Moreover, replacing (ei(t(z))+xi(t(z)), π(z\|t)p(t(z))) by (ei(si(z))+xAD i (z), pAD (z)) in (14) and (15) gives (22) and (23) respectively, given that ei(t(z)) = ∑ z∈Ft π(z\|t)ei(si(z)) and ∑N i=1 ei(t(z)) = ∑N i=1 ei(si(z)). Thus, (ei(t(z)) + xi(t(z)), π(z\|t)p(t(z)), ψi) and (ei(si(z)) + xAD i (z), pAD(z), ψAD i ) solve the same equations, which implies that com- modity consumptions ei(t(z)) + xi(t(z)) and ei(si(z)) + xAD i (z) are identical. max xAD i Z ∑ z=1 π(z)vi ( ei(si(z)) + xAD i (z)) s.t. Z ∑ z=1 pAD(z)xAD i (z) ≤0, (19) N ∑ i=1 xAD i (z) = 0, ∀z ∈{1, . . . , Z}. (20) (19) N ∑ i 1 xAD i (z) = 0, ∀z ∈{1, . . . , Z}. (20) (20) References An equilibrium allocation in the Arrow–Debreu framework exists and is Pareto optimal. This is also the classic result of Arrow and Debreu (1954). As previously said, the existence of an equilibrium allocation is shown in Proposition 17.C.1 in Chapter 17 of Mas- Colell et al. (1995). The Pareto optimality is demonstrated as follows. xAD and pAD solve (19) and (20) if and only if xAD, pAD, and ψAD solve: An equilibrium allocation in the Arrow–Debreu framework exists and is Pareto optimal. This is also the classic result of Arrow and Debreu (1954). As previously said, the existence of an equilibrium allocation is shown in Proposition 17.C.1 in Chapter 17 of Mas- Colell et al. (1995). The Pareto optimality is demonstrated as follows. xAD and pAD solve (19) and (20) if and only if xAD, pAD, and ψAD solve: An equilibrium allocation in the Arrow–Debreu framework exists and is Pareto optimal. This is also the classic result of Arrow and Arrow, K., 1964. The role of securities in the optimal allocation of risk-bearing. Rev. Econom. Stud. 31, 91–96. Debreu (1954). As previously said, the existence of an equilibrium allocation is shown in Proposition 17.C.1 in Chapter 17 of Mas- Colell et al. (1995). The Pareto optimality is demonstrated as follows. xAD and pAD solve (19) and (20) if and only if xAD, pAD, and ψAD solve: Arrow, K., Debreu, G., 1954. Existence of an equilibrium for a competitive economy. Econometrica 22 (3), 265–290. Borch, K., 1962. Equilibrium in a reinsurance market. Econometrica 30 (3), 424–444. Cass, D., Chichilnisky, G., Wu, H.-M., 1996. Individual risk and mutual insurance. Econometrica 64 (2), 333–341. ψAD i ∇vi ( ei(si(z)) + xAD i (z)) = pAD(z) π(z) , ∀i ∈{1, . . . , N}, ∀z ∈{1, . . . , Z}, Debreu, G., 1959. Theory of Value, an Axiomatic Analysis of Economic Equilibrium. Yale University Press. Duffie, D., 2010. Dynamic Asset Pricing Theory. Princeton University Press. Gorton, G., Winton, A., 2003. Financial intermediation. In: Handbook of the Economics of Finance. vol. 1, Elsevier, pp. 431–552. (21) pp Heathcote, J., Storesletten, K., Violante, G.L., 2009. Quantitative macroeconomics with heterogeneous households. Annu. Rev. Econ. 1 (1), 319–354. li d k f h i h i di id l i k Z ∑ z=1 pAD(z)xAD i (z) = 0, ∀i ∈{1, . . . , N}, Z ∑ z=1 pAD(z)xAD i (z) = 0, ∀i ∈{1, . Appendix. Proof of Proposition 1 Given that yi(z) = xi(t(z))+ ei(t(z)), this problem is equivalent to: max xi T ∑ t=1 Π(t)vi ( ei(t) + xi(t)) s.t. T ∑ t=1 p(t)xi(t) ≤0, (11) N ∑ i=1 xi(t) = 0, ∀t ∈{1, . . . , T}, (12) (11) N ∑ i=1 xi(t) = 0, ∀t ∈{1, . . . , T}, (12) (12) Third part of the proof. I finally show that the equilibrium allocation(s) with the features defined in the first part of the proof is (are) identical to the equilibrium allocation(s) in the classic Arrow–Debreu framework, in terms of commodity consumption. The classic Arrow–Debreu framework assumes the existence of which corresponds to an Arrow–Debreu framework in which the extensive social states are reduced to the aggregate endowment states. Following Proposition 17.C.1 in Chapter 17 of Mas-Colell 83 Mathematical Social Sciences 120 (2022) 78–84 A. Goussebaïle C · Z competitive contingent markets, one market per commodity and extensive social state. The price vector for the C commodities contingent on the extensive social state z is denoted by pAD(z). The quantity of commodities contingent on the aggregate endow- ment state z purchased by agent i is denoted by xAD i (z) (which may be either positive or negative, representing a purchase or a sale, respectively). An equilibrium allocation is such that each agent i maximizes her utility under her budget constraint and all contingent markets clear, which can respectively be written: N ∑ i=1 xAD i (z) = 0, ∀z ∈{1, . . . , Z}. (23) N N ∑ i=1 xAD i (z) = 0, ∀z ∈{1, . . . , Z}. (23) (23) With χi = ψAD i and ρ(z) = pAD(z), (21) and (23) correspond respectively to (17) and (18) for any i and z, which means that the equilibrium allocation(s) of the Arrow–Debreu framework is (are) Pareto optimal. Finally, the third part of the proof is con- cluded as follows. Replacing (ei(t(z)) + xi(t(z)), π(z\|t)p(t(z)), ψi) by (ei(si(z)) + xAD i (z), pAD(z), ψAD i ) in (13) gives (21). Moreover, replacing (ei(t(z))+xi(t(z)), π(z\|t)p(t(z))) by (ei(si(z))+xAD i (z), pAD (z)) in (14) and (15) gives (22) and (23) respectively, given that ei(t(z)) = ∑ z∈Ft π(z\|t)ei(si(z)) and ∑N i=1 ei(t(z)) = ∑N i=1 ei(si(z)). References . . , N}, (22) Z ∑ z=1 pAD(z)xAD i (z) = 0, ∀i ∈{1, . . . , N}, (22) (22) Malinvaud, E., 1973. Markets for an exchange economy with individual risks. Econometrica 41 (3), 383–410. Mas-Colell, A., Whinston, M.D., Green, J.R., 1995. Microeconomic Theory. vol. 1, Oxford University Press, New York. 84 84
https://openalex.org/W4312176308	https://www.frontiersin.org/articles/10.3389/fendo.2022.1028805/pdf	English	null	Gasless endoscopic thyroidectomy via modified areola approach with a simple flap-lifting technique	Frontiers in endocrinology	2,022	cc-by	5,211	TYPE Original Research PUBLISHED 23 December 2022 DOI 10.3389/fendo.2022.1028805 TYPE Original Research PUBLISHED 23 December 2022 DOI 10.3389/fendo.2022.1028805 COPYRIGHT © 2022 Guo, Wu, He, Liu, Wan, Li, Peng and Xu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. OPEN ACCESS OPEN ACCESS EDITED BY Kyung Tae, Hanyang University, South Korea REVIEWED BY Sameer Rege, King Edward Memorial Hospital and Seth Gordhandas Sunderdas Medical College, India Servet Celik, Ege University, Turkey CORRESPONDENCE Aman Xu xamhwx1965@126.com †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Thyroid Endocrinology, a section of the journal Frontiers in Endocrinology RECEIVED 26 August 2022 ACCEPTED 29 November 2022 PUBLISHED 23 December 2022 CITATION Guo T, Wu Z, He J, Liu D, Wan H, Li Y, Peng S and Xu A (2022) Gasless endoscopic thyroidectomy via modiﬁed areola approach with a Tao Guo 1,2†, Zehui Wu 1†, Juntong He 1,2, Defeng Liu 1,2, Hong Wan 1,2, Yangyang Li 1,2, Shihao Peng 1,2 and Aman Xu 1 1Department of General Surgery, The First Afﬁliated Hospital of Anhui Medical University, Hefei, Anhui, China, 2Anhui Public Health Clinical Center, Hefei, Anhui, China Objective: Studies have shown that carbon dioxide (CO2) insufﬂation during endoscopic thyroidectomy is associated with many risks. Recently, we have designed a simple lifting tool using Kirschner wire. We aimed to use this tool for ﬂap-lifting in modiﬁed areola approach endoscopic thyroidectomy and compare it with conventional CO2 insufﬂation. Methods: In a prospective study, patients who underwent endoscopic thyroidectomy via modiﬁed areola approach were randomly assigned into gasless (n = 20) or CO2 groups (n = 22). Pre-operative variables included age, gender, tumor diameter, and clinical diagnosis. Intra-operative hemodynamic monitoring included mean arterial pressure, heart rate, pulse oximetry, end-tidal carbon dioxide (ET-CO2) and arterial pH. Other intra- operative details included total operative time, operative blood loss, conversion from endoscopic surgery to open surgery, intra-operative events, and endoscope video score. Postoperatively, the hospital stay, drainage volume, and complications were recoded. CITATION Guo T, Wu Z, He J, Liu D, Wan H, Li Y, Peng S and Xu A (2022) Gasless endoscopic thyroidectomy via modiﬁed areola approach with a simple ﬂap-lifting technique. Front. Endocrinol. 13:1028805. doi: 10.3389/fendo.2022.1028805 COPYRIGHT © 2022 Guo, Wu, He, Liu, Wan, Li, Peng and Xu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. Introduction long-time operation (3); medullary thyroid carcinoma or undifferentiated thyroid carcinoma. long-time operation (3); medullary thyroid carcinoma or undifferentiated thyroid carcinoma. All patients routinely underwent clinical examination, ultrasonography, ﬁne needle aspiration cytology, laryngoscopy and vocal cord examination, serum thyroid proﬁle tests, including T3, T4 and thyroid-stimulating hormone (TSH) levels. Whenever a malignant nodule was suspected or diagnosed by preoperative pathology, contrast-enhanced computed tomography (CT) scans of the neck were recommended. All patients provided informed written consent, and ethical approval was obtained from the Research Ethics Committee of the University of Anhui Medical University (North District), Anhui Public Health Clinical Center (2021-LL- 05). The endoscopic thyroidectomies were all performed by one surgeon. During the operation, the patients were randomly assigned to the use of either CO2 insufﬂation or ﬂap- lifting technique. Because of its good cosmetic effect, scarless (in the neck) endoscopic thyroidectomy (SET) has developed rapidly in the past two decades (1). A number of innovative surgical approaches have been proposed, including the axillary approach, anterior chest approach, areolar approach, transoral approach, retroauricular approach, and various combinations of these approaches. However, successful endoscopic thyroidectomy also depends on sufﬁcient operating space, which is a necessary condition for clear vision. Currently, there are two main methods to maintain the operating space: carbon dioxide (CO2) insufﬂation and ﬂap- lifting technique (gasless). Insufﬂation with CO2 is more prevalent, but it carries many risks, including hypercarbia, acidosis, pneumomediastinum, pneumothorax, subcutaneous emphysema, and cardiac arrhythmias (2–6). Animal experiments have shown that prolonged CO2 insufﬂation at high pressure will decrease cervical venous blood ﬂow, and increase intracranial pressure and cerebral edema during endoscopic thyroidectomy (7, 8). The gasless technique, however, eliminates all these gas-related complications, enabling more patients to tolerate SET. Besides, it reduces the cost of CO2 insufﬂation as well as intensive end-tidal carbon dioxide (ET-CO2) and arterial blood gas monitoring during operation. At present, the gasless technique is widely used in SET with the transaxillary approach. While in other SET approaches, the gasless technique is less used, possibly due to a lack of suitable commercial suspension equipment. Pre-operative variables included age, gender, tumor diameter, and clinical diagnosis. The clinical diagnosis was obtained by comprehensive evaluation of ﬁne needle aspiration cytology, sonography and CT results. Intra-operative hemodynamic monitoring included mean arterial pressure, heart rate, pulse oximetry, ET-CO2 and arterial pH. OPEN ACCESS No use, distribution or reproduction is permitted which does not comply with these terms. Results: Patient characteristics were not different between the two groups. During the operation, ET-CO2 levels were signiﬁcantly higher in the CO2 group (P < 0.05), whereas arterial pH levels were signiﬁcantly lower (P < 0.05). The CO2 group had longer operation time and higher endoscope clarity VAS score than gasless group. Hospital stay, drainage volume, and postoperative complications did not differ signiﬁcantly between the two groups (P > 0.05). Conclusions: The gasless endoscopic thyroidectomy we performed via our Kirschner wire hook was safe, feasible, and yielded good results. endoscopic thyroidectomy, areola approach, ﬂap lifting, CO2 insufﬂation, Kirschner wire, complication endoscopic thyroidectomy, areola approach, ﬂap lifting, CO2 insufﬂation, Kirschner wire, complication Frontiers in Endocrinology frontiersin.org 01 Guo et al. 10.3389/fendo.2022.1028805 Introduction All these parameters were recorded at the following time points: before operation (T0), 60 min after trocar placement (T1), and at the end of surgery (T2). Other intra-operative details included total operative time, operative blood loss, conversion from endoscopic surgery to open surgery, intra-operative events, and endoscope video clarity score. The endoscope video clarity score was evaluated with a visual analog scale (VAS) which consists of a 10-cm line with anchor statements on the left (worst visual ﬁeld) and on the right (excellent visual ﬁeld) (9). Another doctor graded the score on the VAS based on intraoperative image deﬁnition, smoke effects on laparoscopic visibility, and endoscope cleaning times during the procedures. The hospital stay, drainage volume, as well as postoperative complications were recoded, including subcutaneous emphysema, pneumomediastinum, pneumothorax, hypocalcemia, transient recurrent laryngeal nerve (RLN) palsy, permanent RLN palsy, hemorrhage, skin ecchymosis, subcutaneous ﬂuid retention, and wound infection. Kirschner wire (K-wire) is widely used for the ﬁxation of fractures and dislocations due to its easy accessibility, reliability, and affordability. Recently, we have designed a simple lifting tool for SET using K-wire. The purpose of this study is to investigate the safety, feasibility, and postoperative outcomes of gasless endoscopic thyroidectomy via a modiﬁed areola approach with this lifting tool. Surgical procedures Between August 2021 and May 2022, a total of 42 patients with thyroid nodules underwent endoscopic thyroidectomy at The First Afﬁliated Hospital of Anhui Medical University (North District). Inclusion criteria (1): patient having a strong desire for cosmesis (2); benign tumor less than 4 cm in diameter, or differentiated thyroid carcinomas that is smaller than 2 cm in diameter and do not invade adjacent organs (3); no lateral cervical lymph node or distant metastasis was detected before the operation. Exclusion criteria (1): previous history of thyroid surgery (2); patients who cannot tolerate general anesthesia or The surgical steps are similar to the procedure described by Wang (10). Under general anesthesia with endotracheal intubation, the patient is placed in the supine position with a shoulder pillow placed behind the neck, so the neck can be slightly extended. The surgeon stands between the two legs of the patient. The camera assistant stands to the right of the patient, while the scrub nurse stands to the left. The second assistant stands by the patient’s neck. Frontiers in Endocrinology frontiersin.org 02 Guo et al. 10.3389/fendo.2022.1028805 face-down endoscope is inserted for observation. Under direct vision, a 5-mm trocar is placed at the 11 o’clock position of the left areola border as the ﬁrst operation hole. To not interfere with the observation hole, we position another 5-mm trocar at 3 cm above the right nipple as the second operation hole. Because this kind of trocar placement is slightly different from the conventional complete areola approach in that all three incisions are close to the areola (10), we name our method the modiﬁed areola approach. Then, grasping forceps and an electric As shown in Figure 1A, the operation area and incision design are marked preoperatively. From 2 o’clock to 4 o’clock position of the right areola border, a 12-mm curved incision is made as an observation hole. Through it, about 20 mL of diluted epinephrine solutions (prepared with 1 mL adrenaline in 500 mL normal saline) are injected subcutaneously to create an anatomical plane and reduce intraoperative bleeding. A tunnel is then made by bluntly dissecting the subcutaneous tissue with ovary forceps. Then, a 12-mm trocar is placed and a 30-degree FIGURE 1 (A) Operation area and incision design; (B) Appearance of K-wire hook; (C) External schematic diagram of K-wire hook suspension device; (D) Overall surgical scene (yellow arrowheads: smoke suction tube). Postoperative management The patients were carefully checked for signs of bleeding, RLN injury, or difﬁculty breathing. RLN injury is deﬁned as impaired movement of the vocal cords on laryngoscopy postoperatively. On the day of surgery, a liquid diet was provided. Then, soft oral foods were permitted on the second day after operation. Postoperative oral antibiotics were prescribed for 3 days. Surgical procedures RE 1 Operation area and incision design; (B) Appearance of K-wire hook; (C) External schematic diagram of K-wire hook suspension device Overall surgical scene (yellow arrowheads: smoke suction tube). FIGURE 1 (A) Operation area and incision design; (B) Appearance of K-wire hook; (C) External schematic diagram of K-wire hook suspension device; (D) Overall surgical scene (yellow arrowheads: smoke suction tube). Frontiers in Endocrinology frontiersin.org 03 Guo et al. 10.3389/fendo.2022.1028805 neck compartment are dissected. A drainage tube is routinely placed at the end of the surgery. neck compartment are dissected. A drainage tube is routinely placed at the end of the surgery. hook (or harmonic scalpel) are inserted to sharply separate the cord-like connective tissue in the cavity and enlarge the subcutaneous operation space of the anterior chest wall. Next, the bilateral sternocleidomastoid muscles are used as anatomical markers to dissect the subplatysmal space. At this time, for the CO2 group, we insufﬂate CO2 at the pressure of 6–8 mmHg to maintain the working space, while for the gasless group, we place the self-made hook to lift the ﬂap. Figure 1B shows the appearance of the K-wire hook. The hook is made based on the anticipated size of the operation space during the operation. Fold the K-wire as needed, then form one end into an “O” shape and the other end into an inverted “J” shape. At the level of the suprasternal fossa, a 1-mm incision is made using #11 surgical blade at the anterior cervical ﬂap. Then, the “O” shape end of the hook is inserted through the incision. Under the monitor of endoscopy, slowly insert the entire “O” shape ring along its arc into the deep surface of the ﬂap. After that, the cervical ﬂap is lifted vertically upwards. The inverted “J” shape end of the hook is connected to reversed L-shaped bar, which is ﬁxed to the operating table near the patient’s head. The height of the bar can be adjusted until the ﬂap is just tight (Figure 1C). To minimize the smoke generated when the energy equipment works, for both groups we connected a suction device to the trocar (Figure 1D). Statistical analysis Statistical analysis is performed with SPSS 26.0 software (SPSS Inc., Chicago, IL, USA). Continuous data are presented as mean ± standard deviation (SD) or median (range), and categorical data are presented as number (%). Student’s t-test or Mann–Whitney U test is used for comparing continuous data, and the chi-square test or Fisher’s exact test is adopted for comparing categorical data. Pearson’s correlation method is used to assess the relationship between two continuous variables. P < 0.05 is considered statistically signiﬁcant. By using the harmonic scalpel, the anatomical plane is extended from caudal to cephalad. The space scope is as follows: the lower boundary reached the superior sternum fossa, the upper boundary reached the level of the thyroid cartilage, and both sides reached the sternocleidomastoid muscle. Results The strap muscles are dissected along the midline and retracted laterally for exposure of the thyroid gland. Longitudinally cut the isthmus and dissect upward along the gap between thyroid gland and trachea until the identiﬁcation of the lateral branch of the superior laryngeal nerve. The superior artery of thyroid gland and its surrounding veins are coagulated as close as possible to the gland to ensure the blood supply to the superior parathyroid glands. Then turn to the inferior pole of the thyroid gland, expose and protect the inferior parathyroid glands. Tracheoesophageal groove areas are bluntly separated to expose the RLN. Upon conﬁrming the path of the RLN toward its entry point into the larynx, dissect the thyroid lobe. The specimens are taken out through the central trocar and sent to frozen section. If it is malignant, lymph nodes of the central Intraoperative variables (P > 0.05, Table 4). Neither transient or permanent RLN palsy was found postoperatively in both groups. However, there was one case of subcutaneous emphysema and one case of pneumomediastinum among the CO2 group. Patients in the gasless group do not display signiﬁcant surgical scar on the anterior neck during postoperative follow-up (Figure 2). All patients underwent endoscopic thyroidectomy successfully. There was no conversion to open surgery in both groups. Hemodynamic parameters were measured before surgery (T0), 60 minutes after trocar placement (T1), and at the end of surgery (T2) (Table 2). At all these time points, mean arterial pressure, heart rate, and pulse oximetry were not different between the two groups (P > 0.05). There was no signiﬁcant difference between the two groups in ET-CO2 and arterial pH before surgery (P > 0.05). However, after 60 minutes of trocar placement, ET-CO2 levels were signiﬁcantly higher in the CO2 group than in the gasless group (P < 0.05), whereas arterial pH levels were signiﬁcantly lower (P < 0.05). This kind of difference still existed at the end of the operation (P < 0.05). As shown in Table 3, the CO2 group had a longer total operation time than the gassless group, and its endoscope clarity VAS score was lower than that of the gassless group. In terms of operative blood loss, conversion to open surgery, intra-operative events, and RLN not identiﬁed event, there was no signiﬁcant difference between the two groups. All patients underwent endoscopic thyroidectomy successfully. There was no conversion to open surgery in both groups. Hemodynamic parameters were measured before surgery (T0), 60 minutes after trocar placement (T1), and at the end of surgery (T2) (Table 2). At all these time points, mean arterial pressure, heart rate, and pulse oximetry were not different between the two groups (P > 0.05). There was no signiﬁcant difference between the two groups in ET-CO2 and arterial pH before surgery (P > 0.05). However, after 60 minutes of trocar placement, ET-CO2 levels were signiﬁcantly higher in the CO2 group than in the gasless group (P < 0.05), whereas arterial pH levels were signiﬁcantly lower (P < 0.05). This kind of difference still existed at the end of the operation (P < 0.05). Patient baseline characteristics A total of 42 patients with thyroid nodules underwent modiﬁed areola approach endoscopic thyroidectomy during this period. They were randomly assigned to the CO2 group (n=22) and the gasless group (n=20). The preoperative demographics and clinical characteristics are shown in Table 1. Both groups were comparable in terms of age and gender (P > 0.05, Table 1). There was no signiﬁcant difference between the two groups in terms of tumor diameter and preoperative clinicopathological diagnosis (P > 0.05). TABLE 1 Demographics and clinical characteristics of the two groups. TABLE 1 Demographics and clinical characteristics of the two groups. CO2 group (n = 22) Gasless group (n = 20) P Mean age (years) 31.1 ± 5.6 32.0 ± 8.5 0.680 Gender 1.000 Female (%) 20 (95.5%) 19 (95.0%) Tumor diameter (cm, average diameter) 1.0 ± 0.3 1.0 ± 0.2 0.494 Pre-operative clinicopathological diagnosis 0.753 Benign 4 2 Malignant 18 18 Frontiers in Endocrinology frontiersin.org 04 Frontiers in Endocrinology Frontiers in Endocrinology 04 10.3389/fendo.2022.1028805 Guo et al. Discussion Thyroid tumors are the most common malignant tumors of the endocrine system and occur more often in females. The main treatment for thyroid tumors is surgical resection. Traditional open thyroidectomy (OT), considered the gold standard, is widely performed around the world. However, OT will inevitably leave a visible scar on the anterior neck, which negatively affects body image or even the quality of life (11, 12). Endoscopic surgery was ﬁrst described by Gagner et al. in 1996 for secondary hyperparathyroidism. Then, in 1997, Yeung in Hong Kong and Hüscher in Italy published the ﬁrst reports on endoscopic thyroid surgery (13). After two decades of development, endoscopic thyroidectomy has become more and more perfect and popularized, and now a variety of surgical methods have become available. As shown in Table 3, the CO2 group had a longer total operation time than the gassless group, and its endoscope clarity VAS score was lower than that of the gassless group. In terms of operative blood loss, conversion to open surgery, intra-operative events, and RLN not identiﬁed event, there was no signiﬁcant difference between the two groups. TABLE 2 Intra-operative hemodynamic parameters of the two groups. Post-operative outcomes CO2 group (n = 22) Gasless group (n = 20) P Hospital stay (days) 4.5 4.6 0.729 Drainage volume (ml) 20 19.5 0.536 Postoperative complications 0 0 – Subcutaneous emphysema 1 0 – Pneumomediastinum 1 0 – Pneumothorax 0 0 – Hypocalcemia 0 0 – Transient RLN palsy 1 1 1.000 Permanent RLN palsy 0 0 – Hemorrhage 0 0 – Skin ecchymosis 0 0 – Subcutaneous ﬂuid 0 0 – Wound infection 0 0 – Tracheal injury 0 0 – Esophagus injury 0 0 – RLN, recurrent laryngeal nerve. Gasless group (n = 20) Gasless group (n = 20) Gasless group (n = 20) RLN, recurrent laryngeal nerve. thyroidectomy. In 1998, Shimizu horizontally inserted two pieces of K-wires into the subcutaneous tissue in front of the neck and connected them to an abdominal wall lifting device (16). The method obtained a good operation ﬁeld of vision for video- assisted neck surgery (16–18). For scarless endoscopic thyroidectomy, gasless techniques are mostly used in the axillary approach. When it comes to the bilateral areolar approach that can provide good access to bilateral thyroid lobes, gasless techniques are rarely reported. This may be due to the lack of suitable commercial lifting equipment. implementation of follow-up procedures. CO2 insufﬂation is a widely used method to maintain the operating space. However, CO2 is highly soluble in blood and diffuses easily in loose areolar and rough tissue, such as in the neck (14). During endoscopic thyroidectomy surgery, special attention should be paid to CO2 insufﬂation pressure. Otherwise, high CO2 pressure or excessive absorption of CO2 may adversely affect patients’ respiratory, circulatory and other systems and increase the possibility of related complications. It is worth noting that even if CO2 insufﬂation pressure is < 8 mmHg, CO2 embolism can still occur (15). In this study, we devised a simple ﬂap-lifting tool using a piece of K-wire. Using this tool, we performed gasless bilateral areolar approach endoscopic thyroidectomy. Our gasless group do not require CO2 insuffulation throughout the entire operation process. The results showed that the vital signs were stable and arterial pH were within the physiologic range during surgery in both groups. However, following CO2 insufﬂation, ET-CO2 levels were signiﬁcantly higher in the CO2 group than in the gasless group, whereas arterial pH levels were signiﬁcantly lower in the CO2 group. In addition, the CO2 group had a longer total operation time than the gasless group. Post-operative outcomes There was no signiﬁcant difference between the two groups in hospital stay, drainage volume and postoperative complications Creating and maintaining an operating space is the ﬁrst step in endoscopic thyroidectomy, which is critical to the smooth TABLE 2 Intra-operative hemodynamic parameters of the two groups. TABLE 2 Intra-operative hemodynamic parameters of the two groups. CO2 group (n = 22) Gasless group (n = 20) P T0 T1 T2 T0 T1 T2 P1 P2 P3 Mean arterial pressure (mmHg) 73 78 79 74 77 77 0.240 0.612 0.085 Heart rate (beats/min) 70 73 74 70 71 71 1.000 0.213 0.071 Pulse oximetry 99% 99% 99% 99% 99% 99% 0.977 0.763 0.982 ET-CO2 (mmHg) 32 40 40 33 35 35 0.379 <0.001 <0.001 Arterial pH 7.44 7.38 7.35 7.43 7.42 7.41 0.509 <0.001 <0.001 T0: before surgery. T1: at 60 min after trocar placement. T2: at the end of surgery. P1, P2, P3: p value for difference between the groups at T0,T1 and T2, respectively. ET-CO2: End-tidal carbon dioxide. TABLE 3 Intraoperative variables of the two groups. CO2 group (n = 22) Gasless group (n = 20) P Total operative time (min) 135 115 <0.001 Operative blood loss (ml) 60 55 0.051 Conversion 0 0 – Intra-operative events 0 0 – RLN not identiﬁed 0 0 – Endoscope clarity VAS 6.8 8.1 <0.001 RLN, recurrent laryngeal nerve. VAS, endoscope visual analogue score. TABLE 3 Intraoperative variables of the two groups. CO2 group (n = 22) Gasless group (n = 20) P Total operative time (min) 135 115 <0.001 Operative blood loss (ml) 60 55 0.051 Conversion 0 0 – Intra-operative events 0 0 – RLN not identiﬁed 0 0 – Endoscope clarity VAS 6.8 8.1 <0.001 RLN, recurrent laryngeal nerve. VAS, endoscope visual analogue score. TABLE 3 Intraoperative variables of the two groups. P Frontiers in Endocrinology frontiersin.org 05 Frontiers in Endocrinology 05 Guo et al. 10.3389/fendo.2022.1028805 TABLE 4 Post-operative outcomes of the two groups. TABLE 4 Post-operative outcomes of the two groups. TABLE 4 Post-operative outcomes of the two groups. Publisher’s note The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Funding This study was supported by grants from the Basic and Clinical Cooperative Research and Promotion Program of Anhui Medical University (2020xkjT045) and the National and Provincial Key Specialty Construction Plan (No. Z155080000004). Post-operative outcomes Smoke and vapor plumes generated by energy devices easily interfere with the operation ﬁeld of view. Consequently, surgeons have to suction and clear endoscopes frequently, which affects operating cavity pressure and interferes with the procedure. In contrast, the gasless technique allows surgeons to do unlimited suction for bleeding or smoke clearance without loss of exposure. This explains the longer operation time and higher endoscope clarity VAS score in the CO2 group. In contrast, the gasless technique eliminates all the CO2-related complications, enabling more patients to tolerate edoscopic FIGURE 2 Three months after surgery, an anterior neck image of a patient in the gasless group. There was a slight increase in intraoperative blood loss in the CO2 insufﬂation group, but the difference is not statistically signiﬁcant (P>0.05). This is likely due to the relatively small Three months after surgery, an anterior neck image of a patient in the gasless group. Three months after surgery, an anterior neck image of a patient in the gasless group. frontiersin.org Frontiers in Endocrinology 06 Guo et al. 10.3389/fendo.2022.1028805 University (North District). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any identiﬁable images or data included in this article. University (North District). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any identiﬁable images or data included in this article. thyroid lesions in most of the study patients, which decreases the difﬁculty of surgery. In our study, all these operations were performed by one surgeon who had overcome the learning curve and was proﬁcient in the treatment of perithyroid blood vessels. As shown in Table 3, the mean operation time for both groups was about 2 hours. Thus, there is no signiﬁcant difference in operative blood loss between the two groups. Acknowledgments We thank American Journal Experts (AJE) for English language editing of this paper. Author contributions We used orthopedic surgical pliers for twisting and bending K-wire to make the lifting hook. In our experience, the curvature of the “O” shape ring must be smooth to facilitate the placement of the hook. To reduce the risk of damaging other tissues, we used endoscopy to monitor the placement of the lifting hook, which we consider to be extremely important. Collaboration with the manufacturer to improve and commercialize the device may reduce surgical risk and save operation time. TG and AX conceived the study. ZW, JH and DL collected the data. TG and ZW analyzed the data. HW, YL and SP provided guidance on experimental design, data analysis, and presentation of results. TG wrote the paper. ZW and AX revised the paper. All authors contributed to the article and approved the submitted version. It would be ﬁtting to acknowledge the limitations of our study. First, the sample size of our study is relatively small, which decreases the statistical power. Large sample size is needed to get a more reliable conclusion. Second, most cases in our study were microcarcinomas of the thyroid gland, therefore the results may not apply to all thyroid disorders. We hope to include more patients with other thyroid disorders in the future. Third, compared with open thyroidectomy, endoscopic thyroidectomy has a longer learning curve. As a result, the success of endoscopic thyroid surgery is greatly inﬂuenced by the experience of the operators. In our study, we relied on the experience of two surgeons (GT and HJ) rather than many surgeons with different levels, therefore the result may be biased. Conclusion Our study showed that gasless endoscopic thyroidectomy via a modiﬁed areola approach with this lifting tool is safe, feasible, and has good postoperative results. Moreover, K-wire is cheap and easy to ﬁnd. Consequently, our method is easy to popularize, reducing the difﬁculty of endoscopic thyroid surgery. 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https://openalex.org/W4283376772	https://www.frontiersin.org/articles/10.3389/fendo.2022.891039/pdf	English	null	Single-Cell Sequencing Analysis of the db/db Mouse Hippocampus Reveals Cell-Type-Specific Insights Into the Pathobiology of Diabetes-Associated Cognitive Dysfunction	Frontiers in endocrinology	2,022	cc-by	10,134	Single-Cell Sequencing Analysis of the db/db Mouse Hippocampus Reveals Cell-Type-Speciﬁc Insights Into the Pathobiology of Diabetes- Associated Cognitive Dysfunction Shizhan Ma 1, Wenkai Bi 1, Xueying Liu 1, Shangbin Li 2, Yaxin Qiu 1, Chengcheng Huang 3, Renjun Lv 2* and Qingqing Yin 4* 1 Department of Endocrinology, Shandong Provincial Hospital Afﬁliated to Shandong First Medical University, Jinan, China, 2 Department of Geriatrics, Shandong Provincial Hospital Afﬁliated to Shandong First Medical University, Jinan, China, 3 Clinical Education Administration, Afﬁliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China, 4 Department of Geriatric Neurology, Shandong Provincial Hospital Afﬁliated to Shandong First Medical University, Jinan, China ORIGINAL RESEARCH published: 01 June 2022 doi: 10.3389/fendo.2022.891039 ORIGINAL RESEARCH published: 01 June 2022 doi: 10.3389/fendo.2022.891039 Keywords: diabetes-associated cognitive dysfunction, hippocampus, microglia, inﬂammation, single- cell transcriptomics Edited by: Xinguo Hou, Shandong University, China Edited by: Xinguo Hou, Shandong University, China y Xinguo Hou, Shandong University, China Reviewed by: Evelyne Gozal, University of Louisville, United States Hua Xiang Zhuang, Shandong University, China Correspondence: Renjun Lv renjunlv@foxmail.com Qingqing Yin yinyunqing11@126.com Reviewed by: Evelyne Gozal, University of Louisville, United States Hua Xiang Zhuang, Shandong University, China Reviewed by: Evelyne Gozal, University of Louisville, United States Hua Xiang Zhuang, Shandong University, China Correspondence: Renjun Lv renjunlv@foxmail.com Qingqing Yin yinyunqing11@126.com Correspondence: Renjun Lv renjunlv@foxmail.com Qingqing Yin yinyunqing11@126.com Specialty section: This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology Received: 07 March 2022 Accepted: 27 April 2022 Published: 01 June 2022 Specialty section: This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology Specialty section: This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology Received: 07 March 2022 Accepted: 27 April 2022 Published: 01 June 2022 Diabetes-associated cognitive decline (DCD), is one of the complications of diabetes, which is characterized by a series of neurophysiological and pathological abnormalities. However, the exact pathogenesis of DCD is still unknown. Single-cell RNA sequencing (scRNA-seq) could discover unusual subpopulations, explore functional heterogeneity and identify signaling pathways and potential markers. The aim of this research was to provide deeper opinion into molecular and cellular changes underlying DCD, identify different cellular types of the diabetic mice hippocampus at single-cell level, and elucidate the factors mediating the pathogenesis of DCD. To elucidate cell speciﬁc gene expression changes in the hippocampus of diabetic encephalopathy. Single-cell RNA sequencing of hippocampus from db/m and db/db mice was carried out. Subclustering analysis was performed to further describe microglial cell subpopulations. Interestingly using immunohistochemistry, these ﬁndings were conﬁrmed at the protein level. Single cell analysis yielded transcriptome data for 14621 hippocampal cells and deﬁned 11 different cell types. Analysis of differentially expressed genes in the microglia compartments indicated that infection- and immune system process- associated terms, oxidative stress and inﬂammation play vital roles in the progression of DCD. Compared with db/ m mouse, experiments at the protein level supported the activation of microglia, increased expression of inﬂammatory factors and oxidative stress damage in the hippocampus of db/db mouse. In addition, a major ﬁnding of our research was the subpopulation of microglia that express genes related to pro-inﬂammatory disease-associated microglia (DAM). Our research reveals pathological alterations of inﬂammation and oxidative stress mediated hippocampal damage in the db/db mice, and may provide potential diagnostic biomarkers and therapeutic interventions for DCD. Morris Water Maze Test (MWM) ( ) Using the MWM test, we assessed spatial learning and memory ability of eighteen-week mice (17). The maze apparatus includes a circular plastic pool ﬁlled with water added with black in kat approximately 22 ± 1°C. The circular plastic pool is divided into 4 quadrants, one of which includes an escape platform (diameter, 5 cm; height, 15 cm) was placed 1 cm underwater and at a ﬁxed position. The test composed of a 5-day acquisition phase trial and a probe trail on day 6. During acquisition phase trials, the animals were placed in the water, were able to reach to the platform within 60 s, which were remained on the platform for 10 s, and each animal was trained four times every day. However, if the mice failed to ﬁnd the platform within 60 s, they were gently guided to the platform and remained there for 10 s, and the escape latency was recorded as 60 s. On the sixth day, the spatial exploration test with the platform removed was carried out, and rats were permitted to swim for 60 s. Data of the time spent and the number of platform crossings in the target quadrant within 60 s were recorded. The mice movement of the escape latency was recorded using a computerized video system and analyzed by a computer system. Analysis of gene expression in hippocampal cells is an appropriate approach to decipher pathological changes in DCD, but there is a high degree of cellular heterogeneity in hippocampal cells. The current advent of scRNA-seq technology can discover new cell subpopulations and further explore gene regulatory mechanisms to reveal heterogeneity in genes and functions of each cell. Recent studies have shown that scRNA-seq in a mouse model of AD (5xFAD) identiﬁed a novel disease-associated microglia type (DAM), and revealed signiﬁcant molecular heterogeneity within DAM, including pro-inﬂammatory and anti- inﬂammatory phenotypes (12, 13). Thus, this technology provides the opportunity to dissect different cell types in complex tissues, such as the hippocampus at single-cell resolution, which provides insights into the transcriptional signature in individual cells from a more detailed and microscopic perspective (14–16). Hippocampus Dissection and Dissociation The protocol for dissection and single-cell dissociation were performed as previously described (18). After Morris Water Maze testing was completed, mice were immediately euthanized with sodium pentobarbital (50 mg/kg, i.p.) and systemically perfused with cell culture grade saline (0.9%, sigma). Citation: Ma S, Bi W, Liu X, Li S, Qiu Y, Huang C, Lv R and Yin Q (2022) Single-Cell Sequencing Analysis of the db/db Mouse Hippocampus Reveals Cell-Type-Speciﬁc Insights Into the Pathobiology of Diabetes-Associated Cognitive Dysfunction. Front. Endocrinol. 13:891039. doi: 10.3389/fendo.2022.891039 June 2022 \| Volume 13 \| Article 891039 1 Frontiers in Endocrinology \| www.frontiersin.org ScRNA-Seq of T2DM Mouse Hippocampus Ma et al. MATERIALS AND METHODS Hippocampus single-cell suspensions from db/db and db/m mice were added to the Chromium Single Cell Controller Instrument (10x Genomics, Shanghai Genechem Co.,Ltd.) to create single-cell gel beads. According to the manufacturer’s protocol, the scRNA-seq libraries were prepared with the Chromium Single-cell 3′ Reagent V3 Kits, and sequenced on Morris Water Maze Test (MWM) The brains were removed and samples of the hippocampus of the brain were immediately isolated from the ipsilateral side of the brain on ice, which dissociated into a viable single-cell suspension. Brieﬂy, using scalpels, we mince hippocampus samples, which further were digested in Earle’s Balanced Salt Solution (EBSS) containing DNAse I (0.01 mg/ml) and papain (1 mg/ml) for 60 minutes at 37°C. Hyaluronidase prevents dissociation. Intact cells were isolated on a single step discontinuous density gradient and resuspended in phosphate buffered saline containing 0.04% weight/volume bovine serum albumin (BSA), for single cell transcriptomic analysis processing. To explore the pathophysiology of DCD, we used db/db mouse, a model of type 2 diabetes (T2DM), characterized by the homozygous mice express deﬁcient leptin receptors, which results in T2DM phenotypes including hyperglycemia, severe obesity, hyperphagia, polyuria and metabolic syndrome. In our present research, we applied scRNA-seq technology captures T2DM-induced gene changes in a large number of hippocampal cells and provides a comprehensive and detailed view of cell alterations occurring in the hippocampus to decipher the pathology of DCD. INTRODUCTION approved by the Animal Care and Use Committee of Shandong Provincial Hospital Afﬁliated to Shandong First Medical University. Twelve-week male db/db mice (BKS.Cg-Dock7m+/+ Leprdb/J, n=8) and db/m (Dock7m +/+ Leprdb, n=8) were obtained from Changzhou Cavens Laboratory Animal Co., Ltd (Jiangsu, China). They were kept in a temperature-controlled room (23 ± 1°C) under a 12 h light/dark cycle and allowed free access to chow and water. Animals with fasting plasma glucose levels >300 mg/dl were classiﬁed as diabetic. Metabolic diseases such as diabetes mellitus impairs the function of the brain and are called diabetic encephalopathy, including functional impairment of cognition, neuronal signal transduction, synaptic plasticity and neurophysiological changes, and potential structural damage associated with diabetes mellitus (1–3). Diabetes-associated cognitive dysfunction (DCD), a major complication of diabetes, is gaining more attention (4). The impairment of cognition in diabetes includes impaired learning, memory, problem−solving, attention and reduced information processing speed (1). Additionally, numerous clinical studies have shown that diabetes mellitus is closely related to vascular dementia, as well as Alzheimer’s disease (AD) (5, 6). Multiple studies have demonstrated that factors such as neuronal apoptosis, oxidative stress, neuroinﬂammation and altered neurogenesis may play a role in DCD (7, 8), in which oxidative stress and neuroinﬂammation are early-onset mechanism in diabetic encephalopathy (9, 10). The hippocampus plays a vital role in learning and memory, and the impairment of its function is linked to DCD (11). However, the mechanism by which diabetes impairs cognitive function have not yet been clearly established, including changes in these individual cellular compartments of the hippocampus and how they interact with each other. Animals Animal studies were performed in compliance with the requirements of the National Laboratory Animal Use Act of the People’s Republic of China and in accordance with protocols June 2022 \| Volume 13 \| Article 891039 Frontiers in Endocrinology \| www.frontiersin.org 2 ScRNA-Seq of T2DM Mouse Hippocampus Ma et al. an Illumina HiSeq X Ten System. We obtained 150 bp paired- end reads. temperature; Nuclei were dyed with 4, 6- diaminido-2- phenylindole (DAPI) before 10 min of mounting. Sections were then analyzed under a laser scanning confocal microscope (Olympus, Tokyo, Japan). We analyzed the images using Image-Pro Plus 6.0 software (Media Cybernetics, USA). The results were analyzed using GraphPad Prism 5.0 software and are showed as the mean ± standard error of mean. Statistically signiﬁcant differences were determined using a two-tailed unpaired Student’s t test. P<0.05 was considered statistically signiﬁcant. A two-tailed unpaired Student’s t-test was used to determine statistically signiﬁcant differences. P < 0.05 was considered statistically signiﬁcant. ScRNA-Seq Data Analysis Raw sequencing data, cellular barcodes were demultiplexed by using Cell Ranger Software Pipeline (version 3.0), using the STAR aligner (version 3.1.0), we map reads to the mouse reference genome and transcriptome. To produce a matrix of gene counts versus cells, down-sample of reads was required to produce normalized aggregate data across samples. The unique molecular identiﬁer (UMI) count matrix was processed by using R package Seurat (version 3.1.1) (19). In order to eliminate low- quality cells and likely multiplet captures, the cells with >200 genes and <8000 genes; >400 UMIs and <20% mitochondrial RNA (mtRNA) were retained for subsequent analyses. The ﬁltered matrix was normalized for library size to gain normalized counts in Seurat. ScRNA-Seq Analysis Resulted in 26 Clusters and Identiﬁed Ten Cell Types ScRNA-Seq Analysis Resulted in 26 Clusters and Identiﬁed Ten Cell Types Isolated hippocampus from db/m and db/db mouse were separated into single-cell suspensions. After quality control ﬁlters, A total of 14621 cells were used for a following analysis. Of these, 4667 cells (32%) originated from db/db mice and 9954 cells (68%) from db/m mice. After the completion of quality control, PCA and t-SNE analysis was conducted. The workﬂow of this study is shown in Figure 2A. Based on expression of well- established markers, the cells were classiﬁed into 26 transcriptionally distinct clusters (Figures 2B–E), and divided into 10 distinct cell types based on cell-type-speciﬁc gene expression and annotated as follows: B cells, Endothelial cells, Ependymal cells, Fibroblast, Microglia, Mural cells, Neurons, NKT, Oligodendrocyte, Oligodendrocyte Precursor Cell (OPC). The abundance of db/m vs db/db cells per cluster is represented in Figures 2C–E shows the relative abundance of cell types for each genotype. In this scRNA-Seq data, compared with db/m Impairments of Learning and Memory In db/db Mice Identiﬁcation of the top variable genes in individual cells was performed (20). In summary, principal component analysis (PCA) was performed on the highly variable genes to lessen the dimensionality, and the top principal components (PCs) were chosen for cell clustering using a graph-based clustering method. We visualised the clustering results using t-Distributed Stochastic Neighbor Embedding (t-SNE) technique. t-SNE dimensional reduction was performed in R using the Seurat package. Compared with classical cell type markers, the identiﬁed 26 cell clusters correspond to 10 cell types. In order to quantify changes in hippocampal intercellular communication during diabetes mellitus, CellPhoneDB (V2.0) was used to identify biologically relevant ligand receptor partners (21). To characterize intercellular correspondence organizations, we connected any two cell types in which ligands and receptors were communicated in the previous and last cell types, separately. The Morris water maze test is commonly used to study spatial learning and memory loss in animals. During the acquisition phase trials, escape latencies did not differ signiﬁcantly between groups on day 1 (P> 0.05; Figure 1A), in addition, the time to ﬁnd the hidden platform was declined progressively from the second day to the ﬁfth day. Interestingly, the db/db group spent signiﬁcantly more time ﬁnding the hidden platform compared to the db/m group (P < 0.05; Figure 1A). The typical swimming traces of mice is illustrated in Figure 1B, the db/db mouse showed a more disorganized and longer swimming paths. The hidden platform was removed during the spatial probe trial. As expected, compared with that of the db/m group, the time spent in the target quadrant of db/db group and the number of platform crossings were was markedly reduced (P < 0.05; Figures 1C, D). In summary, these results based on the MWM test demonstrated impaired learning and memory in db/db mice. In the current study, all db/db mice developed concomitant cognitive decline. Hence, we performed single-cell transcriptome analysis of hippocampal tissues from db/m mice and db/db mice. The Find Markers function (test.use = bimod) of Seurat package was used to identify differentially expressed genes. P- value < 0.05 and \|log2foldchange \| > 0.58 were set as the differential expression thresholds. Differentially gene expression (DEGs) was subjected to GO enrichment and KEGG pathway enrichment analysis using hypergeometric distribution in R package, and functional association networks were constructed and deﬁned using STRING version 11.0. Immunoﬂuorescence Staining PFA ﬁxed brain tissues were cut into cryosections. Nrf2, HO-1, Nlrp3, Iba-1, Trem2 and Cmklr1 expression levels in brain tissue sections were characterized using immunoﬂuorescence. Cryosections were air dried at room temperature, rinsed with PBS, supplemented with 0.4% Triton X-100, and submitted for antigen retrieval. Sections were blocked with 10% normal goat serum for 1 h at 37°C. The sections were incubated with anti- Nrf2 (GB113808, Abcam; 1:500), anti-HO-1 (GB11845, Abcam; 1:200), anti-NLRP3(GB11300, Abcam; 1:200), anti-TREM2 (bs- 2723R, Abcam; 1:200), anti- Cmklr1 (bs-10185R, Abcam; 1:200) or anti-Iba-1 (GB12105, Abcam; 1:500) at 4°C overnight. The next day, sections were washed and incubated with tetramethylrhodamine labeled anti mouse IgG for 1 h at room June 2022 \| Volume 13 \| Article 891039 Frontiers in Endocrinology \| www.frontiersin.org 3 ScRNA-Seq of T2DM Mouse Hippocampus Ma et al. A B C D FIGURE 1 \| Learning and memory impairment in db/db mouse. In the MWM test, day 1 represents performance on the ﬁrst trial, and subsequent scores show average of all daily trials. (A) Escape latency of the 5-day acquisition trials in MWM. (B) Representative swim pathways of respective groups in the spatial probe test. (C) Time spent in the target quadrant during the probe trial. (D) The numbers of the target platform crossings in the probe trial. Data are represented mean ± SEM for 3 mice in each group. ⁎P < 0.05 vs. db/m group. observed several markers of inﬂammatory response, including Il7ra, Il6ra, Irf2bpl, Cmklr1, Mafg, Map2k3, Ncf1, Nlrp3 and Trem2. To determine glial activation and inﬂammation factors, immunoﬂuorescent staining for in the hippocampus of mice were performed. Iba-1 is highly expressed in activated microglia. We found that the expression of Nlrp3, Cmklr1, compared with db/dm group, the number of positive cells labeled by TREM2 and Iba-1 in hippocampus of db/db group was signiﬁcantly increased (Figure 4), suggesting that T2DM facilitates microglial activation and increases levels of inﬂammatory factors in the hippocampus. Additionally, Immunoﬂuorescence was also used to analyze the expression of Nrf2/HO-1 system, which has beneﬁcial effects by protecting against oxidative damage (22). As seen from Figure 5, the db/db group rats showed signiﬁcantly lower levels of Nrf2 and HO-1 in the hippocampus (P<0.05) compared to the db/m group rats, suggesting the antioxidant system was damaged in the db/db mice. Downregulated genes in db/db mice microglia associated into OXPHOS, myelin and glial cell differentiation (Figure 3; Supplementary Table 1). Immunoﬂuorescence Staining A A B B pp y ) Compared with the db/dm mice microglia, 768 differential genes were expressed in the db/db mice microglia, of which 386 genes were up-regulated and 382 genes were down-regulated (Supplementary Table 2). Functional enrichment analysis of upregulated genes was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG). The top 10 most remarkably enriched KEGG pathways were exhibited in microglia. KEGG pathways analysis suggested that compared with the db/dm mice microglia, the enrichment items of the db/db mice microglia were mainly concentrated in infection- and inﬂammation-associated pathways, such as pathways in Coronavirus disease-COVID-19, Epstein-Barr virus infection, phagosome and NF-kB signaling pathway (Figure 6A; Supplementary Table 3). Previous studies demonstrated that brain microglial overactivation can induce proinﬂammatory gene expression by activation of NF-kB signaling pathway following stroke (23), The down-regulated genes were evaluated by KEGG analysis, and the KEGG pathways were mainly enriched for terms involved in Parkinson disease, AD, oxidative phosphorylation, Huntington disease and Pathways of neurodegeneration-multiple diseases (Figure 6B; Supplementary Table 4). GO term analysis of the upregulated genes of microglia revealed enrichment of expected biological processes such as immune system process, inﬂammatory response, cellular response to lipopolysaccharide, defense response to Gram−positive bacterium and positive regulation of interleukin-6 (Figure 6C), and it has also been reported that microglial inﬂammatory activation, stimulated by diabetes mellitus (DM), which caused memory deﬁcits, gene alterations in brain endothelium (24). Furthermore, GO analysis was performed on the down-regulated genes (fold change > 1.5), and the gene functions were primarily enriched in the items related to myelination, microtubule cytoskeleton organization, neuron projection development, nervous system development and oligodendrocyte differentiation. (Figure 6D). Consistent with our ﬁndings, microglial plays a crucial role in neurodegeneration, probably as a result of the different pathological substrates associated with neurodegenerative diseases as well as systemic inﬂammation and factors inﬂuencing insulin C D D C FIGURE 1 \| Learning and memory impairment in db/db mouse. In the MWM test, day 1 represents performance on the ﬁrst trial, and subsequent scores show average of all daily trials. (A) Escape latency of the 5-day acquisition trials in MWM. (B) Representative swim pathways of respective groups in the spatial probe test. (C) Time spent in the target quadrant during the probe trial. (D) The numbers of the target platform crossings in the probe trial. Data are represented mean ± SEM for 3 mice in each group. Immunoﬂuorescence Staining ⁎P < 0.05 vs. db/m group. mice, the relative abundance of microglia and oligodendrocyte in db/db mice changed signiﬁcantly (Figure 2E), so we ﬁrst analyzed this type of cells, and further performed DEGs and/or sub cluster analysis on these data. June 2022 \| Volume 13 \| Article 891039 DEGs Analysis in db/db vs db/dm Mice Microglia Based on DEGs, the following functional networks upregulation were found in db/db microglia (Figure 3; Supplementary Table 1): immune system and response, inﬂammation, neuronal functions, OXPHOS, Cell cycle, NF-kB signal, nucleic acid binding and RNA binding, chromosome maintenance, cytoskeleton, DNA Repair, glucose/lipid metabolism, GTP binding and GTPase activity. Notably, microglia are immune cells that participate in inﬂammatory responses in central nervous system (CNS), we June 2022 \| Volume 13 \| Article 891039 Frontiers in Endocrinology \| www.frontiersin.org ScRNA-Seq of T2DM Mouse Hippocampus Ma et al. A B C D E FIGURE 2 \| Schematic diagram of the experimental workﬂow, and scRNA-seq analysis of db/db vs db/m mouse hippocampus. The workﬂow is illustrated in (A), represent barcode sequence labeled cells. (B) t-SNE plot of 14621 cells isolated from db/db and db/m hippocampus, exhibiting 26 clusters that were identiﬁed. C) t-SNE plot of 14621 cells isolated from the db/db and db/m hippocampus, colour coded according to genotype. (D) Number of db/db (orange) and db/m (cyan) ippocampus cells per cluster. (E) Relative abundances of the different hippocampus cell types per genotype. a et al. ScRNA-Seq of T2DM Mouse Hippocampus A A C B D B D E D E FIGURE 2 \| Schematic diagram of the experimental workﬂow, and scRNA-seq analysis of db/db vs db/m mouse hippocampus. The workﬂow is illustrated in (A), represent barcode sequence labeled cells. (B) t-SNE plot of 14621 cells isolated from db/db and db/m hippocampus, exhibiting 26 clusters that were identiﬁed. (C) t-SNE plot of 14621 cells isolated from the db/db and db/m hippocampus, colour coded according to genotype. (D) Number of db/db (orange) and db/m (cyan) hippocampus cells per cluster. (E) Relative abundances of the different hippocampus cell types per genotype. resistance, such as T2DM (25). Taken together, the results of GO and KEGG analysis reveal that the T2DM could induce the activation of microglia which in turn might cause learning and memory, as well as an increased risk of neurodegenerative diseases. Activated microglia are observed in nearly all neurological disorders, including neurodevelopmental and neurodegenerative diseases. However, it is unclear whether or how microglia regulate their response to speciﬁc types of injury (T2DM induced brain injury). Next, we assessed whether microglial cells in our data could be subdivided into subpopulations based on distinct activation states, cell markers and biological pathways and determined twelve subclusters with a microglial transcriptional proﬁle (Figures 7A, B; Supplementary Table 5). DEGs Analysis in db/db vs db/dm Mice Microglia Subsequently, we sought to determine the proportion of each subpopulation Diverse Microglial Activation Responses Are Triggered in db/db Mice Interestingly, subpopulation 0, db/db mice had an increased proportion of inﬂammatory related subpopulations of microglia, such as subpopulation 1, subpopulation 3, subpopulation 7 and subpopulation 9 (Figure 7C, D). We identiﬁed the marker genes [sorted by average log2(fold change)] for each subpopulation relative to all other microglia subpopulations, and these genes were plotted using a heatmap (Figure 7E; Supplementary Table 6). inﬂammatory response and positive regulation of NF-kB transcription factor activity (Figures 8A–C). Additionally, the cluster 10 expressed the highest levels of the two best-known inﬂammation genes, Gpx1 and Cat (30) (Supplementary Figures 1A–D), the GO terms, including hydrogen peroxide catabolic process, heme biosynthetic process, response to oxidative stress and response to hydrogen peroxide (Supplementary Figure 1E). It is well known that persistent hyperglycemia causes oxidative stress, which leads to brain damage (31). Our gene expression analysis revealed that most of the analyzed cells highly expressed marker microglia genes (C1qa, Fcrls, Cx3cr1, P2ry12/P2ry13, and Trem2) (26), but not all cells (Supplementary Table 6), indicating that existing detection techniques and marker deﬁnitions require further exploration. Except for the highly expressed marker genes, genes signiﬁcantly upregulated were also found in speciﬁc microglial states, such as AD risk genes (Supplementary Table 6) (Ctsb, Ctsd, Trem2, and Tyrobp) are enriched in the activated response microglia (ARM), which was identiﬁed main activated microglia states by Sala Frigerio et al. (26). This highlights cognitive dysfunction in diabetes and the pathogenesis of AD may share some commonalities. We also observed upregulation of neurodegeneration- and inﬂammation- related genes such as Csf2ra, Jun, Cox6c, Tlr7, and Ttr. We found that cluster 1 expressed the higher levels of Gadd45b (Growth arrest and DNA-damage-inducible 45 beta) gene, which acts as an apoptosis factor and mediator of neuroinﬂammation (13, 27, 28), and inﬂammation makers (Cd14 and Tlr2) (13, 29), and showed enrichment for the GO term regulation of immune system process, Interestingly, increased transcripts were observed for other Ms4a family members (Ms4a6b, Ms4a6d, Tmem176a, and Tmem176b) expressed by microglia (Cluster 0, 1, 3, 5, 6, 8, 9) (Supplementary Table 6). Genes such as macrophage migration inhibitory factor (Mif), lactate dehydrogenase A (Ldha), and triosephosphate isomerase 1 (Tpi1) were found to be upregulated in cluster 5 microglia (Supplementary Table 6). Of note, we found the most signiﬁcantly enriched genes in cluster 7 microglia expressed many inﬂammatory signals that were not normally expressed in other microglial clusters (Figures 9A-B; Supplementary Table 6). Diverse Microglial Activation Responses Are Triggered in db/db Mice Study has often referred to the response of microglia to injury or pathology as ‘‘activated,’’ an umbrella term that refers to biochemical and physiological deviations from homeostasis. June 2022 \| Volume 13 \| Article 891039 Frontiers in Endocrinology \| www.frontiersin.org 5 Ma et al. ScRNA-Seq of T2DM Mouse Hippocampus FIGURE 3 \| STRING analysis of DEGs networks in db/db with db/m microglia. Shapes represent up - and downregulated messages. Triangles represent upregulated gene networks and circles represent downregulated gene networks; Different colors represent different cluster, the size of the dots represents the degree. FIGURE 3 \| STRING analysis of DEGs networks in db/db with db/m microglia. Shapes represent up - and downregulated messages. Triangles represent upregulated gene networks and circles represent downregulated gene networks; Different colors represent different cluster, the size of the dots represents the degree. June 2022 \| Volume 13 \| Article 891039 June 2022 \| Volume 13 \| Article 891039 Frontiers in Endocrinology \| www.frontiersin.org ScRNA-Seq of T2DM Mouse Hippocampus Ma et al. A B C D E FIGURE 4 \| The changes of microglial activation and neuroinﬂammation in hippocampal CA1 sections from from db/m and db/db group. (A) Representative immunoﬂuorescence staining of Iba-1, Nlrp3, Cmklr1 and Trem2. Representative images of Iba-1 (red) staining and representative images of Nlrp3, Cmklr1, Trem2 (green) staining. Nuclei are stained with DAPI (blue). (scale bar = 50 mm). Quantiﬁcation of Iba1- (B), Nlrp3- (C), Cmklr1- (D), Trem2- (E) positive cells in the db/db mice hippocampus. All data are represented as means ± S.E.M for 8 mice in each group. P < 0.05, P < 0.01 vs. db/m group. A B FIGURE 4 \| The changes of microglial activation and neuroinﬂammation in hippocampal CA1 sections from from db/m and db/db group. (A) Representative immunoﬂuorescence staining of Iba-1, Nlrp3, Cmklr1 and Trem2. Representative images of Iba-1 (red) staining and representative images of Nlrp3, Cmklr1, Trem2 (green) staining. Nuclei are stained with DAPI (blue). (scale bar = 50 mm). Quantiﬁcation of Iba1- (B), Nlrp3- (C), Cmklr1- (D), Trem2- (E) positive cells in the db/db mice hippocampus. All data are represented as means ± S.E.M for 8 mice in each group. P < 0.05, *P < 0.01 vs. db/m group. (Figures 7C, D), and perform DEGs and gene set enrichment analysis. Frontiers in Endocrinology \| www.frontiersin.org Diverse Microglial Activation Responses Are Triggered in db/db Mice They upregulated various pro-inﬂammatory chemokines Nlrp3, Lgals3, Cxcl2, Cxcl10, Cxcl16 and inﬂammatory cytokine interleukin 1 (Il1a, Il1b), as well as Ccl4 and Ccl3 (32, 33). The ligand for chemokine receptor type 5 (Ccr5) is Ccl4, also referred to as macrophage inﬂammatory protein-1b (Mip-1b), which adjusts the transmission and responder functions of different immune cells (34). Additionally, cluster 7 showed enrichment for the GO term regulation of immune system process, inﬂammatory response, June 2022 \| Volume 13 \| Article 891039 Frontiers in Endocrinology \| www.frontiersin.org 7 ScRNA-Seq of T2DM Mouse Hippocampus Ma et al. A B C FIGURE 5 \| The changes of expression of Nrf2/HO-1 system in hippocampal CA1 sections from from db/m and db/db group. (A) Immunoﬂuorescence analysis of Nrf2 and HO-1. Representative images of Nrf2 and HO-1 (green) staining in the hippocampus. Nuclei are stained with DAPI (blue). (scale bar = 50 mm). Quantiﬁcation of Nrf2- (B) and HO-1- (C) positive cells in the db/db mice hippocampus. All data are represented as means ± S.E.M for 8 mice in each group. P < 0.05 vs. db/m group. FIGURE 5 \| The changes of expression of Nrf2/HO-1 system in hippocampal CA1 sections from from db/m and db/db group. (A) Immunoﬂuorescence analysis of Nrf2 and HO-1. Representative images of Nrf2 and HO-1 (green) staining in the hippocampus. Nuclei are stained with DAPI (blue). (scale bar = 50 mm). Quantiﬁcation of Nrf2- (B) and HO-1- (C) positive cells in the db/db mice hippocampus. All data are represented as means ± S.E.M for 8 mice in each group. *P < 0.05 vs. db/m group. positive regulation of I-kb kinase/NF-kB signaling and NF-kB transcription factor activity (Figure 9C), leading us to postulate that these cells represent pro-inﬂammatory lesional microglia, which might be deﬁned as into the phenotypes M1 (proinﬂammatory) (35). Recently Rangaraju et al. found signiﬁcant molecular heterogeneity in DAM (disease-associated microglia) (13). Interestingly, genes related to pro-inﬂammatory DAM, such as Tlr2, Ptgs2, Il1b and regulators (NF-kb, Stat, and RelA), were mainly enriched in cluster 7 microglia (Supplementary Table 6). Moreover, cluster 6 microglia also expressed upregulated the inﬂammatory signals genes in cluster 7 microglia, indicating an overlap in transcriptional signaling and may be some association between these two states. Diverse Microglial Activation Responses Are Triggered in db/db Mice These ﬁndings could be translated to human disease, and novel microglial pathogenic markers might serve as biomarkers or therapeutic targets, which also help to further deﬁne how we distinguish microglial activation states in vivo. contribute to oxidative stress, amyloid- b accumulation, tau hyperphosphorylation, formation of advanced glycation end products (36). Whereas, the multifactorial pathogenesis of diabetic encephalopathy remains unclear. The diabetic db/db mouse is a ideal animal model to study the pathophysiology of diabetes mellitus, because this organism, like humans, develops diabetes and its associated complications, including oxidative stress, obesity, and hyperglycemia. Furthermore, db/db mouse shows neurobehavioral deﬁcits, autonomic behavior and memory impairment are important roles of hippocampal damage caused by T2DM (37). In the present study, our MWM results suggested that db/db mice suffered from impaired in memory ability and spatial learning, indicating db/db mice had deﬁcits in long-term and spatial memory functions (38, 39). The complexity of hippocampus structural makes it vulnerable to a variety of pathological conditions such as diabetes and is one of the most sensitive regions of the brain to microenvironmental changes (1). Next, we explored the different cell types and their unique transcriptional features in the mouse hippocampus at single-cell level using scRNA-seq technology. Overall, the results of analysis based on changes in cell abundance and intercellular communication suggest different hippocampus cells exhibit different degrees of sensitivity to DCD. Analysis of DEGs between the db/m and db/db groups showed that gene transcription in each cell type underwent widespread changes in db/db mice. Frontiers in Endocrinology \| www.frontiersin.org DISCUSSION Cognitive impairment is an important complication in the CNS of diabetic patients, which could reﬂect diabetes induced changes in the brain. There are some studies suggesting the link between AD and T2DM, such as hyperglycemia, leading to glutamate induced excitotoxicity in neuronal cells and may June 2022 \| Volume 13 \| Article 891039 Frontiers in Endocrinology \| www.frontiersin.org 8 ScRNA-Seq of T2DM Mouse Hippocampus Ma et al. A B C D FIGURE 6 \| DEGs analysis reveal altered pathways in microglial cells in db/db mice. Representative GO (A) and KEGG (C) terms of upregulated gene in db/db mice microglial cells versus db/m microglial cells and representative GO (B) and KEGG (D) terms of downregulated gene in db/db mice microglial cells versus db/m microglial cells. Signiﬁcance is indicated as a P value calculated using the Fisher exact test (P< 0.05) and expressed as -log10 (P value). A D FIGURE 6 \| DEGs analysis reveal altered pathways in microglial cells in db/db mice. Representative GO (A) and KEGG (C) terms of upregulated gene in db/db mice microglial cells versus db/m microglial cells and representative GO (B) and KEGG (D) terms of downregulated gene in db/db mice microglial cells versus db/m microglial cells. Signiﬁcance is indicated as a P value calculated using the Fisher exact test (P< 0.05) and expressed as -log10 (P value). The pathogenesis of DCD is complex and not very clear, among which the more deﬁnite view is that persistent inﬂammation caused by the secretion of a large number of pro-inﬂammatory factors and pro-oxidant substances is the main contributing factor to DCD (40). Activated microglia of the CNS are major cellular responders to injury or infection, which may drive or perpetuate CNS inﬂammation by increasing the expression of inﬂammatory molecules, thereby exacerbating some of these detrimental processes (41). GO and KEGG pathway analyses showed that inﬂammation-, immunity-, and infection-associated terms were enriched in db/db mice microglia, such as Il17ra, Il6ra, Cmklr1, Nlrp3, Trem2, Coronavirus disease-COVID-19, Epstein-Barr virus infection, phagosome and NF-kB signaling pathway. Among immune cells in the CNS, which has a vital role in propagating neuroinﬂammation (42). Numerous evidences suggested that the activation of Nlrp3 plays a vital role in DCD and other neurodegenerative diseases (43, 44). Nlrp3 is mainly involved in neuroinﬂammation in vivo and in vitro. Frontiers in Endocrinology \| www.frontiersin.org DISCUSSION For example, recent studies have reported that Nlrp3 can activate Tlr4 signaling, leading to the neuroinﬂammatory responses (45), and illustrated the possible mechanism related to Nlrp3 inﬂammasome and peripheral inﬂammation in microglia of mice, and the aggravation of diabetes neuroinﬂammation in the cortex and hippocampus (46, 47). Additionally, research shows that the chemokine-like receptor 1 (Cmklr1) is a chemokine like G protein-coupled receptor expressed on speciﬁc cell populations in mice and humans, including inﬂammatory mediators, dendritic cells, neurons and microglia, regulating chemotaxis to the sites of inﬂammation and activation state (48, 49). Lately, it has been reported that Cmklr1 axis contributed to the development of diabetic cardiomyopathy on inﬂammation, which was primarily mediated by Nlrp3 inﬂammasome (50). Triggering receptor expressed on myeloid cells 2 (Trem2) is a single pass transmembrane receptor that activates a range of signaling pathways associated with immune function through ligand binding (51). Trem2 has been used as a microglial activation marker and a general requirement for its activation (29, 52). Galectin-3 can bind to the Trem2, activate microglia, and induce neuroinﬂammatory responses in an AD mouse model, our recent study conﬁrmed that inhibition of endogenous Trem2 ligand ameliorates DCD by inhibiting oxidative stress and neuroinﬂammation (53). In addition, studies have shown that hyperglycemia leads to NF‐kB activation and triggering the release of proinﬂammatory cytokines (54). Cytokines (IL and TNF‐a) expressed and secreted by microglia in the CNS and are involved in the regulation of neuroimmune endocrine networks (55). The histological features of many neurological diseases are mostly characterized as ‘‘microgliosis’’, which includes alterations in microglial morphology, change of gene expression, but also migration, growth and proliferation in response to injury (56). June 2022 \| Volume 13 \| Article 891039 Frontiers in Endocrinology \| www.frontiersin.org 9 ScRNA-Seq of T2DM Mouse Hippocampus Ma et al. A B C D E \| Subclustering of microglial cells. (A) t-SNE plot showing microglial cells subclusters. (B) t-SNE plot of microglial cell, colour coded according to C) Percentage of cells in each microglial cell cluster. (D) Cell cluster percentages across two experimental groups (db/db and db/m). (E) The heatmap e expression levels of speciﬁc genes in microglial cells subclusters. A B C D A B B D C D C E FIGURE 7 \| Subclustering of microglial cells. (A) t-SNE plot showing microglial cells subclusters. (B) t-SNE plot of microglial cell, colour coded according to genotype. DISCUSSION (C) Percentage of cells in each microglial cell cluster. (D) Cell cluster percentages across two experimental groups (db/db and db/m). (E) The heatmap showing the expression levels of speciﬁc genes in microglial cells subclusters. E RE 7 \| Subclustering of microglial cells. (A) t-SNE plot showing microglial cells subclusters. (B) t-SNE plot of microglial cell, colour coded according to ype. (C) Percentage of cells in each microglial cell cluster. (D) Cell cluster percentages across two experimental groups (db/db and db/m). (E) The heatmap FIGURE 7 \| Subclustering of microglial cells. (A) t-SNE plot showing microglial cells subclusters. (B) t-SNE plot of microglial cell, colour coded according to genotype. (C) Percentage of cells in each microglial cell cluster. (D) Cell cluster percentages across two experimental groups (db/db and db/m). (E) The heatmap showing the expression levels of speciﬁc genes in microglial cells subclusters. June 2022 \| Volume 13 \| Article 891039 Frontiers in Endocrinology \| www.frontiersin.org 10 ScRNA-Seq of T2DM Mouse Hippocampus Ma et al. A B C FIGURE 8 \| Gene expression pattern analyses in cluster 1. (A) Feature plot of inﬂammation marker genes Gadd45b, Cd14 and Tlr2 in cluster 1. (B) Violin plots revealed the expression of inﬂammation marker genes for 1 cluster. (C) The enriched GO terms of cluster 1 are shown. A B B C FIGURE 8 \| Gene expression pattern analyses in cluster 1. (A) Feature plot of inﬂammation marker genes Gadd45b, Cd14 and Tlr2 in cluster 1. (B) Violin plots revealed the expression of inﬂammation marker genes for 1 cluster. (C) The enriched GO terms of cluster 1 are shown. C FIGURE 8 \| Gene expression pattern analyses in cluster 1. (A) Feature plot of inﬂammation marker genes Gadd45b, Cd14 and Tlr2 in cluster 1. (B) Violin plots revealed the expression of inﬂammation marker genes for 1 cluster. (C) The enriched GO terms of cluster 1 are shown. “alternative activation,” and “acquired deactivation” (58). Classical activation is related to the production of pro- inﬂammatory cytokines such as TNF-a, nitric oxide (NO) and proteases and are also called “M1 microglia,”, whereas alternative activation and acquired deactivation are termed “M2 microglia” (58). In brief, the M1 activation state is deﬁned as the pro- inﬂammatory M1 phenotypes, and the M2 activation state is considered to anti-inﬂammatory or neuroprotective M2 phenotypes. Frontiers in Endocrinology \| www.frontiersin.org DISCUSSION In our results, compared with db/m mice, the relative abundance of microglia in db/db mice changed signiﬁcantly, further analysis of differentially expressed genes revealed, the inﬂammation related genes were found to be upregulated in db/db microglia, which leading us to postulate that these cells represent pro-inﬂammatory lesional microglia, which might be deﬁned as into the phenotypes M1 (proinﬂammatory). However, simple classiﬁcation scheme Iba-1, a microglia speciﬁc marker, has been reported to be widely used for microglial detection, and increased expression of Iba-1 could suggest microglial activation (57), which was also conﬁrmed by our Iba-1 immunoﬂuorescent staining results. In addition, Microglial activation was closely associated with neurodegenerative diseases, such as AD, Parkinson’s disease, and multiple sclerosis (40). Interestingly, our KEGG pathway analyses revealed that Parkinson disease, AD, Huntington disease and Pathways of neurodegeneration-multiple diseases were enriched in microglia. The state of microglia in the CNS changes rapidly with the change of environment, but its molecular and functional characteristics are not clear (41). Accumulating evidence suggests that CNS microglial activation is heterogeneous, depending on the factors that microglia become activated, there are three states of microglia, “classical activation,” June 2022 \| Volume 13 \| Article 891039 Frontiers in Endocrinology \| www.frontiersin.org 11 ScRNA-Seq of T2DM Mouse Hippocampus Ma et al. A B C FIGURE 9 \| Gene expression pattern analyses in cluster 7. (A) Feature plot of inﬂammation marker genes Nlrp3, Il1a, Il1b and Cxcl2 in cluster 7. (B) Violin plots revealed the expression of inﬂammation marker genes for 1 cluster. (C) The enriched GO terms of cluster 7 are shown. A A B B C FIGURE 9 \| Gene expression pattern analyses in cluster 7. (A) Feature plot of inﬂammation marker genes Nlrp3, Il1a, Il1b and Cxcl2 in cluster 7. (B) Violin plots revealed the expression of inﬂammation marker genes for 1 cluster. (C) The enriched GO terms of cluster 7 are shown. might lump together heterogenous sets of microglia, the information obtained from histological research might be limited or even misunderstood. transcription factor activity were enriched in cluster 7. We speculate that they might be a specialized group that uniquely initiates the inﬂammatory response. For example, Il1b can cause neurotoxicity (61), chemokines Ccl4 attracts inﬁltrating immune cells can exacerbate pathology. Frontiers in Endocrinology \| www.frontiersin.org DISCUSSION Lgals3 is the gene of galectin-3, our recent study showed that serum galectin-3 levels were remarkably increased not only in the of T2DM patients with mild cognitive impairment (MCI), but also in the serum and brain of T2DM mice, and we also conﬁrmed galectin-3 was associated with neuroinﬂammation, oxidative stress, impaired learning and memory (53, 62). Our results suggested that galectin-3 could be a promising therapeutic target candidate for treating DCD. The functions of Ms4a family genes found in cluster (0, 1, 3, 5, 6, 8, 9) are currently not well deﬁned and are primarily are transmembrane chemosensors (63), some of which regulate immune cell functions (64). Ms4a family members are a key modulator of Alzheimer’s disease risk (65), nevertheless, the functions of Ms4a family members in the disease is not understood. In addition, Mif in cluster 5 microglia is closely linked to the growth, motility, inﬂammation and immune regulation of immune cells in the CNS. Ldha and Tpi1 were Next, we identiﬁed twelve microglial subpopulations according to microglia with different activation states/biological pathways, which possessed unique molecular signatures in response to injury. We found that cluster 1 expressed the higher levels of Gadd45b gene, which was a member of the gene family related to apoptosis, and DNA damage repair (13). Studies have demonstrated that Gadd45b might be critically involved in neuroinﬂammation related neurological diseases (59, 60). Interestingly, our previous study conﬁrmed Gadd45b modulated hippocampal neuroinﬂammation in animal model of post-stroke depression (PSD), Gadd45b may have therapeutic potential for CNS diseases through neuroinﬂammation (27). Of note, we found that inﬂammation associated terms were enriched in cluster 7, and express Nlrp3, Lgals3, Cxcl2, Cxcl10, Cxcl16 and inﬂammatory cytokine interleukin 1 (Il1a, Il1b), as well as chemokines Ccl4 and Ccl3. Additionally, regulation of immune system process, inﬂammatory response, positive regulation of I-kb kinase/NF-kB signaling and NF-kB June 2022 \| Volume 13 \| Article 891039 Frontiers in Endocrinology \| www.frontiersin.org 12 ScRNA-Seq of T2DM Mouse Hippocampus Ma et al. related to glycolysis, indicating that the metabolic proﬁle of these cells has changed. A change from oxidative phosphorylation to glycolysis and metabolism occurs when microglia are stimulated by adverse factors (40), and they regulate glucose metabolic proﬁle after the transition to the active state, presumably to meet increased energy requirements. relevance in humans, in patient samples. DISCUSSION Moreover, further evaluation of the differentially expressed pathways in functional experiments is required to assess whether they play a deleterious or protective role in DCD. SUPPLEMENTARY MATERIAL In conclusion, through single-cell RNA sequencing, we gained a deeper insight into the heterogeneity of microglial cell populations in the db/db mice hippocampus. We observed changes in the abundance of hippocampal cell populations, most notably microglial cell populations, which conﬁrm the novel opinion that microglia play a vital role in DCD. Our ﬁndings in this article should be veriﬁed in additional early/ intermediate stage DCD models, and to determine their The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo.2022.891039/ full#supplementary-material Supplementary Figure 1 \| Gene expression pattern analyses in cluster 10. Feature plot of inﬂammation marker genes Gpx1 (A) and Cat (C) in cluster 10. Violin plots showing the expression of inﬂammation marker genes Gpx1 (B) and Cat (D) for 10 cluster. (E) The enriched GO terms of cluster 10 are shown. DATA AVAILABILITY STATEMENT gy q A potential subtype of protective microglia, disease-associated microglia (DAM) were recently proposed in mice model of AD (5xFAD), and decipher their dynamics during AD progression (12). Interestingly, the conversion of microglia from a stable state to an activation status is thought to be a continual process, which includes two phases. The Trem2 independent stage (DAM1), involving activation of Tyrobp, Apoe, and B2m, and downregulation of the Cx3cr1 and P2ry12/P2ry13 genes, followed by the Trem2 dependent stage (DAM2) including upregulation of Cst7, Lpl, and CD9 genes (12). Research showed that DAM mitigates the disease through enhancing phagocytosis in the late stage of AD (12). In addition, single-cell analysis conﬁrmed DAM have been identiﬁed in normal aging and in models of neurodegenerative disease, suggesting that DAM represent a general response to neurodegenerative diseases (66). Interestingly, we did not detect protective microglia associated with neurodegeneration in diabetes. DCD may be an early stage of dementia with MCI, or cognitive impairment due to diabetes related cerebrovascular damage could serve as an explanation. Whereas, our study found pro-inﬂammatory DAM emerges in hippocampus of T2DM mouse model and are characterized by expression of pro-inﬂammatory genes (Tlr2, Ptgs2, and Il1b) and regulators (NF-kB, Stat, and RelA) (13). Over all, our study discovers previously unknown heterogeneity of microglia, a potential detrimental microglia type associated with DCD. Additionally, understanding the heterogeneity within the DAM may ﬁnd new biological opinions into diversity of microglia and might contribute to the discovery of immunomodulatory therapeutic targets and drugs for DCD. The original contributions presented in the study are publicly available. This data can be found here: NCBI, GSE201644. FUNDING This work was supported by National Natural Science Foundation of China (Grant No.82000771), Shandong Provincial Natural Science Foundation (Grant No. ZR2021MH014, ZR2019PH017), and Jinan Municipal Science and Technology Project (grant no. 202134033). ETHICS STATEMENT The animal study was reviewed and approved by Ethics Committee of Shandong Provincial Hospital Afﬁliated to Shandong First Medical University. AUTHOR CONTRIBUTIONS SM, RL, QY, and WB designed the experiments, analysis was carried out by XL, SL, YQ, and CH. SM and QY supervised the study. RL and QY wrote the article. 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https://openalex.org/W4210471953	https://hull-repository.worktribe.com/file/4099651/1/Published%20article	English	null	Data-Driven Techniques for Low-Cost Sensor Selection and Calibration for the Use Case of Air Quality Monitoring	Sensors	2,022	cc-by	22,077	Data-Driven Techniques for Low-Cost Sensor Selection and Calibration for the Use Case of Air Quality Monitoring Rameez Raja Kureshi 1 , Bhupesh Kumar Mishra 1, Dhavalkumar Thakker 1,, Reena John 1, Adrian Walker 2 Sydney Simpson 2, Neel Thakkar 3 and Agot Kirsten Wante 4 Rameez Raja Kureshi 1 , Bhupesh Kumar Mishra 1, Dhavalkumar Thakker 1,, Reena Joh Sydney Simpson 2, Neel Thakkar 3 and Agot Kirsten Wante 4 1 Faculty of Engineering and Informatics, University of Bradford, Bradford BD7 1DP, UK; krameezr@bradford.ac.uk (R.R.K.); b.mishra@bradford.ac.uk (B.K.M.); rjohn1@bradford.ac.uk (R.J.) 2 City of Bradford Metropolitan District Council, Bradford BD1 1HX, UK; adrian.walker@bradford.gov.uk (A.W.); sydney.simpson@bradford.gov.uk (S.S.) 3 IBM India Pvt Ltd., Kolkata 700156, India; neelthakkar@acm.org 4 IVL Swedish Environmental Research Institute, 114 28 Stockholm, Sweden; agot.watne@ivl.se * Correspondence: d.thakker@bradford.ac.uk g 4 IVL Swedish Environmental Research Institute, 114 28 Stockholm, Sweden; agot.watne@ivl. * C d d th kk @b df d k * Correspondence: d.thakker@bradford.ac.uk Abstract: With the emergence of Low-Cost Sensor (LCS) devices, measuring real-time data on a large scale has become a feasible alternative approach to more costly devices. Over the years, sensor technologies have evolved which has provided the opportunity to have diversity in LCS selection for the same task. However, this diversity in sensor types adds complexity to appropriate sensor selection for monitoring tasks. In addition, LCS devices are often associated with low conﬁdence in terms of sensing accuracy because of the complexities in sensing principles and the interpretation of monitored data. From the data analytics point of view, data quality is a major concern as low-quality data more often leads to low conﬁdence in the monitoring systems. Therefore, any applications on building monitoring systems using LCS devices need to focus on two main techniques: sensor selection and calibration to improve data quality. In this paper, data-driven techniques were presented for sensor calibration techniques. To validate our methodology and techniques, an air quality monitoring case study from the Bradford district, UK, as part of two European Union (EU) funded projects was used. For this case study, the candidate sensors were selected based on the literature and market availability. The candidate sensors were narrowed down into the selected sensors after analysing their consistency. To address data quality issues, four different calibration methods were compared to derive the best-suited calibration method for the LCS devices in our use case system. In the calibration, meteorological parameters temperature and humidity were used in addition to the observed readings. Citation: Kureshi, R.R.; Mishra, B.K.; Thakker, D.; John, R.; Walker, A.; Simpson, S.; Thakkar, N.; Wante, A.K. Data-Driven Techniques for Low-Cost Sensor Selection and Calibration for the Use Case of Air Quality Monitoring. Sensors 2022, 22, 1093. https://doi.org/ 10.3390/s22031093 Academic Editors: Raffaele Bruno and Manuel Aleixandre Received: 30 September 2021 Accepted: 25 January 2022 Published: 31 January 2022 Citation: Kureshi, R.R.; Mishra, B.K.; Thakker, D.; John, R.; Walker, A.; Simpson, S.; Thakkar, N.; Wante, A.K. Data-Driven Techniques for Low-Cost Sensor Selection and Calibration for the Use Case of Air Quality Monitoring. Sensors 2022, 22, 1093. https://doi.org/ 10.3390/s22031093 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Keywords: Low-Cost Sensor (LCS); calibration; data-driven techniques; drift analysis; air quality sensors sensors sensors Data-Driven Techniques for Low-Cost Sensor Selection and Calibration for the Use Case of Air Quality Monitoring Moreover, we uniquely considered Absolute Humidity (AH) and Relative Humidity (RH) as part of the calibration process. To validate the result of experimentation, the Coefﬁcient of Determination (R2), Root Mean Square Error (RMSE), and Mean Absolute Error (MAE) were compared for both AH and RH. The experimental results showed that calibration with AH has better performance as compared with RH. The experimental results showed the selection and calibration techniques that can be used in designing similar LCS based monitoring systems. 1. Introduction Low-Cost Sensors (LCSs) are changing the conventional way to monitor and measure instances in real-time with the help of micro-scale sensing techniques [1]. The use of LCSs has beneﬁts in terms of cost-effectiveness, compactness, and portability which make these devices an efﬁcient alternative against the high-cost monitoring systems [2]. For example, building LCS-based air quality monitoring devices (use case used in this study) to deploy against high-cost air quality monitoring stations in a city is more feasible in terms of high spatio-temporal and instantaneous data monitoring to the user at any speciﬁc location. Additionally, LCSs have appeared as an economical substitute for high-cost Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/sensors Sensors 2022, 22, 1093. https://doi.org/10.3390/s22031093 Sensors 2022, 22, 1093 2 of 23 sensors devices in many applications including air quality monitoring (Indoor–Outdoor), ﬂood monitoring, and observing health status. In recent years, with the enhancement in sensor technology, there have been many alternative sensors to perform the same tasks while developing any LCS-based applications. However, there has not been a universal single type of LCS implementation as the LCSs have different working principles such as electrochemical, optical particle counters (OPC), non-dispersive infra-red (NDIR), metal- oxide-semiconductor, or solid-state microsensors designed to monitor air pollutants [3–5]. This diversity in working principles of sensors adds complexity to the process of LCS selection while building the monitoring systems using LCS-based devices. Apart from the working principle of LCS devices, meteorological parameters such as temperature and humidity make LCS data unreliable and less accurate when they have been used in an open environment [6,7]. Furthermore, LCSs are monitoring various components from the environment according to their function which can cause sensitivity issues [8,9]. For example, when the LCS is used to monitor air pollutants, the sensor also responds more to other gas compounds in addition to the actual gas detection [10]. Moreover, LCSs have characteristics that cannot give a stable performance over a certain period due to drift of sensitivity and ageing which can cause increased data inaccuracy [9,11]. Apart from the issues discussed earlier, LCSs also often face challenges to detect measured levels below a point where it would not be able to differentiate between sensor noise and actual sensed values in the environment [9,12,13]. 1. Introduction This is because the sensor is designed in a range called the dynamic boundary. When the sensor faces the actual data level near or below the dynamic boundary, it often fails to monitor the data accurately. To overcome this, it is a prerequisite to calibrate LCSs before the on-ﬁeld application is required [14]. In addition to these challenges, LCS data quality and their consistency under the same environment are other factors that make the LCS-based applications even more complex. pp p Considering the lower feasibility of high-cost air quality monitoring sensors to de- ploy at many strategic locations within a small area, LCS-based devices become a feasible solution for many applications. However, the use of LCSs brings forth its challenges in terms of sensor selection, quality of data and accuracy of measurement [9,15]. There have been different applications that have used LCSs for real-time monitoring, but these appli- cations do not talk about the sensor selection and their calibration process [3,5,8–10,16]. Furthermore, the market availability of sensors in different forms to measure the same component adds a challenge to LCS selection. To our best knowledge, there have been challenges in terms of how to select, calibrate, and build LCS-based devices considering twin factors of sensors’ availability and data quality. To improve the data quality issue, calibration methods have been used to improve LCS-based devices’ performances. Over the years, there have been different calibration methods such as Multiple Linear Regres- sion (MLR) [5,17], Random Forest (RF) [18,19], Support Vector Regression (SVR) [3], and Artiﬁcial Neural Networks (ANN) [20] have been used to improve data quality. Finding an accurate calibration method for the LCS selection based on the LCS deployment en- vironment has made the calibration task further complex as various LCSs have different working principles and conﬁgurations [9]. Although different calibration methodologies have been applied to improve data quality to the LCS-based monitoring applications, there is a need for a more precise and comparable approach on how to calibrate LCSs to ascertain data quality assurance with respect to industrial scale, high-cost, high-quality reference data. Since the calibration is dependent on the types of LCS-based devices, applications, deployment environment, and meteorological parameters, there is a need for a well-deﬁned methodology for sensor calibration. gy In this paper, a data-driven LCS calibration methodology was presented to address the challenges of LCS-based device building for an application. 2. Literature Review With the increase in the availability of micro-sensor technology in recent years, the use of LCS-based devices has been boosted in monitoring and measurements applications with wider spatial coverage in different domains [22]. Comparing LCSs with high-cost monitoring stations, LCSs are less expensive and easy to access and deploy. Though, the data from LCSs are generally less reliable with low quality [9,15,23]. Predominantly, the data collected from LCS-based devices are easy to handle, process, and can be analysed by experts allowing sharing the monitoring outcomes with the public or stakeholders to spread awareness and other purposes [1]. There has been a wide range of application domains such as air quality [16], road trafﬁc [24], water quality [25] and human health [26] which makes the LCS-based devices more demanding. Castell et al. [10] evaluated the performance of the LCS (AQMesh) and analysed data quality. They also listed several funded applications based on LCS-based applications such as OpenSense (https://gitlab.ethz.ch/tec/public/ opensense, accessed on 15 February 2021), Everyaware (http://www.everyaware.eu/, accessed on 20 February 2021), Citi-Sense-MOB and Citi-Sense [27] where the devices mount on vehicles or any stationary location and start sending real-time data to their web platform from eight European cities for further processing. These devices have individual gas sensors which cost around EUR 20–100 each and the whole monitoring device cost range is EUR 500-5000 approximately. Chojer et al. [16] reviewed various works from 2012 to 2019 based on LCS-based monitoring systems and ﬁnalised the most relevant 35 research applications with meteorological parameters such as temperature and RH. Additionally, this study has shown that out of 35 applications, only 10 applications were focused on sensor performance including validation, calibration, and testing. The main beneﬁt of LCS-based applications is their affordability and availability in the market [28]. Kumar et al. [29] showed the beneﬁts of LCS-based applications in terms of accessing real-time data, increased spatial resolution, reduced uncertainty, identiﬁcation of emitting sources from indoor activities, and health beneﬁts compared with the traditional monitoring systems. This work also highlighted some challenges such as data quality and performance evaluation for LCS-based device development. Some of the studies [30–32] showed that neither temperature nor RH has any inﬂuence on LCS performance. Zou et al. 1. Introduction In this proposed methodol- ogy, at ﬁrst, candidate LCS selection was applied based on literature reviews and sensors’ market availability. Data-driven statistical analyses [21] give the conﬁdence to select the most consistent LCSs for building LCS-based devices to improve data quality. In this paper, considering the air quality monitoring application, a comparative analysis was explored Sensors 2022, 22, 1093 3 of 23 among the widely used calibration methods by applying Absolute Humidity (AH) and Relative humidity (RH) along with temperature and measured pollutants. Four different calibration methods, (i) Multivariate Linear Regression, (ii) Multi-Layer Perceptron (MLP), (iii) Convolution Neural Network (CNN), and (iv) Random Forest (RF) were compared with both AH and RH as calibration parameters to ﬁnd the best-suited calibration method for the selected LCS in a real-world use case application in two EU projects. The calibration of selected LCSs was accomplished with the reference of a high-cost air quality monitor- ing station from Urban Observatory, Shefﬁeld (https://urbanﬂows.ac.uk/, accessed on 1 March 2021). Among different calibration models, the RF model has better performance in terms of the coefﬁcient of determination, root mean square error, and mean absolute error. In addition, it was observed that calibration accuracy has better performance when AH was used over RH as one of the calibration parameters. p The rest of the paper is organised as follows. In Section 2, a comprehensive review of LCSs and LCS-based applications is presented. In Section 3, the data-driven calibration techniques are presented. In Section 4, the experimental result analysis and discussion are presented. In Section 5, the paper is completed with a conclusion along with future work. 2. Literature Review This raises an issue when selecting appropri- ate LCSs that can minimise this trade-off and provide more reliable data. All applications based on LCSs have common problems that are data reliability and data quality [5,18]. Maag et al. [9] surveyed LCS problems and found that LCS data quality can be inﬂuenced and relied upon by several factors such as sensor types and working principles, meteoro- logical parameters, low sensitivity, and sensor consistency. Kotsev et al. [39] explain the approaches for reliable data quality from LCSs and mention some of the known parameters which can affect electrochemical sensor responses such as temperature, humidity and cross-sensitivity. y As a solution to address all of these challenges of LCSs, sensor calibration is re- quired [40,41]. Some of the calibrations have been conducted in a lab environment such as the research of Wang et al. [42] who calibrated LCS PM in lab conditions whereas Spinelle et al. [37] applied on-ﬁeld calibration of LCSs against a reference station. Choosing a calibration model depends upon certain parameters such as the type of sensor, type of phenomena of the device, resources required for that device, storage, computation, and communication capabilities of those sensors [38]. Multivariate Linear Regression (MLR) has been widely used for calibration [5,17]. For the calibration using MLR, two or more covariates are mainly used to get the targeted variables outcome. The MLR model is easy to implement but has limitations as well since the MLR used a linear equation with coefﬁcients based on some assumptions such as linearity, residual error, and co-linearity. Another model that has been also widely used is Random Forest (RF) [18,19]. The RF-based model improved the stability by randomly selecting several observations from the dataset during training leaving some of the datasets for testing the model. The predictions are based on the mean of the results coming from a number of trees. However, the tree size grows with the increase in the dataset size, which increases time complexity while training the model. Support Vector Regression (SVR) appeared as another method used for the calibration [3]. An SVR-based calibration model uses kernel functions to train the model with the given datasets. SVM generates an optimal hyperplane to distinguish different classes to predict the outcomes. However, the SVM-based model required users to deﬁne the number of support vectors. Artiﬁcial neural networks (ANN) [20] are also among the commonly used calibration models. 2. Literature Review [33] experimented with eight different low-cost PM sensors to ﬁnd the relation between temperature and humidity in a lab environment and found that the temperature does not have any signiﬁcant effect on LCS functioning. Additionally, their study highlighted that the RH in the range of 10–90% may affect the magnitude of the sensor’s output and it can be improved through calibration. Zamora et al. [34] show that the meteorological parameter RH has a signiﬁcant impact on LCS performance. Similarly, Jayaratne et al. [35] have experimented with different Particulate Matter (PM) Sensors 2022, 22, 1093 4 of 23 sensors and found that if RH exceeds 75% then it affects the sensor’s performance adversely. Laurent et al. [36,37] used AH instead of RH and temperature for the LCS calibration process. This study raised the signiﬁcance of environmental parameters and working principles of LCSs, which required more sophisticated calibration efforts for data reliability. p p q p y Sensor calibration is the method of adjustment performed on the sensor to make the functionality of the sensor as accurate as possible and error-free. Maag et al. [9] surveyed various calibration methods and categorised calibration types into two parts: pre-deployment calibration (calibration model) and post-deployment calibration (network recalibration strategies). Additionally, this literature review listed an overview of sensor calibration models classiﬁed as follows: i. Offset and Gain Calibration; ii. Temperature and Humidity Correction; and iii. Sensor Array Calibration. Offset and gain calibration gather calibration errors due to uncertain boundaries and discard any possible non-linear responses. Temperature and humidity correction extends the recorded values with existing values to calibrate the LCS. Sensor array calibration is an extension of temperature and humidity correction where it applies interfering gases and environmental factors. Sensor calibration can be done either in a controlled environment or an uncontrolled environment. A controlled environment implies calibration of the sensor either with high-end instruments or with already calibrated sensors. Whereas, an uncontrolled environment implies that sensor parameters are regulated according to other sensors because it cannot measure the data in a controlled environment which can lead to erroneous data monitoring [36,38]. In general, the monitoring stations with high-cost equipment are deployed at static lo- cations. The high-cost devices are reliable but infeasible to deploy in different locations [10]. However, there is always a trade-off between high cost and generally high-ﬁdelity devices with low-cost and often low-ﬁdelity devices. 2. Literature Review ANN-based models are mostly used when datasets have noise. Sensors 2022, 22, 1093 5 of 23 However, the ANN training requires a number of iterations with certain user-deﬁned parameters such as the number of nodes, hidden layers, activation function, and weights. The performance of the ANN model depends on the user-deﬁned parameters However, the ANN training requires a number of iterations with certain user-deﬁned parameters such as the number of nodes, hidden layers, activation function, and weights. The performance of the ANN model depends on the user-deﬁned parameters. parameters such as the number of nodes, hidden layers, activation function, and weigh The performance of the ANN model depends on the user-deﬁned parameters. parameters such as the number of nodes, hidden layers, activation function, and weights. The performance of the ANN model depends on the user-deﬁned parameters. p p p In these calibration processes, experts have used statistical calibration models as men- tioned above. The selection of the right sensors based on the literature and market accessi- bility is also challenging due to the wide-ranging availability of LCSs. Williams et al. [43] provide a guidebook that can help with LCS selection, however, their studies suggested that the sensor selection fully relies on the user preferences based on the sensor manual. In a similar study [44], it has been explained that the sensor selection process fully depends upon the end-user and it is an application that can help to deﬁne the scope. Similarly, Sousan et al. [45] checked the consistency of PM sensors (Sharp GP sensors) using the average slope method where it has been found that calculating the average of multiple measurements over the large time-frequency can decrease the random noise and hence increase the data quality. Going through the literature, it has been observed that many applications have been developed using LCSs. The literature also highlights the increase in LCS-based applications over the years. The applications areas of LCSs are not limited to only a few domains, rather LCS-based applications have been covered in wider domains suggesting that LCSs have been extensively used in recent years. The enhancement in the use of LCSs in different applications is due to the emergence of a wide range of LCSs and alternatives for similar tasks. 2. Literature Review From the literature, it has been also noted that there has been the use of different sensors in different applications which raises the challenge of sensor selection while developing LCS-based devices. With the appearance of more alternative LCSs for the same tasks, the sensor selection challenge will increase further in the coming years. Therefore, there is a need for the sensor selection strategy to build effective LCS-based applications. Data quality has been another concern that is argued in many LCS-based applications. Different calibration methods have been applied in different applications. However, how different meteorological parameters affect the calibration method and which calibration method is more efﬁcient remains a challenging aspect as the calibration process efﬁciency depends on the parameters being used for calibration, the sensor working principles, and the application domain. In general, to build an effective LCS-based device for an application, sensor selection and calibration are crucial for making the LCS-based system more effective with higher data quality. 3. Data-Driven Sensor Calibration: Our Methodology For the development of LCS devices, a data-driven sensor calibration methodology, as shown in Figure 1, was presented. At ﬁrst, several sensors were selected as candidate sensors based on literature and market availability. From the literature, we enlisted widely used candidate sensors such as OPC-R1, PMS5003, PM Nova SDS011, and Particulate Matter Sensor SPS30 for PM monitoring, SGP30, CJMCU-611, and CU-1106 for CO2 monitoring and similarly for other air pollutants monitoring sensors. While deciding on the candidate sensors, we considered the sensor’s availability in the UK market and their suppliers. The suppliers were chosen based on the procurement criteria set by our organisation. Following this, we also explored the datasheets provided by the manufacturer and available libraries to support sensor implementation with programming. By applying these three factors, we shortlisted candidate sensors to examine for our use case. After that, sensors were selected based on the statistical analysis presented in our previous work [21]. In the next step, the calibration process for the selected sensors was applied to improve data quality in the selected use case. Use case: Bradford district, UK, air quality (indoor and outdoor) monitoring is pre- sented as the use case in this study. There have been more than 1500 air quality monitoring stations, such as Automatic Urban and Rural Network (AURN) (https://uk-air.defra.gov. uk/networks/network-info?view=aurn, accessed on 17 January 2021), deployed across the UK. These stations have installed large, expensive, and calibrated sensors devices that Sensors 2022, 22, 1093 6 of 23 can monitor several air pollutants such as oxides of nitrogen (NOx), sulphur dioxide (SO2), particles (PM10 and PM2.5), carbon monoxide (CO), and ozone (O3). Additionally, in many cases, these stations are located away from trafﬁc areas or a small distance from city centres which can put a limitation on this station’s coverage to monitor air quality. The limitation on coverage area from the high-cost monitoring stations in Bradford city allows deploying LCS-based devices as an effective alternative to monitor air quality (indoor-outdoor) across the city and also allows real-time exposure assessment from many locations. This work contributes to part of two EU projects, Smart Cities and Open data Reuse (SCORE) and LifeCritical. For both projects, an area of Bradford needs to be covered with sensors indoors and outdoors to support constant monitoring of air quality over a year. The focus is to have sufﬁcient geographical coverage of this area to support a granular level of monitoring. 3. Data-Driven Sensor Calibration: Our Methodology The high-cost and immobile devices, used, for example, as part of AURN, is completely imprac- tical. Hence, we needed to rely on the use of LCS-based devices for this purpose where the accuracy of monitoring is paramount in order to support analytics and policymaking. W 6 of 23 Figure 1. Process diagram for data-driven sensor calibration methodology to build air quality mon- itoring devices. Figure 1. Process diagram for data-driven sensor calibration methodology to build air quality monitoring devices. Figure 1. Process diagram for data-driven sensor calibration methodology to build air quality mon- itoring devices. Figure 1. Process diagram for data-driven sensor calibration methodology to build air quality monitoring devices. Use case: Bradford district, U 3.1. Selection of Candidate Sensors q y g p sented as the use case in this study. There have been more than 1500 air quality monitoring stations, such as Automatic Urban and Rural Network (AURN) (https://uk-air.de- fra.gov.uk/networks/network-info?view=aurn, accessed on 17 January 2021), deployed across the UK. These stations have installed large, expensive, and calibrated sensors de- vices that can monitor several air pollutants such as oxides of nitrogen (NOx), sulphur dioxide (SO2), particles (PM10 and PM2.5), carbon monoxide (CO), and ozone (O3). Addi- tionally, in many cases, these stations are located away from traffic areas or a small dis- tance from city centres which can put a limitation on this station’s coverage to monitor air quality. The limitation on coverage area from the high-cost monitoring stations in Brad- ford city allows deploying LCS-based devices as an effective alternative to monitor air quality (indoor-outdoor) across the city and also allows real-time exposure assessment from many locations. This work contributes to part of two EU projects, Smart Cities and Open data Reuse (SCORE) and LifeCritical. For both projects, an area of Bradford needs to be covered with sensors indoors and outdoors to support constant monitoring of air In the LCS-based use case, the aim is to build a reliable air quality monitoring system that can measure several pollutants such as PM, CO2, NO2, NH3, CO, and Volatile Organic Compounds (VOCs) as these pollutants are heavily dependent on various activities carried out by humans [46]. The pollutant concentration depends not only on the emissions of a pollutant but also on meteorological conditions like wind speed (WS), relative humidity (RH), and turbulence. The meteorological parameters and other atmospheric compounds can inﬂuence the sensor measurements. For the gas sensors, there can be a cross-sensitivity. In other words, the concentration of a particular pollutant measured by a sensor can be affected by the concentration of a different pollutant due to the measurement techniques. However, the cross-sensitivity of sensors was not considered when selecting the gas sensors for this study. We relied on the information provided by the manufacturer to measure particular gases. For the use case, the following candidate sensors, as listed in Table 1, were considered to measure air pollutants either outdoors or indoors. These candidate sensors were selected based on the existing studies and market availability [47–49]. quality over a year. The focus is to have sufficient geographical coverage support a granular level of monitoring The high-cost and immobile devi 3.2. Narrowing down the Selection of Low-Cost Air Quality Monitoring Sensors Description Sensor Specification Image s sensor can measure erature humidity baro- Temp in Celsius (°C); Hu- s sensor can measure erature, humidity, baro- Temp in Celsius (°C); Hu- idit % b t i Table 1. List of candidate sensors Candidate Sensor Name Description BME680 This sensor can measure temperature, humidity, barometric pressure, and VOC gas. Table 1. List of candidat Candidate Sensor Name Description Sen BME680 This sensor can measure temperature, humidity, baro- metric pressure, and VOC gas. Temp midity CJMCU-811 This sensor can be used for detecting eCO2, VOC gases. It is a digital gas sensor inte- grated CCS801 sensor and 8- bit analogue-to-digital con- verter (ADC). eCO2 i SGP-30 This gas sensor is mainly used to monitor eCO2 and TVOC. eCO2 i Envio+ This pHAT is a collection of multiple sensors such as BME280 which can measure temperature, humidity, and pressure, MICS6814 ana- logue gas sensor is responsi- ble for measuring CO, NO2, and ammonia (NH3) and LTR-559 is light and proxim- ity sensor. Additionally, it has a built-in ADS1015 ana- logue-to-digital convertor and 0.96 “colour LCD for display”. BME2 pressur 婉LTR- ity sen logue SDS011 This sensor is used to meas- ure PM2.5 and PM10 air pollu- tants. This sensor is an infra- red-based laser sensor and has a fan to provide self-air- flow. PM2.5: PMS5003 It is used to measure PM1, PM2.5, and PM10. PM2.5: CJMCU-811 This sensor can be used for detecting eCO2, VOC gases. It is a digital gas sensor integrated CCS801 sensor and 8-bit analogue-to-digital converter (ADC). Table 1. List of candidat Candidate Sensor Name Description Sen BME680 This sensor can measure temperature, humidity, baro- metric pressure, and VOC gas. Temp midity CJMCU-811 This sensor can be used for detecting eCO2, VOC gases. It is a digital gas sensor inte- grated CCS801 sensor and 8- bit analogue-to-digital con- verter (ADC). eCO2 i SGP-30 This gas sensor is mainly used to monitor eCO2 and TVOC. eCO2 i Envio+ This pHAT is a collection of multiple sensors such as BME280 which can measure temperature, humidity, and pressure, MICS6814 ana- logue gas sensor is responsi- ble for measuring CO, NO2, and ammonia (NH3) and LTR-559 is light and proxim- ity sensor. Additionally, it has a built-in ADS1015 ana- logue-to-digital convertor and 0.96 “colour LCD for display”. BME2 pressur 婉LTR- ity sen logue SDS011 This sensor is used to meas- ure PM2.5 and PM10 air pollu- tants. quality over a year. The focus is to have sufficient geographical coverage support a granular level of monitoring The high-cost and immobile devi 3.2. Narrowing down the Selection of Low-Cost Air Quality Monitoring Sensors This sensor is an infra- red-based laser sensor and has a fan to provide self-air- flow. PM2.5: PMS5003 It is used to measure PM1, PM2.5, and PM10. PM2.5: SGP-30 This gas sensor is mainly used to monitor eCO2 and TVOC. Table 1. List of candidat Candidate Sensor Name Description Sen BME680 This sensor can measure temperature, humidity, baro- metric pressure, and VOC gas. Temp midity CJMCU-811 This sensor can be used for detecting eCO2, VOC gases. It is a digital gas sensor inte- grated CCS801 sensor and 8- bit analogue-to-digital con- verter (ADC). eCO2 i SGP-30 This gas sensor is mainly used to monitor eCO2 and TVOC. eCO2 i Envio+ This pHAT is a collection of multiple sensors such as BME280 which can measure temperature, humidity, and pressure, MICS6814 ana- logue gas sensor is responsi- ble for measuring CO, NO2, and ammonia (NH3) and LTR-559 is light and proxim- ity sensor. Additionally, it has a built-in ADS1015 ana- logue-to-digital convertor and 0.96 “colour LCD for display”. BME2 pressur 婉LTR- ity sen logue SDS011 This sensor is used to meas- ure PM2.5 and PM10 air pollu- tants. This sensor is an infra- red-based laser sensor and has a fan to provide self-air- flow. PM2.5: PMS5003 It is used to measure PM1, PM2.5, and PM10. PM2.5: Envio+ This pHAT is a collection of multiple sensors such as BME280 which can measure temperature, humidity, and pressure, MICS6814 analogue gas sensor is responsible for measuring CO, NO2, and ammonia (NH3) and LTR-559 is light and proximity sensor. Additionally, it has a built-in ADS1015 analogue-to-digital convertor and 0.96 “colour LCD for display”. Sensor Name Description Sen BME680 This sensor can measure temperature, humidity, baro- metric pressure, and VOC gas. Temp midity CJMCU-811 This sensor can be used for detecting eCO2, VOC gases. It is a digital gas sensor inte- grated CCS801 sensor and 8- bit analogue-to-digital con- verter (ADC). eCO2 i SGP-30 This gas sensor is mainly used to monitor eCO2 and TVOC. eCO2 i Envio+ This pHAT is a collection of multiple sensors such as BME280 which can measure temperature, humidity, and pressure, MICS6814 ana- logue gas sensor is responsi- ble for measuring CO, NO2, and ammonia (NH3) and LTR-559 is light and proxim- ity sensor. Additionally, it has a built-in ADS1015 ana- logue-to-digital convertor and 0.96 “colour LCD for display”. quality over a year. The focus is to have sufficient geographical coverage support a granular level of monitoring The high-cost and immobile devi 3.2. Narrowing down the Selection of Low-Cost Air Quality Monitoring Sensors BME2 pressur 婉LTR- ity sen logue SDS011 This sensor is used to meas- ure PM2.5 and PM10 air pollu- tants. This sensor is an infra- red-based laser sensor and has a fan to provide self-air- flow. PM2.5: PMS5003 It is used to measure PM1, PM2.5, and PM10. PM2.5: SDS011 This sensor is used to measure PM2.5 and PM10 air pollutants. This sensor is an infrared-based laser sensor and has a fan to provide self-airﬂow. BME680 p , y, metric pressure, and VOC gas. midity CJMCU-811 This sensor can be used for detecting eCO2, VOC gases. It is a digital gas sensor inte- grated CCS801 sensor and 8- bit analogue-to-digital con- verter (ADC). eCO2 i SGP-30 This gas sensor is mainly used to monitor eCO2 and TVOC. eCO2 i Envio+ This pHAT is a collection of multiple sensors such as BME280 which can measure temperature, humidity, and pressure, MICS6814 ana- logue gas sensor is responsi- ble for measuring CO, NO2, and ammonia (NH3) and LTR-559 is light and proxim- ity sensor. Additionally, it has a built-in ADS1015 ana- logue-to-digital convertor and 0.96 “colour LCD for display”. BME2 pressur 婉LTR- ity sen logue SDS011 This sensor is used to meas- ure PM2.5 and PM10 air pollu- tants. This sensor is an infra- red-based laser sensor and has a fan to provide self-air- flow. PM2.5: PMS5003 It is used to measure PM1, PM2.5, and PM10. PM2.5: PMS5003 It is used to measure PM1, PM2.5, and PM10. BME680 metric pressure, and VOC gas. midity CJMCU-811 This sensor can be used for detecting eCO2, VOC gases. It is a digital gas sensor inte- grated CCS801 sensor and 8- bit analogue-to-digital con- verter (ADC). eCO2 i SGP-30 This gas sensor is mainly used to monitor eCO2 and TVOC. eCO2 i Envio+ This pHAT is a collection of multiple sensors such as BME280 which can measure temperature, humidity, and pressure, MICS6814 ana- logue gas sensor is responsi- ble for measuring CO, NO2, and ammonia (NH3) and LTR-559 is light and proxim- ity sensor. Additionally, it has a built-in ADS1015 ana- logue-to-digital convertor and 0.96 “colour LCD for display”. BME2 pressur 婉LTR- ity sen logue SDS011 This sensor is used to meas- ure PM2.5 and PM10 air pollu- tants. This sensor is an infra- red-based laser sensor and has a fan to provide self-air- flow. PM2.5: PMS5003 It is used to measure PM1, PM2.5, and PM10. quality over a year. The focus is to have sufficient geographical coverage support a granular level of monitoring The high-cost and immobile devi 3.2. Narrowing down the Selection of Low-Cost Air Quality Monitoring Sensors support a granular level of monitoring. The high cost and immobile devices, used, for example, as part of AURN, is completely impractical. Hence, we needed to rely on the use of LCS-based devices for this purpose where the accuracy of monitoring is paramount in order to support analytics and policymaking. 3.1. Selection of Candidate Sensors In the LCS-based use case, the aim is to build a reliable air quality monitoring system that can measure several pollutants such as PM, CO2, NO2, NH3, CO, and Volatile Organic C d (VOC ) th ll t t h il d d t i ti iti The next challenge was to ﬁnd the most feasible sensors from the candidate sensors. To achieve this, statistical analysis presented in our earlier work [21] was applied that gives the most feasible sensors among the candidate sensors. For example, to measure PM2.5 and PM10, three different sensors SDS011, PMS5003, and OPC-R1 were compared in the lab environment for 48 h to ﬁnd the best feasible sensor among these three for PM measurements. If the sensors from the same type and same manufacturer are not consistent among themselves when they are exposed to the same environment, then they need to be discarded from consideration in the next step of calibration. Compounds (VOCs) as these pollutants are heavily dependent on various activities car- ried out by humans [46]. The pollutant concentration depends not only on the emissions of a pollutant but also on meteorological conditions like wind speed (WS) relative humid All selected LCSs have working limitations, mentioned in the manufacturer’s datasheet, e.g., SDS011 has a particle measurement range of 0.00–999.99 µg/m3. In the lab experimen- Sensors 2022, 22, 1093 Sensors 2022, 7 of 23 7 of 23 tation, we considered the general case scenarios that reﬂe under the general working environment. Table 1. List of candidate sensors and their details. Candidate Sensor Name Description Sensor Speciﬁcation BME680 This sensor can measure temperature, humidity, barometric pressure, and VOC gas. Temp in Celsius (◦C); Humidity—%; barometric pressure—hPa Sensors 2022, 22, x FOR PEER REVIEW Table 1. List of candidate sensors and their details. Candidate Sensor Name Description Sensor Specification BME680 This sensor can measure temperature, humidity, baro- metric pressure, and VOC gas. Temp in Celsius (°C); Hu- midity—%; barometric pres- sure—hPa CJMCU-811 This sensor can be used for detecting eCO2, VOC gases. quality over a year. The focus is to have sufficient geographical coverage support a granular level of monitoring The high-cost and immobile devi 3.2. Narrowing down the Selection of Low-Cost Air Quality Monitoring Sensors It is a digital gas sensor inte- grated CCS801 sensor and 8- bit analogue-to-digital con- verter (ADC). eCO2 in ppm, VOC gases in ppb. SGP-30 This gas sensor is mainly used to monitor eCO2 and TVOC. eCO2 in ppm, VOC gases in ppb. Envio+ This pHAT is a collection of multiple sensors such as BME280 which can measure temperature, humidity, and pressure, MICS6814 ana- logue gas sensor is responsi- ble for measuring CO, NO2, and ammonia (NH3) and LTR-559 is light and proxim- ity sensor. Additionally, it has a built-in ADS1015 ana- logue-to-digital convertor and 0.96 “colour LCD for display”. BME280: temperature (°C); pressure (hPa), humidity (%) 婉LTR-559 light and proxim- ity sensor婉MICS6814 ana- logue gas sensor (CO, NO2, NH3) SDS011 This sensor is used to meas- ure PM2.5 and PM10 air pollu- tants. This sensor is an infra- red-based laser sensor and has a fan to provide self-air- flow. PM2.5: ug/m3婉PM10: ug/m3 PMS5003 It is used to measure PM1, PM2.5, and PM10. PM2.5: ug/m3婉PM10: ug/m3 CJMCU-811 This sensor can be used for detecting eCO2, VOC gases. It is a digital gas sensor integrated CCS801 sensor and 8-bit analogue-to-digital converter (ADC). eCO2 in ppm, VOC gases in ppb. Sensors 2022, 22, x FOR PEER REVIEW Table 1. List of candidate sensors and their details. Candidate Sensor Name Description Sensor Specification BME680 This sensor can measure temperature, humidity, baro- metric pressure, and VOC gas. Temp in Celsius (°C); Hu- midity—%; barometric pres- sure—hPa CJMCU-811 This sensor can be used for detecting eCO2, VOC gases. It is a digital gas sensor inte- grated CCS801 sensor and 8- bit analogue-to-digital con- verter (ADC). eCO2 in ppm, VOC gases in ppb. SGP-30 This gas sensor is mainly used to monitor eCO2 and TVOC. eCO2 in ppm, VOC gases in ppb. Envio+ This pHAT is a collection of multiple sensors such as BME280 which can measure temperature, humidity, and pressure, MICS6814 ana- logue gas sensor is responsi- ble for measuring CO, NO2, and ammonia (NH3) and LTR-559 is light and proxim- ity sensor. Additionally, it has a built-in ADS1015 ana- logue-to-digital convertor and 0.96 “colour LCD for display”. BME280: temperature (°C); pressure (hPa), humidity (%) 婉LTR-559 light and proxim- ity sensor婉MICS6814 ana- logue gas sensor (CO, NO2, NH3) SDS011 This sensor is used to meas- ure PM2.5 and PM10 air pollu- tants. quality over a year. The focus is to have sufficient geographical coverage support a granular level of monitoring The high-cost and immobile devi 3.2. Narrowing down the Selection of Low-Cost Air Quality Monitoring Sensors This sensor is an infra- red-based laser sensor and has a fan to provide self-air- flow. PM2.5: ug/m3婉PM10: ug/m3 PMS5003 It is used to measure PM1, PM2.5, and PM10. PM2.5: ug/m3婉PM10: ug/m3 SGP-30 This gas sensor is mainly used to monitor eCO2 and TVOC. eCO2 in ppm, VOC gases in ppb. Sensors 2022, 22, x FOR PEER REVIEW Table 1. List of candidate sensors and their details. Candidate Sensor Name Description Sensor Specification BME680 This sensor can measure temperature, humidity, baro- metric pressure, and VOC gas. Temp in Celsius (°C); Hu- midity—%; barometric pres- sure—hPa CJMCU-811 This sensor can be used for detecting eCO2, VOC gases. It is a digital gas sensor inte- grated CCS801 sensor and 8- bit analogue-to-digital con- verter (ADC). eCO2 in ppm, VOC gases in ppb. SGP-30 This gas sensor is mainly used to monitor eCO2 and TVOC. eCO2 in ppm, VOC gases in ppb. Envio+ This pHAT is a collection of multiple sensors such as BME280 which can measure temperature, humidity, and pressure, MICS6814 ana- logue gas sensor is responsi- ble for measuring CO, NO2, and ammonia (NH3) and LTR-559 is light and proxim- ity sensor. Additionally, it has a built-in ADS1015 ana- logue-to-digital convertor and 0.96 “colour LCD for display”. BME280: temperature (°C); pressure (hPa), humidity (%) 婉LTR-559 light and proxim- ity sensor婉MICS6814 ana- logue gas sensor (CO, NO2, NH3) SDS011 This sensor is used to meas- ure PM2.5 and PM10 air pollu- tants. This sensor is an infra- red-based laser sensor and has a fan to provide self-air- flow. PM2.5: ug/m3婉PM10: ug/m3 PMS5003 It is used to measure PM1, PM2.5, and PM10. PM2.5: ug/m3婉PM10: ug/m3 Envio+ This pHAT is a collection of multiple sensors such as BME280 which can measure temperature, humidity, and pressure, MICS6814 analogue gas sensor is responsible for measuring CO, NO2, and ammonia (NH3) and LTR-559 is light and proximity sensor. Additionally, it has a built-in ADS1015 analogue-to-digital convertor and 0.96 “colour LCD for display”. BME280: temperature (◦C); pressure (hPa), humidity (%) LTR-559 light and proximity sensor MICS6814 analogue gas sensor (CO, NO2, NH3) Table 1. List of candidate sensors and their details. Candidate Sensor Name Description Sensor Specification BME680 This sensor can measure temperature, humidity, baro- metric pressure, and VOC gas. Temp in Celsius (°C); Hu- midity—%; barometric pres- sure—hPa CJMCU-811 This sensor can be used for detecting eCO2, VOC gases. quality over a year. The focus is to have sufficient geographical coverage support a granular level of monitoring The high-cost and immobile devi 3.2. Narrowing down the Selection of Low-Cost Air Quality Monitoring Sensors It is a digital gas sensor inte- grated CCS801 sensor and 8- bit analogue-to-digital con- verter (ADC). eCO2 in ppm, VOC gases in ppb. SGP-30 This gas sensor is mainly used to monitor eCO2 and TVOC. eCO2 in ppm, VOC gases in ppb. Envio+ This pHAT is a collection of multiple sensors such as BME280 which can measure temperature, humidity, and pressure, MICS6814 ana- logue gas sensor is responsi- ble for measuring CO, NO2, and ammonia (NH3) and LTR-559 is light and proxim- ity sensor. Additionally, it has a built-in ADS1015 ana- logue-to-digital convertor and 0.96 “colour LCD for display”. BME280: temperature (°C); pressure (hPa), humidity (%) 婉LTR-559 light and proxim- ity sensor婉MICS6814 ana- logue gas sensor (CO, NO2, NH3) SDS011 This sensor is used to meas- ure PM2.5 and PM10 air pollu- tants. This sensor is an infra- red-based laser sensor and has a fan to provide self-air- flow. PM2.5: ug/m3婉PM10: ug/m3 PMS5003 It is used to measure PM1, PM2.5, and PM10. PM2.5: ug/m3婉PM10: ug/m3 SDS011 This sensor is used to measure PM2.5 and PM10 air pollutants. This sensor is an infrared-based laser sensor and has a fan to provide self-airﬂow. PM2.5: ug/m3 PM10: ug/m3 Sensor Name Description Sensor Specification BME680 This sensor can measure temperature, humidity, baro- metric pressure, and VOC gas. Temp in Celsius (°C); Hu- midity—%; barometric pres- sure—hPa CJMCU-811 This sensor can be used for detecting eCO2, VOC gases. It is a digital gas sensor inte- grated CCS801 sensor and 8- bit analogue-to-digital con- verter (ADC). eCO2 in ppm, VOC gases in ppb. SGP-30 This gas sensor is mainly used to monitor eCO2 and TVOC. eCO2 in ppm, VOC gases in ppb. Envio+ This pHAT is a collection of multiple sensors such as BME280 which can measure temperature, humidity, and pressure, MICS6814 ana- logue gas sensor is responsi- ble for measuring CO, NO2, and ammonia (NH3) and LTR-559 is light and proxim- ity sensor. Additionally, it has a built-in ADS1015 ana- logue-to-digital convertor and 0.96 “colour LCD for display”. BME280: temperature (°C); pressure (hPa), humidity (%) 婉LTR-559 light and proxim- ity sensor婉MICS6814 ana- logue gas sensor (CO, NO2, NH3) SDS011 This sensor is used to meas- ure PM2.5 and PM10 air pollu- tants. This sensor is an infra- red-based laser sensor and has a fan to provide self-air- flow. PM2.5: ug/m3婉PM10: ug/m3 PMS5003 It is used to measure PM1, PM2.5, and PM10. quality over a year. The focus is to have sufficient geographical coverage support a granular level of monitoring The high-cost and immobile devi 3.2. Narrowing down the Selection of Low-Cost Air Quality Monitoring Sensors PM2.5: OPC-R1 This sensor is used to detect PM1, PM2.5, and PM10 with the help of laser scattering technology. Sensors 2022, 22, x FOR PEER REVIEW OPC-R1 This sensor is used to detect PM1, PM2.5, and PM10 with the help of laser scattering technology. PM2.5: • L Sensor Speciﬁcation Specification Specification Specification ; barometric pres- Temp in Celsius (◦C); Humidity—%; barometric pressure—hPa n Celsius (°C); Hu- %; barometric pres- sure—hPa n Celsius (°C); Hu- %; barometric pres- sure—hPa n Celsius ( C); Hu- %; barometric pres- sure—hPa ppm VOC gases in ppm, VOC gases in ppb. ppm, VOC gases in ppb. ppm, VOC gases in ppb. eCO2 in ppm, VOC gases in ppb. ppm, VOC gases in ppb. ppm, VOC gases in ppb. ppm, VOC gases in ppb. ppm, VOC gases in ppb. quality over a year. The focus is to have sufficient geographical coverage support a granular level of monitoring The high-cost and immobile devi 3.2. Narrowing down the Selection of Low-Cost Air Quality Monitoring Sensors PM2.5: ug/m3婉PM10: ug/m3 PMS5003 It is used to measure PM1, PM2.5, and PM10. PM2.5: ug/m3 PM10: ug/m3 Sensor Name Description Sensor Specification BME680 This sensor can measure temperature, humidity, baro- metric pressure, and VOC gas. Temp in Celsius (°C); Hu- midity—%; barometric pres- sure—hPa CJMCU-811 This sensor can be used for detecting eCO2, VOC gases. It is a digital gas sensor inte- grated CCS801 sensor and 8- bit analogue-to-digital con- verter (ADC). eCO2 in ppm, VOC gases in ppb. SGP-30 This gas sensor is mainly used to monitor eCO2 and TVOC. eCO2 in ppm, VOC gases in ppb. Envio+ This pHAT is a collection of multiple sensors such as BME280 which can measure temperature, humidity, and pressure, MICS6814 ana- logue gas sensor is responsi- ble for measuring CO, NO2, and ammonia (NH3) and LTR-559 is light and proxim- ity sensor. Additionally, it has a built-in ADS1015 ana- logue-to-digital convertor and 0.96 “colour LCD for display”. BME280: temperature (°C); pressure (hPa), humidity (%) 婉LTR-559 light and proxim- ity sensor婉MICS6814 ana- logue gas sensor (CO, NO2, NH3) SDS011 This sensor is used to meas- ure PM2.5 and PM10 air pollu- tants. This sensor is an infra- red-based laser sensor and has a fan to provide self-air- flow. PM2.5: ug/m3婉PM10: ug/m3 PMS5003 It is used to measure PM1, PM2.5, and PM10. PM2.5: ug/m3婉PM10: ug/m3 OPC-R1 This sensor is used to detect PM1, PM2.5, and PM10 with the help of laser scattering technology. PM2.5: ug/m3 PM10: ug/m3 Sensors 2022, 22, x FOR PEER REVIEW OPC-R1 This sensor is used to detect PM1, PM2.5, and PM10 with the help of laser scattering technology. PM2.5: ug/m3婉PM10: ug/m3 • LPG and propane— 200 5000 ppm tation, we considered the general case scenarios that reﬂect that all sensors’ ranges’ fall under the general working environment. EER REVIEW EER REVIEW EER REVIEW Table 1. List of candidate sensors and their details. Description Sensor Specification Image s sensor can measure erature, humidity, baro- Temp in Celsius (°C); Hu- s sensor can measure erature, humidity, baro- Temp in Celsius (°C); Hu- idity % ba o et i e tation, we considered the general case scenarios that reﬂect that all sensors’ ranges’ fall under the general working environment. EER REVIEW EER REVIEW EER REVIEW Table 1. List of candidate sensors and their details. ppm, VOC gases in ppb. ppm, VOC gases in ppb. eCO2 in ppm, VOC gases in ppb. ppm, VOC gases in ppb. 0: temperature (°C); 0: temperature (°C); ppm, VOC gases in ppb. ppm, VOC gases in ppb. eCO2 in ppm, VOC gases in ppb. ppm, VOC gases in ppb. 0: temperature (°C); 0: temperature (°C); 0: temperature (°C); (hPa), humidity (%) 59 li ht a d o i 0: temperature (°C); (hPa), humidity (%) 59 light and proxim- 0: temperature (°C); (hPa), humidity (%) 59 light and proxim- BME280: temperature (◦C); pressure (hPa), humidity (%) LTR-559 light and proximity sensor MICS6814 analogue gas sensor (CO, NO2, NH3) 0: temperature ( C); (hPa), humidity (%) 59 light and proxim- or婉MICS6814 ana- as sensor (CO, NO2, NH3) or婉MICS6814 ana- as sensor (CO, NO2, NH3) or婉MICS6814 ana as sensor (CO, NO2, NH3) MICS6814 ana- nsor (CO, NO2, NH3) MICS6814 ana- nsor (CO, NO2, NH3) MICS6814 ana nsor (CO, NO2, NH3) 婉PM10: ug/m3 PM2.5: ug/m3 PM10: ug/m3 3婉PM10: ug/m3 3婉PM10: ug/m3 MICS6814 ana- nsor (CO, NO2, H3) MICS6814 ana- nsor (CO, NO2, H3) MICS6814 ana nsor (CO, NO2, H3) 婉PM10: ug/m3 PM2.5: ug/m3 PM10: ug/m3 婉PM10: ug/m3 婉PM10: ug/m3 ug/m3婉PM10: ug/m3 ug/m3婉PM10: ug/m3 ug/m3婉PM10: ug/m3 / 3婉PM / 3 ug/m3婉PM10: ug/m3 PM2.5: ug/m3 PM10: ug/m3 ug/m3婉PM10: ug/m3 婉PM10: ug/m3 婉PM10: ug/m3 婉PM10: ug/m3 PM2.5: ug/m3 PM10: ug/m3 婉PM10: ug/m3 8 of 23 Sensors 2022, 22, 1093 Table 1. Cont. Candidate Sensor Name Description Sensor Speciﬁcation Image MQ-2 This gas sensor is mainly used to detect CO, methane, butane, LPG, smoke. • LPG and propane—200–5000 ppm • Butane—300–5000 ppm • Methane—5000–20,000 ppm • Hydrogen—300–5000 ppm • Alcohol—100–2000 ppm OPC-R1 PM1, PM2.5, and PM10 with the help of laser scattering technology. PM2.5: ug/m3婉PM10: ug/m3 MQ-2 This gas sensor is mainly used to detect CO, methane, butane, LPG, smoke. • LPG and propane— 200–5000 ppm • Butane—300–5000 ppm • Methane—5000–20,000 ppm • Hydrogen—300–5000 ppm • Alcohol—100–2000 3.2. Narrowing down the Selection of Low-Cost Air Quality Monitoring Sensors The next challenge was to find the most feasible sensors from the candidate sensors. To achieve this, statistical analysis presented in our earlier work [21] was applied that gives the most feasible sensors among the candidate sensors. For example, to measure PM2.5 and PM10, three different sensors SDS011, PMS5003, and OPC-R1 were compared in the lab environment for 48 h to find the best feasible sensor among these three for PM measurements. 3 2 Narrowing d 3.3. Sensor Calibration 3.3. Sensor Calibration 3.2. Narrowing down the Selection of Low-Cost Air Quality Monitoring Sensors The next challenge was to find the most feasible sensors from the candidate se To achieve this, statistical analysis presented in our earlier work [21] was applie gives the most feasible sensors among the candidate sensors. For example, to m PM2.5 and PM10, three different sensors SDS011, PMS5003, and OPC-R1 were compa the lab environment for 48 h to find the best feasible sensor among these three f measurements If the sensors from the same type and same manufacturer are no After implementation of previous steps, BME680, SGP30, Enviro+, MQ-2, and SDS011 sensors were selected. These sensors were used to build an Air Quality (AQ) monitoring device for measuring air pollutants, as shown in Figure 2. For computational and connec- tivity purposes, selected sensors were assembled with Raspberry Pi 3B+ (RPi) which gives remote access control of the whole device and sends data to the cloud-based web server for data storage, analysis, and visualisation. After implementation of previous steps, BME680, SGP30, Enviro+, MQ-2, and SDS011 sensors were selected. These sensors were used to build an Air Quality (AQ) monitoring device for measuring air pollutants, as shown in Figure 2. For computational and connec- tivity purposes, selected sensors were assembled with Raspberry Pi 3B+ (RPi) which gives remote access control of the whole device and sends data to the cloud-based web server for data storage, analysis, and visualisation. measurements. If the sensors from the same type and same manufacturer are no sistent among themselves when they are exposed to the same environment, the need to be discarded from consideration in the next step of calibration. All selected LCSs have working limitations, mentioned in the manufac datasheet, e.g., SDS011 has a particle measurement range of 0.00–999.99 μg/m3. In t experimentation, we considered the general case scenarios that reflect that all se ranges’ fall under the general working environment. 3.3. Sensor Calibration After implementation of previous steps, BME680, SGP30, Enviro+, MQ-2, and S sensors were selected. These sensors were used to build an Air Quality (AQ) mon device for measuring air pollutants, as shown in Figure 2. For computational and c tivity purposes, selected sensors were assembled with Raspberry Pi 3B+ (RPi) which remote access control of the whole device and sends data to the cloud-based web for data storage, analysis, and visualisation. (a) (b) Figure 2. ppm, VOC gases in ppb. ppm, VOC gases in ppb. eCO2 in ppm, VOC gases in ppb. ppm, VOC gases in ppb. 0: temperature (°C); 0: temperature (°C); If the sensors from the same type and same manufacturer are not con- sistent among themselves when they are exposed to the same environment, then they need to be discarded from consideration in the next step of calibration. All selected LCSs have working limitations, mentioned in the manufacturer’s datasheet, e.g., SDS011 has a particle measurement range of 0.00–999.99 μg/m3. In the lab experimentation, we considered the general case scenarios that reflect that all sensors’ ranges’ fall under the general working environment Table 1. Cont. , , help of laser sca a o i The nex 3 2 Narrowing d 3.3. Sensor Calibration 3.3. Sensor Calibration (a) Final LCS-based AQ monitoring device using RPi 3B+. (b) Block diagram of the ﬁnal device with Raspberry Pi 3B+ and other LCS components. sistent among need to be dis All selec datasheet, e.g experimentati ranges’ fall un 3.3. Sensor Cal After imp sensors were device for me tivity purpose remote access for data storag (a) nsidered the general case scenarios that reflect that neral working environment. on of previous steps, BME680, SGP30, Enviro+, MQ-2, hese sensors were used to build an Air Quality (AQ) pollutants, as shown in Figure 2. For computational a sensors were assembled with Raspberry Pi 3B+ (RPi) the whole device and sends data to the cloud-based s, and visualisation. (b) (a) (b) Figure 2. (a) Final LCS-based AQ monitoring device using RPi 3B+. (b) Block diagram of the ﬁnal device with Raspberry Pi 3B+ and other LCS components. (a) (b) After building the air quality monitoring LCS-based device, data quality aspects were applied. Studies have argued that [5,9,18] sensor calibration is required to increase the data quality of LCS-based systems. Considering this, from the calibration point of view, the two AQ devices were deployed at “The Urban Flows Observatory (https://urbanﬂows.ac.uk/, accessed on 1 March 2021), Shefﬁeld” (Figure 3) for one month. Both the devices had all selected sensors that monitor air pollutants such as CO2, NO2, CO, NH3, TVOC, and PM (PM2.5 and PM10). Additionally, these devices had sensors that can measure meteorological parameters (temperature and humidity). In this paper, the calibration methods for PM2.5 and PM10 are presented. For PM2.5 and PM10 data calibration, SDS011 PM sensors were calibrated against the “high-end Palas Fidas 200” instrument which was installed at the remote van, as shown in Figure 3, by Shefﬁeld City Council. This station monitors data at the 30 min interval whereas our AQ devices monitor data at every 10 min interval. Data pre-processing was applied to the AQ data to convert into 30 min data using the mean value of three 10 min readings. All the measured data were received from the AQ devices in every 10 min time interval that was sent to the cloud-based web server Sensors 2022, 22, 1093 9 of 23 (http://smartbradford.co.uk:7201/, accessed on 12 January 2021) and also stored in the AQ devices in CSV (Comma-Separated Values) format. EW 10 of 26 Figure 3. LCS-based AQ monitoring devices using RPi 3B+ at Urban Observatory, Sheffield Site. Figure 3. 3 2 Narrowing d 3.3. Sensor Calibration 3.3. Sensor Calibration LCS-based AQ monitoring devices using RPi 3B+ at Urban Observatory, Shefﬁeld Site. Figure 3. LCS-based AQ monitoring devices using RPi 3B+ at Urban Observatory, Sheffield Site. Figure 3. LCS-based AQ monitoring devices using RPi 3B+ at Urban Observatory, Shefﬁeld Site. Different calibration models were experimented with to compare and select the most accurate model. For example, we took PM2.5 and PM10 data for experimental purposes and undertook experiments on four calibration models: Multivariate Linear Regression (MLR), Multi-Layer Perceptron (MLP), Convolution Neural Network (CNN), and Ran- dom Forest (RF). The literature has argued that AH and RH act differently with different LCSs in the calibration process. Mead et al. [50] show that the RH greatly depends on temperature, therefore, fluctuations can be observed in RH throughout the day. In con- trast, AH is observed to be constant as it is independent of temperature. Due to this factor, the calibration process was adapted based on AH as the corrections were constant and li b d i h i AH Pi d hi l [51] b d h h Different calibration models were experimented with to compare and select the most accurate model. For example, we took PM2.5 and PM10 data for experimental purposes and undertook experiments on four calibration models: Multivariate Linear Regression (MLR), Multi-Layer Perceptron (MLP), Convolution Neural Network (CNN), and Random Forest (RF). The literature has argued that AH and RH act differently with different LCSs in the calibration process. Mead et al. [50] show that the RH greatly depends on temperature, therefore, ﬂuctuations can be observed in RH throughout the day. In contrast, AH is ob- served to be constant as it is independent of temperature. Due to this factor, the calibration process was adapted based on AH as the corrections were constant and linear based on Sensors 2022, 22, 1093 10 of 23 10 of 23 per unit change in AH. Piedrahita et al. [51] observed that temperature has a signiﬁcant impact on this sensor signal response, but the impact of AH is lower on the signal response as it has been observed to be almost constant, contradictory to the RH impact on sensor signal response. However, they still consider AH in calibration modelling to improve the model performance. Additionally, some of the studies [52,53] show that temperature and humidity have a non-linear relationship with particle concentrations. e = Exponential function e = Exponential function Using Equation (6), AH was calculated based on the two observations T and RH coming from the BME680 sensor and the exponential function. This AH was used as one of the parameters for the calibration process. R2 = SSRES SSTOT = 1 −∑i(yi −ˆyi)2 ∑i(yi −y)2 RMSE = v u u t n ∑ i=1 ( ˆyi −yi)2 n MAE = n ∑ i=1 \|( ˆyi −yi)\| n R2 = SSRES SSTOT = 1 −∑i(yi −ˆyi)2 ∑i(yi −y)2 (2) RMSE = v u u t n ∑ i=1 ( ˆyi −yi)2 n (3) MAE = n ∑ i=1 \|( ˆyi −yi)\| n (4) (2) (3) (4) T = Temperature (C) 3 2 Narrowing d 3.3. Sensor Calibration 3.3. Sensor Calibration Research also shows that PM2.5 and PM10 values have a positive correlation with RH but a negative correlation with temperature and AH [37]. Considering these previous studies, in this work, temperature and humidity along with the pollutant were applied for the calibration. For humidity as a factor in calibration, both AH and RH were examined to determine which among these two humidity measures gives better results for the data quality. g y g q y To ﬁnd AH, we used the Clausius Clapeyron equation [54] as shown in Equation (1), AH = 6.112 ∗e[ 17.67∗T T+243.5 ] ∗RH ∗2.1674 273.15 + T (1) (1) where, T = Temperature (C) RH = Relative Humidity (%) where where ŷr ˆyref = reference data from Palas Fidas 200, Shefﬁeld. b0, b1, b2, and b3 = Regression coefficients. T T (C) RH H idi (%) f h B f b0, b1, b2, and b3 = Regression coefﬁcients. T = Temperature (C), RH = Humidity (%) f AH Ab l H idi ( / 3) g T = Temperature (C), RH = Humidity (%) from the BME680 sensor. AH = Absolute Humidity (g/m3). PM M PM d (f SDS011) AH = Absolute Humidity (g/m3). PM2.5raw = Mean PM data (from SDS AH = Absolute Humidity (g/m3). PM2.5raw = Mean PM data (from SDS PM2.5raw = Mean PM data (from SDS011). For the calibration analysis, line and h PM2.5raw = Mean PM data (from SDS011). For the calibration analysis, line and For the calibration analysis, line and scatter plots were presented for both PM2.5 and PM10 as shown in Figures 4–7. Figures 4 and 6 present the line plots for PM2.5 and PM10, respectively, whereas Figures 5 and 7 shows scatter plots for PM2.5 and PM10, respectively. Further detailed statistical analysis was also undertaken (presented in Section 4) to validate the graphical analysis. From the line plots, for PM2.5 and PM10, it can be observed that calibrated values are closer to the reference data when AH was used. From the scatter plots, it can be also observed that the regression fit line is closer to the line of equality when AH was used for the calibration in comparison to the RH for both PM2.5 and PM10. Analysis of these plots infers that AH has better performance than RH in the calibration process. PM10 as shown in Figures 4–7. Figures 4 and 6 present the line plots for PM2.5 and PM10, respectively, whereas Figures 5 and 7 shows scatter plots for PM2.5 and PM10, respectively. Further detailed statistical analysis was also undertaken (presented in Section 4) to vali- date the graphical analysis. From the line plots, for PM2.5 and PM10, it can be observed that calibrated values are closer to the reference data when AH was used. From the scatter plots, it can be also observed that the regression fit line is closer to the line of equality when AH was used for the calibration in comparison to the RH for both PM2.5 and PM10. Analysis of these plots infers that AH has better performance than RH in the calibration process. where R2 = R-squared. R2 = R-squared. R2 = R-squared. RMSE = Root Mean Square Error. RMSE = Root Mean Square Error. MAE = Mean Absolute Error. MAE = Mean Absolute Error. SSRES = Residual sum of squared errors of our regression model. SSTOT = Total sum of squared errors. SSTOT = Total sum of squared errors. yi = Observed value from our kit. yi = Observed value from our kit. yi = Mean value of pollutants value from our kit ˆyi = Values predicted by the model. n = Number of observations. For comparative analysis between RH and AH, we experimented with selected models and analysed their impacts on the results. Additionally, for model evaluation, the following statistical measures were used as shown in Equations (2)–(4). Using these equations, performance measures such as R2, RMSE, and MAE were calculated using observed values (yi) recorded using our LCS-based device and (yi) recorded as a mean value of pollutant values from our devices. R2 was calculated as a ratio of the residual sum of squared errors (SSRES) of our regression model and the total sum of squared errors (SSTOT). Sensors 2022, 22, 1093 11 of 23 11 of 23 3.3.1. Multivariate Linear Regression 3.3.1. Multivariate Linear Regression Multivariate Linear Regression (MLR) is one of the widely used calibration methods applied for two or more independent variable dependencies with one single targeted variable by adjusting coefﬁcients in linear equations, as represented in Equation (5) [55]. EW 12 of 26 (5) yi = ap xip + . . . + a1 * xi1 + a0 + zi (5) yi = ap * xip + . . . + a1 * xi1 + a0 + zi li d i f MLR h Equation (5) is the generalised representation of MLR where ap, a1, and a0 are co- efﬁcients, xip, xi1 are dependent variables, zi is constant, and yi is the calibrated tar- geted variable. In this study, the MLR model was applied for the selected sensors using Equations (6) and (7). Equation (5) is the generalised representation of MLR where ap, a1, and a0 are coeffi- cients, xip, xi1 are dependent variables, zi is constant, and yi is the calibrated targeted varia- ble. In this study, the MLR model was applied for the selected sensors using Equations (6) and (7). ˆyref = b0 + b1 * T + b2 * PMraw + b3 * AH (6) ˆyref = b0 + b1 * T + b2 * PMraw + b3 * RH (7) ŷref = b0 + b1T + b2 PMraw + b3* AH (6) ŷref = b0 + b1T + b2 PMraw + b3* RH (7) (6) (6) (7) (7) where where ŷr (b) (a) (b) Figure 4. (a) MLR calibration plot for PM2.5 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) MLR calibration plot for PM2.5 using RH. Blue col- our represents the reference data and orange colour represents the calibrated data. Figure 4. (a) MLR calibration plot for PM2.5 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) MLR calibration plot for PM2.5 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. (a) (a) (b) Figure 4. (a) MLR calibration plot for PM2.5 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) MLR calibration plot for PM2.5 using RH. Blue col- our represents the reference data and orange colour represents the calibrated data. Figure 4. (a) MLR calibration plot for PM2.5 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) MLR calibration plot for PM2.5 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. Sensors 2022, 22, 1093 12 of 23 12 of 23 (a) (b) Figure 5. (a) MLR calibration scatter plot for PM2.5 with the line of equality, regression fitted lin and the calibrated data using AH; (b) MLR calibration scatter plot for PM2.5 with the line of equalit regression fitted line, and the calibrated data using RH. Figure 5. (a) MLR calibration scatter plot for PM2.5 with the line of equality, regression ﬁtted line, and the calibrated data using AH; (b) MLR calibration scatter plot for PM2.5 with the line of equality, regression ﬁtted line, and the calibrated data using RH. (a) (b) Figure 5. (a) MLR calibration scatter plot for PM2.5 with the line of equality, regression fitted line, and the calibrated data using AH; (b) MLR calibration scatter plot for PM2.5 with the line of equality, regression fitted line, and the calibrated data using RH. (b) (b) M2.5 with the line of equality, regression fitted line (a) (a) Figure 5. (a) MLR calibration scatter plot for (b) ality, (a) calibr Figure 5. (a) MLR calibration scatter plot for PM2.5 with the line of equality, regression fitted lin and the calibrated data using AH; (b) MLR calibration scatter plot for PM2.5 with the line of equality regression fitted line, and the calibrated data using RH. where where ŷr Figure 5. (a) MLR calibration scatter plot for PM2.5 with the line of equality, regression ﬁtted line, and the calibrated data using AH; (b) MLR calibration scatter plot for PM2.5 with the line of equality, regression ﬁtted line, and the calibrated data using RH. and the calibrated data using AH; (b) MLR calibration scatter plot for PM2.5 with the line of equality, regression fitted line, and the calibrated data using RH. (b) MLR calibration plot for PM10 using AH. Blue colour represents the reference r represents the calibrated data; (b) MLR calibration plot for PM10 using RH. B e reference data and orange colour represents the calibrated data. (b) (a) (b) Figure 6. (a) MLR calibration plot for PM10 using AH. Blue colour represents the reference data an orange colour represents the calibrated data; (b) MLR calibration plot for PM10 using RH. Blue colou represents the reference data and orange colour represents the calibrated data. (a) (b) Figure 6. (a) MLR calibration plot for PM10 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) MLR calibration plot for PM10 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. Figure 6. (a) MLR calibration plot for PM10 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) MLR calibration plot for PM10 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. (a) Figure orange repres (a) present (b) (a) Figure 6. (a) MLR calibration plot for PM10 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) MLR calibration plot for PM10 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. Figure 6. (a) MLR calibration plot for PM10 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) MLR calibration plot for PM10 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. 3.3.2. Statistical Approaches Multi-layer perceptron (MLP) is a forward-structured Artiﬁcial Neural Network that operates on sets of input vectors to give output with a set of output vectors. MLP is one of the efﬁcient calibration methods that has been applied in many problems [56]. The Multi- Layer Perceptron model, as shown in Figure 8, was designed as the second calibration model. In the input layer, four parameters, temperature, humidity (AH and RH), PMref, and PMraw were applied and the calibrated PM value was obtained at the output layer. The same dataset used for MLR was used for MLP as well. The model was designed as a sequential model with relu activation function, Adam optimiser, and mean square error as a loss function with 2000 epochs for training. For the MLP calibration analysis, line and scatter plots were observed for both PM2.5 and PM10 as shown in Figures 9–12, where Figures 9 and 11 present the line plots for PM2.5 and PM10, respectively, and Figures 10 and 12 show scatter plots for PM2.5 and PM10, correspondingly. The same as MLR, the calibrated line plots for PM2.5 and PM10 are closer to the reference value when AH has been used. Similarly, in the scatter plots, the Sensors 2022, 22, 1093 13 of 23 13 of 23 regression ﬁt line is closer to the line of equality when AH has been used for the calibration in comparison to the RH for both PM2.5 and PM10. Analysis of these plots concludes that AH has better performance than RH in the calibration process in the case of MLP as well. nsors 2022, 22, x FOR PEER REVIEW 14 of regression ﬁt line is closer to the line of equality when AH has been used for the calibration in comparison to the RH for both PM2.5 and PM10. Analysis of these plots concludes that AH has better performance than RH in the calibration process in the case of MLP as well. sors 2022, 22, x FOR PEER REVIEW 14 of (b) PM10 with the line of equality, regression fit ibration scatter plot for PM10 with the line of using RH. rward-structured Artificial Neural Netw output with a set of output vectors. 3.3.2. Statistical Approaches MLP has been applied in many problems [5 in Figure 8, was designed as the second ameters, temperature, humidity (AH an alibrated PM value was obtained at the u ed fo MLP a ell The odel a d (a) W Figure 7. (a) MLR calibration scatter plot and the calibrated data using AH; (b) MLR regression fitted line, and the calibrated da 3.3.2. Statistical Approaches Multi-layer perceptron (MLP) is a operates on sets of input vectors to gi of the efficient calibration methods th Multi-Layer Perceptron model, as sho tion model. In the input layer, four p PMref, and PMraw were applied and th Th d t t d f MLR (a) (b) Figure 7. (a) MLR calibration scatter plot for PM10 with the line of equality, regression fitted lin and the calibrated data using AH; (b) MLR calibration scatter plot for PM10 with the line of equalit regression fitted line, and the calibrated data using RH. 3.3.2. Statistical Approaches Figure 7. (a) MLR calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using AH; (b) MLR calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using RH. W Figure 7. (a) MLR calibration scatter plot for PM10 with the line of equality, regression fitte and the calibrated data using AH; (b) MLR calibration scatter plot for PM10 with the line of eq regression fitted line, and the calibrated data using RH. 3.3.2. Statistical Approaches Multi-layer perceptron (MLP) is a forward-structured Artificial Neural Networ operates on sets of input vectors to give output with a set of output vectors. MLP of the efficient calibration methods that has been applied in many problems [56 Multi-Layer Perceptron model, as shown in Figure 8, was designed as the second c tion model. In the input layer, four parameters, temperature, humidity (AH and PMref, and PMraw were applied and the calibrated PM value was obtained at the o layer. The same dataset used for MLR was used for MLP as well. The model was des as a sequential model with relu activation function, Adam optimiser, and mean s error as a loss function with 2000 epochs for training. (b) e wa ll (a) wer d t Figure 7. 3.3.2. Statistical Approaches (a) MLR calibration scatter plot for PM10 with the line of equality, regression fitted lin and the calibrated data using AH; (b) MLR calibration scatter plot for PM10 with the line of equalit regression fitted line, and the calibrated data using RH. 3 3 2 Statistical Approaches Figure 7. (a) MLR calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using AH; (b) MLR calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using RH. ayer. The same dataset used for MLR was used for MLP as well. The model was des as a sequential model with relu activation function, Adam optimiser, and mean s error as a loss function with 2000 epochs for training. Multi-layer perceptron (MLP) is a forward-structured Artifici operates on sets of input vectors to give output with a set of outp of the efficient calibration methods that has been applied in ma Multi-Layer Perceptron model, as shown in Figure 8, was designe tion model. In the input layer, four parameters, temperature, h PMref, and PMraw were applied and the calibrated PM value was layer. The same dataset used for MLR was used for MLP as well. T as a sequential model with relu activation function, Adam optim error as a loss function with 2000 epochs for training. Figure 8. Block diagram of the Multi-Layer Perceptron model. Figure 8. Block diagram of the Multi-Layer Perceptron model. Figure 8. Block diagram of the Multi-Layer Perceptron mo Figure 8. Block diagram of the Multi-Layer Perceptron model. For the MLP calibration analys 3.3.3. Convolution Neural Network For the MLP calibration analy 3.3.3. Convolution Neural Network Figure 8. Block diagram of the Multi-Layer Perceptron model. For the MLP calibration analysis, line and scatter plots were observed for both PM2 and PM10 as shown in Figures 9–12, where Figures 9 and 11 present the line plots for PM2 For the MLP calibration analysis, line and scatter plots were observed for both and PM10 as shown in Figures 9–12, where Figures 9 and 11 present the line plots for and PM10, respectively, and Figures 10 and 12 show scatter plots for PM2.5 and PM1 respondingly. The same as MLR, the calibrated line plots for PM2.5 and PM10 are clo the reference value when AH has been used. Similarly, in the scatter plots, the regre fit line is closer to the line of equality when AH has been used for the calibration in parison to the RH for both PM2.5 and PM10. Analysis of these plots concludes that A better performance than RH in the calibration process in the case of MLP as well. Recently, CNN architectures have been used in various modelling scenarios of sequen- tial data such as time series [57,58]. CNN has appeared as one of the most widely used calibration models as CNN can extract inherent information from the data set [59]. In the calibration, the same as the MLP model, the CNN model has four 3D inputs and reshape was applied that gives one output, two hidden convolutional layers with 64 ﬁlters each, and a window size of 2 was also deﬁned for the CNN model. All layers were activated through the “relu” function with 2000 epochs support with the “adam” optimiser. The output in terms of line plots and scatter plots were analysed for both AH and RH as shown in Figures 13–16. This model also has similar results in both plots that support better calibration performance for AH in comparison to RH. 14 of 23 14 of 23 Sensors 2022, 22, 1093 (a) (b) Figure 9. (a) MLP calibration plot for PM2.5 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) MLP calibration plot for PM2.5 using RH. Blue col- our represents the reference data and orange colour represents the calibrated data. Figure 9. (a) MLP calibration plot for PM2.5 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) MLP calibration plot for PM2.5 using RH. For the MLP calibration analy 3.3.3. Convolution Neural Network Blue colour represents the reference data and orange colour represents the calibrated data. Sensors 2022, 22, x FOR PEER REVIEW 16 of EER REVIEW 16 of 26 (b) 16 o (a) sors 2022, 22, x FOR PEER REVIEW (b) (a) Figure 9. (a) MLP calibration plot for PM2.5 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) MLP calibration plot for PM2.5 using RH. Blue col- our represents the reference data and orange colour represents the calibrated data. Figure 9. (a) MLP calibration plot for PM2.5 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) MLP calibration plot for PM2.5 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. sors 2022, 22, x FOR PEER REVIEW 16 of (b) (b) with the line of equality, regression fitted line, on scatter plot for PM2.5 with the line of equality, (a) (b) Figure 10. (a) MLP calibration scatter plot for PM2.5 with the line of equality, regression fitted lin and the calibrated data using AH; (b) MLP calibration scatter plot for PM2.5 with the line of equali regression fitted line, and the calibrated data using RH. Figure 10. (a) MLP calibration scatter plot for PM2.5 with the line of equality, regression ﬁtted line, and the calibrated data using AH; (b) MLP calibration scatter plot for PM2.5 with the line of equality, regression ﬁtted line, and the calibrated data using RH. (a) (b) Figure 10. (a) MLP calibration scatter plot for PM2.5 with the line of equality, regression fitted line, and the calibrated data using AH; (b) MLP calibration scatter plot for PM2.5 with the line of equality, regression fitted line, and the calibrated data using RH. (a) (a) Figure 10. (a) MLP calibration scatter plot for P and the calibrated data using AH; (b) MLP calib (b) regres th the (a) P calib d data u Figure 10. (a) MLP calibration scatter plot for PM2.5 with the line of equality, regression fitted lin and the calibrated data using AH; (b) MLP calibration scatter plot for PM2.5 with the line of equalit regression fitted line, and the calibrated data using RH. Figure 10. For the MLP calibration analy 3.3.3. Convolution Neural Network (a) MLP calibration scatter plot for PM2.5 with the line of equality, regression ﬁtted line, and the calibrated data using AH; (b) MLP calibration scatter plot for PM2.5 with the line of equality, regression ﬁtted line, and the calibrated data using RH. regression fitted line, and the calibrated data using RH. (b) (b) (a) (b) Figure 11. (a) MLP calibration plot for PM10 using AH. Blue colour represents the reference data an orange colour represents the calibrated data; (b) MLP calibration plot for PM10 using RH. Blue colou represents the reference data and orange colour represents the calibrated data. (a) (b) Figure 11. (a) MLP calibration plot for PM10 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) MLP calibration plot for PM10 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. Figure 11. (a) MLP calibration plot for PM10 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) MLP calibration plot for PM10 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. (a) (a) (a) (b) Figure 11. (a) MLP calibration plot for PM10 using AH. Blue colour represents the reference data an orange colour represents the calibrated data; (b) MLP calibration plot for PM10 using RH. Blue colou represents the reference data and orange colour represents the calibrated data. Figure 11. (a) MLP calibration plot for PM10 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) MLP calibration plot for PM10 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. Figure 11. (a) MLP calibration plot for PM10 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) MLP calibration plot for PM10 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. Sensors 2022, 22, 1093 15 of 23 (b) (b) M10 with the line of equality, regression fitted line, tion scatter plot for PM10 with the line of equality, g RH. n used in various modelling scenarios of se- NN has appeared as one of the most widely inherent information from the data set [59]. del, the CNN model has four 3D inputs and wo hidden convolutional layers with 64 filters ed for the CNN model. For the MLP calibration analy 3.3.3. Convolution Neural Network Convolution Neural Network Recently, CNN architectures have quential data such as time series [57,58 used calibration models as CNN can ex In the calibration, the same as the MLP reshape was applied that gives one outpu each, and a window size of 2 was also d vated through the “relu” function with The output in terms of line plots and sc (b) e “ad b th (a) h the Figure 12. (a) MLP calibration scatter plot for PM10 with the line of equality, regression fitted line, and the calibrated data using AH; (b) MLP calibration scatter plot for PM10 with the line of equality, regression fitted line, and the calibrated data using RH. 3.3.3. Convolution Neural Network Figure 12. (a) MLP calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using AH; (b) MLP calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using RH. The output in terms of line plots and scatter plots were analysed for both AH and RH as shown in Figures 13–16. This model also has similar results in both plots that support better calibration performance for AH in comparison to RH. Recently, CNN architectures have been used in various modelling scenarios of se- quential data such as time series [57,58]. CNN has appeared as one of the most widely used calibration models as CNN can extract inherent information from the data set [59]. In the calibration, the same as the MLP model, the CNN model has four 3D inputs and reshape was applied that gives one output, two hidden convolutional layers with 64 filters each, and a window size of 2 was also defined for the CNN model. All layers were acti- vated through the “relu” function with 2000 epochs support with the “adam” optimiser. The output in terms of line plots and scatter plots were analysed for both AH and RH as shown in Figures 13–16. This model also has similar results in both plots that support better calibration performance for AH in comparison to RH. (a) (b) Figure 13. (a) CNN calibration plot for PM2.5 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) CNN calibration plot for PM2.5 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. For the MLP calibration analy 3.3.3. Convolution Neural Network Sensors 2022, 22, x FOR PEER REVIEW 18 of 26 (a) (b) Figure 13. (a) CNN calibration plot for PM2.5 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) CNN calibration plot for PM2.5 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. CNN architectures have been used in various modelling s uch as time series [57,58]. CNN has appeared as one of th n models as CNN can extract inherent information from th on, the same as the MLP model, the CNN model has four plied that gives one output, two hidden convolutional layers ndow size of 2 was also defined for the CNN model. All la the “relu” function with 2000 epochs support with the “ada erms of line plots and scatter plots were analysed for both res 13–16. This model also has similar results in both plot on performance for AH in comparison to RH. (b) (b) NN lib i l f PM i AH Bl l h Rece quential used cali In the ca reshape w each, and vated thr The outp shown in better cal (a) Sensors 2022, 22, x FOR PEER REVIEW (a) i (b) (a) Figure 13. (a) CNN calibration plot for PM2.5 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) CNN calibration plot for PM2.5 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. Figure 13. (a) CNN calibration plot for PM2.5 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) CNN calibration plot for PM2.5 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. (b) (b) (a) (b) (a) (b) Figure 14. (a) CNN calibration scatter plot for PM2.5 with the line of equality, regression fitted line, and the calibrated data using AH; (b) CNN calibration scatter plot for PM2.5 with the line of equality, regression fitted line, and the calibrated data using RH. Figure 14. (a) CNN calibration scatter plot for PM2.5 with the line of equality, regression ﬁtted line, and the calibrated data using AH; (b) CNN calibration scatter plot for PM2.5 with the line of equality, regression ﬁtted line, and the calibrated data using RH. (a) (a) (b) (a) Figure 14. For the MLP calibration analy 3.3.3. Convolution Neural Network All layers were acti- epochs support with the “adam” optimiser. plots were analysed for both AH and RH as (a) (b) Figure 12. (a) MLP calibration scatter plot for PM10 with the line of equality, regression fitted line, and the calibrated data using AH; (b) MLP calibration scatter plot for PM10 with the line of equality, regression fitted line, and the calibrated data using RH. 3.3.3. Convolution Neural Network Figure 12. (a) MLP calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using AH; (b) MLP calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using RH. (a) (b) Figure 12. (a) MLP calibration scatter plot for PM10 with the line of equality, regression fitted line, and the calibrated data using AH; (b) MLP calibration scatter plot for PM10 with the line of equality, regression fitted line, and the calibrated data using RH. 3.3.3. Convolution Neural Network Recently, CNN architectures have been used in various modelling scenarios of se- quential data such as time series [57,58]. CNN has appeared as one of the most widely used calibration models as CNN can extract inherent information from the data set [59]. In the calibration, the same as the MLP model, the CNN model has four 3D inputs and reshape was applied that gives one output, two hidden convolutional layers with 64 filters each, and a window size of 2 was also defined for the CNN model. All layers were acti- vated through the “relu” function with 2000 epochs support with the “adam” optimiser. The output in terms of line plots and scatter plots were analysed for both AH and RH as shown in Figures 13–16. This model also has similar results in both plots that support better calibration performance for AH in comparison to RH. (a) (a) Figure 12. (a) MLP calibration scatter plot fo and the calibrated data using AH; (b) MLP ca regression fitted line, and the calibrated data 3.3.3. For the MLP calibration analy 3.3.3. Convolution Neural Network (a) CNN calibration scatter plot for PM2.5 with the line of equality, regression fitted line, and the calibrated data using AH; (b) CNN calibration scatter plot for PM2.5 with the line of equality, regression fitted line, and the calibrated data using RH. Figure 14. (a) CNN calibration scatter plot for PM2.5 with the line of equality, regression ﬁtted line, and the calibrated data using AH; (b) CNN calibration scatter plot for PM2.5 with the line of equality, regression ﬁtted line, and the calibrated data using RH. 16 of 23 lity, Sensors 2022, 22, 1093 (a) (b) Figure 15. (a) CNN calibration plot for PM10 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) CNN calibration plot for PM10 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. Figure 15. (a) CNN calibration plot for PM10 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) CNN calibration plot for PM10 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. Sensors 2022, 22, x FOR PEER REVIEW 19 of (b) (a) (b) (a) Figure 15. (a) CNN calibration plot for PM10 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) CNN calibration plot for PM10 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. Figure 15. (a) CNN calibration plot for PM10 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) CNN calibration plot for PM10 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. sors 2022, 22, x FOR PEER REVIEW 19 of (b) (a) (b) Figure 16. (a) CNN calibration scatter plot for PM10 with the line of equality, regression fitted lin and the calibrated data using AH; (b) CNN calibration scatter plot for PM10 with the line of equalit regression fitted line, and the calibrated data using RH. 3.3.4. Random Forest Figure 16. (a) CNN calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using AH; (b) CNN calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using RH. (a) (b) (a) Figure 16. For the MLP calibration analy 3.3.3. Convolution Neural Network (a) CNN calibration scatter plot for PM10 with the line of equality, regression fitted lin and the calibrated data using AH; (b) CNN calibration scatter plot for PM10 with the line of equali regression fitted line, and the calibrated data using RH. 3 3 4 Random Forest Figure 16. (a) CNN calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using AH; (b) CNN calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using RH. i 3.3.4. Random Forest Blue colour represents the reference data and orange colour represents the calibrated data. ors 2022, 22, x FOR PEER REVIEW 20 of (a) (b) Figure 18. (a) RF calibration scatter plot for PM2.5 with the line of equality, regression fitted line, a the calibrated data using AH; (b) RF calibration scatter plot for PM2.5 with the line of equality, gression fitted line, and the calibrated data using RH. Figure 18. (a) RF calibration scatter plot for PM2.5 with the line of equality, regression ﬁtted line, and the calibrated data using AH; (b) RF calibration scatter plot for PM2.5 with the line of equality, regression ﬁtted line, and the calibrated data using RH. (a) (b) Figure 18. (a) RF calibration scatter plot for PM2.5 with the line of equality, regression fitted line, and the calibrated data using AH; (b) RF calibration scatter plot for PM2.5 with the line of equality, re- gression fitted line, and the calibrated data using RH. (b) (b) th the line of equality, regression fitted line, and atter plot for PM2 5 with the line of equality re- (a) (a) Figure 18. (a) RF calibration scatter plot for PM2 the calibrated data using AH; (b) RF calibration (b) ssion line o (a) calibra ta usin Figure 18. (a) RF calibration scatter plot for PM2.5 with the line of equality, regression fitted line, an the calibrated data using AH; (b) RF calibration scatter plot for PM2.5 with the line of equality, r gression fitted line, and the calibrated data using RH. Figure 18. (a) RF calibration scatter plot for PM2.5 with the line of equality, regression ﬁtted line, and the calibrated data using AH; (b) RF calibration scatter plot for PM2.5 with the line of equality, regression ﬁtted line, and the calibrated data using RH. g ; ( ) p q y, gression fitted line, and the calibrated data using RH. (a) (b) Figure 19. (a) RF calibration plot for PM10 using AH. Blue colour represents the reference data an orange colour represents the calibrated data; (b) RF calibration plot for PM10 using RH. Blue colou represents the reference data and orange colour represents the calibrated data. (a) (b) Figure 19. (a) RF calibration plot for PM10 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) RF calibration plot for PM10 using RH. i 3.3.4. Random Forest tion of classification or regression trees which was first introduced by Breiman in 200 [60]. In this experiment, 20 trees were used in the forest for calibration. The experiment results, lines, and scatter plots were analysed as completed for the previous three model Figures 17–20 show the lines and scatter plots obtained for AH and RH for both PM2.5 an PM10. The analysis of the plots shows similar results as the previous three models whic supports the conclusion that AH gives better performance than RH for calibration. The Random Forest (RF) model is a machine learning technique based on a com- bination of classification or regression trees which was first introduced by Breiman in 2001 [60]. In this experiment, 20 trees were used in the forest for calibration. The experimental results, lines, and scatter plots were analysed as completed for the previous three models. Figures 17–20 show the lines and scatter plots obtained for AH and RH for both PM2.5 and PM10. The analysis of the plots shows similar results as the previous three models which supports the conclusion that AH gives better performance than RH for calibration. 17 of 23 17 of 23 Sensors 2022, 22, 1093 (a) (b) Figure 17. (a) RF calibration plot for PM2.5 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) RF calibration plot for PM2.5 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. Figure 17. (a) RF calibration plot for PM2.5 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) RF calibration plot for PM2.5 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. Sensors 2022, 22, x FOR PEER REVIEW 20 of 2 R PEER REVIEW 20 of 26 (b) 20 o (a) sors 2022, 22, x FOR PEER REVIEW (a) (b) Figure 17. (a) RF calibration plot for PM2.5 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) RF calibration plot for PM2.5 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. Figure 17. (a) RF calibration plot for PM2.5 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) RF calibration plot for PM2.5 using RH. p , y , Four calibration models were exami 4. Experimental Results, Analysis, and Discussion at the Sheffield site. Among the 1891 data, we divided it into a 70/30 ratio for training an testing data (number of training data = 1324 and number of testing data = 567) for all fou models. For the comparative analysis, experimented results are summarised in Tables and 3 for PM2.5 and PM10, respectively. In both the tables, five fields are presented: R2 (Co efficient of Determination), RMSE (Root Mean Square Error), and MAE (Mean Absolu Error), Mean PMs’ reading from the reference station, and four calibration models. From Table 2, the comparative analysis for PM2.5, it can be observed that R2 values (Coefficien of Determination) for the four calibration models are nearly the same, ranging from 0.8 to 0.89 for the AH whereas there was more variance in R2 ranging from 0.84 to 0.88 whe RH was used. Among four calibration models, the RF model has the highest R2 of bot AH and RH cases. The next parameters that were compared are RMSE for both AH an RH. Analysing this, it was noted that the RF model has the lowest RMSE for both AH an RH, which tells us that it is able to fit the dataset the best out of the four calibration model The next performance parameter that was compared is MAE. Comparing MAE, the R model has less error than the other models. It has a nearly 47% improvement in errors i comparison with MAE for the MLR model. The calibrated values from all four models were compared with the reference dat Mean values and standard deviations of reference data and calibrated models were com pared. The comparative analysis showed that the mean values of MLR and CNN calibr tion models are closer than the other two models, MLP and RF, to the reference mea when AH was used. On the other hand, for the MLP and RF models mean values are close h h MLR d CNN h f h RH d C i h d Four calibration models were examined with the dataset of 1891 records for 1 month at the Shefﬁeld site. Among the 1891 data, we divided it into a 70/30 ratio for training and testing data (number of training data = 1324 and number of testing data = 567) for all four models. For the comparative analysis, experimented results are summarised in Tables 2 and 3 for PM2.5 and PM10, respectively. i 3.3.4. Random Forest (a) RF calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using AH; (b) RF calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using RH. 4. Experimental Results, Analysis, and Discussion Four calibration models were examined with the dataset of 1891 records for 1 month at the Shefﬁeld site. Among the 1891 data, we divided it into a 70/30 ratio for training and testing data (number of training data = 1324 and number of testing data = 567) for all four models. For the comparative analysis, experimented results are summarised in Tables 2 and 3 for PM2.5 and PM10, respectively. In both the tables, ﬁve ﬁelds are presented: R2 (Coefﬁcient of Determination), RMSE (Root Mean Square Error), and MAE (Mean Ab- solute Error), Mean PMs’ reading from the reference station, and four calibration models. From Table 2, the comparative analysis for PM2.5, it can be observed that R2 values (Coefﬁ- cient of Determination) for the four calibration models are nearly the same, ranging from 0.87 to 0.89 for the AH whereas there was more variance in R2 ranging from 0.84 to 0.88 when RH was used. Among four calibration models, the RF model has the highest R2 of both AH and RH cases. The next parameters that were compared are RMSE for both AH and RH. Analysing this, it was noted that the RF model has the lowest RMSE for both AH and RH, which tells us that it is able to ﬁt the dataset the best out of the four calibration models. The next performance parameter that was compared is MAE. Comparing MAE, the RF model has less error than the other models. It has a nearly 47% improvement in errors in comparison with MAE for the MLR model. Table 2. Statistical performance measures analysis for PM2.5. Model R2 RMSE MAE Mean Reading (After Calibration) Reference Mean = 9.32 µg/m3 Standard Deviation (After Calibration) Reference Standard = 9.26 µg/m3 AH RH AH RH AH RH AH RH AH RH MLR 0.87 0.84 3.32 3.65 2.19 2.58 9.36 9.86 8.72 9.13 MLP 0.88 0.85 3.20 3.48 2.13 2.18 9.60 8.10 9.08 7.94 CNN 0.89 0.88 3.07 3.65 2.01 2.30 9.26 10.29 8.32 9.50 RF 0.89 0.88 3.05 3.07 1.19 1.86 9.75 9.67 9.05 9.02 (a) (b) (a) Figure 20. i 3.3.4. Random Forest (a) RF calibration scatter plot for PM10 with the line of equality, regression fitted line, an the calibrated data using AH; (b) RF calibration scatter plot for PM10 with the line of equality, re gression fitted line, and the calibrated data using RH. 4 Experimental Results Analysis and Discussion Figure 20. (a) RF calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using AH; (b) RF calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using RH. i 3.3.4. Random Forest Blue colour represents the reference data and orange colour represents the calibrated data. Figure 19. (a) RF calibration plot for PM10 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) RF calibration plot for PM10 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. (b) (b) (a) (a) (a) (b) Figure 19. (a) RF calibration plot for PM10 using AH. Blue colour represents the reference data an orange colour represents the calibrated data; (b) RF calibration plot for PM10 using RH. Blue colou represents the reference data and orange colour represents the calibrated data. Figure 19. (a) RF calibration plot for PM10 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) RF calibration plot for PM10 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. Figure 19. (a) RF calibration plot for PM10 using AH. Blue colour represents the reference data and orange colour represents the calibrated data; (b) RF calibration plot for PM10 using RH. Blue colour represents the reference data and orange colour represents the calibrated data. Sensors 2022, 22, 1093 18 of 23 18 of 23 (a) (b) Figure 20. (a) RF calibration scatter plot for PM10 with the line of equality, regression fitted line, an the calibrated data using AH; (b) RF calibration scatter plot for PM10 with the line of equality, r gression fitted line, and the calibrated data using RH. 4. Experimental Results, Analysis, and Discussion Figure 20. (a) RF calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using AH; (b) RF calibration scatter plot for PM10 with the line of equality, regression ﬁtted line, and the calibrated data using RH. (b) (a) (a) (b) Figure 20. (a) RF calibration scatter plot for PM10 with the line of equality, regression fitted line, and the calibrated data using AH; (b) RF calibration scatter plot for PM10 with the line of equality, re- gression fitted line, and the calibrated data using RH. 4. Experimental Results, Analysis, and Discussion Four calibration models were examined with the dataset of 1891 records for 1 month at the Sheffield site. i 3.3.4. Random Forest Among the 1891 data, we divided it into a 70/30 ratio for training and testing data (number of training data = 1324 and number of testing data = 567) for all four models. For the comparative analysis, experimented results are summarised in Tables 2 and 3 for PM2.5 and PM10, respectively. In both the tables, five fields are presented: R2 (Co- efficient of Determination), RMSE (Root Mean Square Error), and MAE (Mean Absolute Error), Mean PMs’ reading from the reference station, and four calibration models. From Table 2, the comparative analysis for PM2.5, it can be observed that R2 values (Coefficient of Determination) for the four calibration models are nearly the same, ranging from 0.87 to 0.89 for the AH whereas there was more variance in R2 ranging from 0.84 to 0.88 when RH was used. Among four calibration models, the RF model has the highest R2 of both AH and RH cases. The next parameters that were compared are RMSE for both AH and RH. Analysing this, it was noted that the RF model has the lowest RMSE for both AH and RH, which tells us that it is able to fit the dataset the best out of the four calibration models. The next performance parameter that was compared is MAE. Comparing MAE, the RF model has less error than the other models. It has a nearly 47% improvement in errors in comparison with MAE for the MLR model. The calibrated values from all four models were compared with the reference data. Mean values and standard deviations of reference data and calibrated models were com- pared. The comparative analysis showed that the mean values of MLR and CNN calibra- tion models are closer than the other two models, MLP and RF, to the reference mean when AH was used. On the other hand, for the MLP and RF models mean values are closer than the MLR and CNN to the reference means when RH was used. Comparing the stand- ard deviation, it was found that the RF model has the closest standard deviation values in both AH and RH cases to the reference standard deviation data. Similarly in Table 3, all four models are compared with each other for PM10. The comparative analysis reflected that there is a wider variance among the measured performance measures’ values for AH Figure 20. p , y , Four calibration models were exami 4. Experimental Results, Analysis, and Discussion Model R2 RMSE MAE Mean Reading (After Calibration) Reference Mean = 12.24 µg/m3 Standard Deviation (After Calibration) Reference Standard = 9.75 µg/m3 AH RH AH RH AH RH AH RH AH RH MLR 0.79 0.75 5.28 4.95 3.69 3.53 12.39 12.52 9.10 8.81 MLP 0.81 0.78 4.43 4.68 3.13 3.26 12.64 12.35 9.55 9.01 CNN 0.80 0.81 4.42 4.71 3.04 3.19 12.45 12.10 9.15 9.09 RF 0.83 0.83 4.03 4.05 2.78 2.77 12.64 12.45 9.43 9.38 The calibrated values from all four models were compared with the reference data. Mean values and standard deviations of reference data and calibrated models were com- pared. The comparative analysis showed that the mean values of MLR and CNN calibration models are closer than the other two models, MLP and RF, to the reference mean when AH was used. On the other hand, for the MLP and RF models mean values are closer than the MLR and CNN to the reference means when RH was used. Comparing the standard deviation, it was found that the RF model has the closest standard deviation values in both AH and RH cases to the reference standard deviation data. Similarly in Table 3, all four models are compared with each other for PM10. The comparative analysis reﬂected that there is a wider variance among the measured performance measures’ values for AH and RH for PM10 in comparison to PM2.5. From Table 3, it can be seen that the RF calibration model has fewer errors than the other three calibration models. It can also be observed that, for MAE, the RF model has a 25% improvement in MAE error measures than the other models. When comparing the mean readings, it is noted that the MLR model is closer to the reference mean and the MLP model is close to the reference standard deviation values for the Standard Deviation. From this comparative analysis of all these parameters, it was observed that the calibration models are performing better when AH was used as com- pared with RH. From the results, it was observed that the RF calibration model (R2 = 0.89, RMSE = 3.05, and MAE = 1.19) appeared as the best calibration output as compared with the other models for PM2.5. For the case of PM10, there was a variance in the performances of the different calibration models. p , y , Four calibration models were exami 4. Experimental Results, Analysis, and Discussion In both the tables, ﬁve ﬁelds are presented: R2 (Coefﬁcient of Determination), RMSE (Root Mean Square Error), and MAE (Mean Ab- solute Error), Mean PMs’ reading from the reference station, and four calibration models. From Table 2, the comparative analysis for PM2.5, it can be observed that R2 values (Coefﬁ- cient of Determination) for the four calibration models are nearly the same, ranging from 0.87 to 0.89 for the AH whereas there was more variance in R2 ranging from 0.84 to 0.88 when RH was used. Among four calibration models, the RF model has the highest R2 of both AH and RH cases. The next parameters that were compared are RMSE for both AH and RH. Analysing this, it was noted that the RF model has the lowest RMSE for both AH and RH, which tells us that it is able to ﬁt the dataset the best out of the four calibration models. The next performance parameter that was compared is MAE. Comparing MAE, the RF model has less error than the other models. It has a nearly 47% improvement in errors in comparison with MAE for the MLR model. ard deviation, it was found that the RF model has the closest standard deviation values i both AH and RH cases to the reference standard deviation data. Similarly in Table 3, a four models are compared with each other for PM10. The comparative analysis reflecte that there is a wider variance among the measured performance measures’ values for AH Table 2. Statistical performance measures analysis for PM2.5. Model R2 RMSE MAE Mean Reading (After Calibration) Reference Mean = 9.32 µg/m3 Standard Deviation (After Calibration) Reference Standard = 9.26 µg/m3 AH RH AH RH AH RH AH RH AH RH MLR 0.87 0.84 3.32 3.65 2.19 2.58 9.36 9.86 8.72 9.13 MLP 0.88 0.85 3.20 3.48 2.13 2.18 9.60 8.10 9.08 7.94 CNN 0.89 0.88 3.07 3.65 2.01 2.30 9.26 10.29 8.32 9.50 RF 0.89 0.88 3.05 3.07 1.19 1.86 9.75 9.67 9.05 9.02 ard deviation, it was found that the RF mode both AH and RH cases to the reference stan Table 2. Statistical performance measures analysis for PM2.5. 19 of 23 Sensors 2022, 22, 1093 Table 3. Statistical performance measures analysis for PM10. p , y , Four calibration models were exami 4. Experimental Results, Analysis, and Discussion The coefﬁcient of determination of the RF (R2 = 0.83) model gives better results. However, it was also observed that the CNN model gives a better result (R2 = 0.81) with the use of RH for calibration, but RMSE and MAE are higher than RF as shown in Table 3 for PM10. The experimental setup in our use case involving LCS-based AQ monitoring, and methodology covering the sensor selection and calibration, are transferable to similar applications across different domains. This methodology has the potential to be considered with its key success factors to make any LCS-based application kit design an innovative solution. This proposed methodology opens the door for efﬁcient and effective practices for LCS-based applications. 5. Conclusions and Future Work LCSs give an alternative solution against the high-cost sensors used for various mea- surement and monitoring purposes as they are compact in size and low-cost. However, it has been also observed that the use of LCS-based applications is challenging due to inconsistency in standard and different alternatives when measuring the same components. Additionally, the LCS has appeared as a multipurpose tool and the system is easy to con- ﬁgure, however, it is difﬁcult to select the right LCS for a speciﬁc task due to the diversity of LCSs available in the market with similar conﬁgurations. Even the sensor selection process has been carefully prepared but it needs to anticipate the possible obstacles such as delay in supply and different measurement units for the same purpose of measurement. Furthermore, some of the LCSs’ parameters can directly impact sensor performance and Sensors 2022, 22, 1093 20 of 23 20 of 23 data reliability, for example, meteorological parameters make the sensor selection and calibration process even harder. By examining the experimental outcomes from different sensors, we found that there have been different environmental responses of individual sensors. We also observed that there have been consistency issues among the sensors from the same manufacturer which appear as a challenging factor in deciding on a sensor during the sensor selection stage. From this study, we also found that the consistency and sensitivity of individual LCSs to environmental factors including temperature and humidity need to be analysed before applying the calibration. y y pp y g The conﬁdence in data from LCSs is lower as it required calibration. Therefore, building a device using LCSs is challenging and required some methodology on sensor selection and data processing. Our data-driven approaches provide a methodology that can help to build LCS-based devices from the sensor selection process and their calibration. To validate this methodology, experimental analysis was performed with different candidate sensors along with data collection. The data-driven approach provides a methodology to enhance data quality. Four widely used calibration models were applied for the LCS-based AQ device to analyse, and hence comparison was performed among the calibration models against the high-cost monitoring station data. Calibration parameters were established at pre-deﬁned locations with a high-cost reference station. This calibration process can also be transferable to other reference stations and sensors depending on the sensor types and their application. 5. Conclusions and Future Work pp The comparison among four commonly used calibration methods was presented to determine the best-suited calibration model in our use case study. In addition, from the analysis, it was also observed that AH has better performance than RH in the sensor calibration. Among the four models, the RF model appeared as the best model for the calibration of LCSs. To bring more conﬁdence to this work, the calibrated LCS devices will be deployed across the different regions in Bradford, UK, as a use case study for 3–6 months in the near future. The data coming from calibrated LCS devices will be analysed against the reference values over a longer duration to analyse the drift in LCS performance. Additionally, techniques and methodologies for re-calibration will be further explored to enhance the data quality of LCS-based monitoring systems. p q y g y The presented methodology did not include any uncertainty analysis as the obser- vation was applied for only a short duration for data analysis for sensor selection and calibration. However, for any LCS-based applications, an acceptable uncertainty needs to be deﬁned during the measurements. Additionally, in this study, the PM sensors were only analysed and calibrated. This appears as a limitation of this study as there are gas sensors that have also been used to monitor gas as a pollutant in AQ. As future work, a longer period of observation and data analysis needs to be applied to add additional conﬁdence and to reduce the uncertainty in monitored data from LCSs. Acceptable uncertainty is required to ensure the measurement uncertainties are lower and are sufﬁcient to make the calibration results valid. Acceptable uncertainty also ensures the uncertainty in data does not affect the LCS-based monitoring objectives. In addition, the same principles can be applied to LCS-based gas sensors. Author Contributions: R.R.K.: conceptualisation, methodology, software, validation, formal analy- sis, investigation, writing—review and editing, B.K.M.: conceptualisation, methodology, software, validation, formal analysis, investigation, writing—review and editing, D.T.: conceptualisation, methodology, validation, formal analysis, investigation, writing—review and editing, supervision, project administration, funding acquisition, R.J.: validation, investigation, writing—review and edit- ing, A.W.: validation, domain expertise, S.S.: validation, project administration, funding acquisition, N.T.: validation, writing—review and A.K.W.: validation, writing—review. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Interreg EC grant as part of the Smart Cities and Open Data Reuse (SCORE) and LifeCritical projects. References 2017, 99, 293–302. [CrossRef] p q y g p 11. Mueller, M.; Meyer, J.; Hueglin, C. 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https://openalex.org/W2132300672	https://hal.univ-reunion.fr/hal-01198475v2/file/1472-6807-8-28.pdf	English	null	CUSP: an algorithm to distinguish structurally conserved and unconserved regions in protein domain alignments and its application in the study of large length variations	BMC structural biology	2,008	cc-by	11,017	To cite this version: Sankaran Sandhya, Barah Pankaj, Kande Madabosse Govind, Bernard Offmann, Narayanaswamy Srinivasan, et al.. CUSP: an algorithm to distinguish structurally conserved and unconserved regions in protein domain alignments and its application in the study of large length variations. BMC Structural Biology, 2008, 8 (1), pp.1-14. 10.1186/1472-6807-8-28. hal-01198475v2 CUSP: an algorithm to distinguish structurally conserved and unconserved regions in protein domain alignments and its application in the study of large length variations Sankaran Sandhya, Barah Pankaj, Kande Madabosse Govind, Bernard Offmann, Narayanaswamy Srinivasan, Ramanathan Sowdhamini Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01198475 https://hal.univ-reunion.fr/hal-01198475v2 Submitted on 12 Jun 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. 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Distributed under a Creative Commons Attribution 4.0 International License BioMed Central BioMed Central Op Research article CUSP: an algorithm to distinguish structurally conserved and unconserved regions in protein domain alignments and its application in the study of large length variations Sankaran Sandhya1, Barah Pankaj1,2, Madabosse Kande Govind1, Bernard Offmann3, Narayanaswamy Srinivasan4 and Ramanathan Sowdhamini1 Open Access Address: 1National Centre for Biological Sciences (TIFR), UAS-GKVK Campus, Bellary Road, Bangalore 560 065, India, 2Mathematical modeling and Computational Biology group, Centre for Cellular and Molecular Biology, Hyderabad, India, 3Laboratoire de Biochimie et Génétique Moléculaire, Université de La Réunion, La Réunion, France and 4Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India Email: Sankaran Sandhya - sandhya@ncbs.res.in; Barah Pankaj - pankaj_b@ccmb.res.in; Madabosse Kande Govind - mkgovind@gmail.com; Bernard Offmann - bernard.offmann@univ-reunion.fr; Narayanaswamy Srinivasan - ns@mbu.iisc.ernet.in; Ramanathan Sowdhamini - mini@ncbs res in * Corresponding author Received: 15 January 2008 Accepted: 31 May 2008 Published: 31 May 2008 BMC Structural Biology 2008, 8:28 doi:10.1186/1472-6807-8-28 Published: 31 May 2008 BMC Structural Biology 2008, 8:28 doi:10.1186/1472-6807-8-28 This article is available from: http://www.biomedcentral.com/1472-6807/8/28 This article is available from: http://www.biomedcentral.com © 2008 Sandhya et al; licensee BioMed Central Ltd. © 2008 Sandhya et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2008 Sandhya et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creative which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cit Background properties and determine if class-distinct trends operate on protein domains. We extend earlier analysis on indel properties further by annotating such regions in terms of their preferred structural types, lengths and biochemical parameters and look for class-specific trends, if any. Such indels also extend functional and structural support to protein domains and this is also discussed briefly for a few superfamilies. g Protein databanks such as the PDB [1], with nearly 47,000 structures in the current year, are growing at a rapid pace. Interestingly, the increase in the number of protein struc- tures in the last decade is not accompanied by a concom- itant rise in the number of novel folds. This suggests that protein folds are resilient to exploit their large degrees of conformational freedom and can tolerate large modifica- tions in sequence and length. Structural comparisons of related proteins show that changes, in the form of substi- tutions, deletions or insertions are accommodated into existing protein scaffolds. Protein domains show from two-three residue variation to over two-fold length varia- tions as in the PDB entries for P-loop NTP hydrolases and the TIM fold. We report an algorithm, CUSP, which identifies conserved units of structure in proteins and distinguishes such regions from indels where length variations are intro- duced. The PASS2 database[4] provides structure-based alignments of non-redundant representatives of protein superfamilies sharing < 40% sequence identity. Since ini- tial equivalences are specified using STAMP 4.0[8] or LSQ- MAN[9], such alignments maximize structurally similar regions amongst related domains and distinguish them from indels that are structurally variable across different members. These alignments derived through COM- PARER[10] have examined protein domains that show not only low sequence conservation but also demonstrate variety in length and thus serve as ideal starting points to describe indel regions. Recent studies correlating domain length variations with the taxonomy spans of domains report that over one-third of all domains tend to increase/decrease in domain size. The fraction of domains that increase in domain size is two-fold larger than domains that decrease in size[2]. Analysis of protein length distributions across the main kingdoms have also shown that mean protein lengths are 40–60% greater in eukaryotes than in prokaryotes[3]. Such expansions in length correlate with the accretion of functional motifs during the evolution of sophisticated regulation networks in higher eukaryotes. Methods Dataset 353 structure-based superfamily alignments from PASS2 database [4], with more than 3 members, at < 40% iden- tity cutoff and nearly equal representation from the four major structural classes (72, 81, 88 and 112 superfamilies from α, β, α/β (AorB) and α+β (AplusB) classes respec- tively) were considered for the analysis. Since PASS2 derives from SCOP hierarchical schemes[12], only non- redundant representatives are considered in the align- ments and biases due to over-representation of similar structures are avoided. In a separate analysis on the study of physical parameters between related domains of a superfamily, "structural templates" were shown to have a strong correlation of physical parameters such as solvent accessibility, hydro- gen bonding patterns, spatial orientations and interac- tions between different members [7]. Such segmental conservation of features suggests that such features are not as well preserved in poorly conserved regions resulting in structural and functional diversity amongst related domains through variable regions. Length accretions are critical in mediating structural and functional variety in proteins and it is, therefore, important to understand their Background CUSP was used to examine length variations in 353 multi- membered superfamily alignments (> 3 sequence diverse relatives) from the PASS2 database [4]. To determine if observed trends are affected by the inclusion of more pro- teins, sequence homologues of the structural entries in PASS2 were included from the GenDis database[11]. In a separate analysis, the CUSP algorithm was also applied to such alignments to detect core features of a domain super- family. Further, we have extended the study to analyze the conservation of a biochemical property such as solvent exposure in structurally conserved and unconserved regions. Structural variation is influenced by the number, length and location of insertions and deletions of residues (indels) [4]. Pascarella and Argos [5], noted that less than 2% of indels are longer than 10 residues suggesting that a gradual accretion of protein length through shorter indels can achieve structural diversity. Reeves and co-workers [6], in an analysis of domain variations in CATH super- families have shown that even at low sequence identities (< 30%), 50% of the domain structure is conserved. How- ever, changes in the form of structural re-orientations and the number of structural elements are high between remotely related proteins. Domain length variations although discontinuous in sequence co-locate in 3D space and mediate functional variety. Abstract Background: Distantly related proteins adopt and retain similar structural scaffolds despite length variations that could be as much as two-fold in some protein superfamilies. In this paper, we describe an analysis of indel regions that accommodate length variations amongst related proteins. We have developed an algorithm CUSP, to examine multi-membered PASS2 superfamily alignments to identify indel regions in an automated manner. Further, we have used the method to characterize the length, structural type and biochemical features of indels in related protein domains. Results: CUSP, examines protein domain structural alignments to distinguish regions of conserved structure common to related proteins from structurally unconserved regions that vary in length and type of structure. On a non-redundant dataset of 353 domain superfamily alignments from PASS2, we find that 'length- deviant' protein superfamilies show > 30% length variation from their average domain length. 60% of additional lengths that occur in indels are short-length structures (< 5 residues) while 6% of indels are > 15 residues in length. Structural types in indels also show class- specific trends. Conclusion: The extent of length variation varies across different superfamilies and indels show class-specific trends for preferred lengths and structural types. Such indels of different lengths even within a single protein domain superfamily could have structural and functional consequences that drive their selection, underlying their importance in similarity detection and computational modelling. The availability of systematic algorithms, like CUSP, should enable decision making in a domain superfamily-specific manner. Page 1 of 14 (page number not for citation purposes) BMC Structural Biology 2008, 8:28 http://www.biomedcentral.com/1472-6807/8/28 http://www.biomedcentral.com/1472-6807/8/28 BMC Structural Biology 2008, 8:28 (see Figure 1). Likewise, solvent accessibility scores com- puted through PSA program [14] are also mapped to every sequence. Each alignment position is scanned and the assignments H, C and E at each position are retained if such a structural type shows > 75% occurrence. This is applied to capture consensus trends observed in the majority of members as described earlier in JOY assign- ments [15] and in the detection of equivalent structures amongst superfamily representatives [6]. Gaps (-) are retained to account for insertions or deletions in any member. A '' replaces a structural type at a position in the alignment when such a structural type shows < 25% occurrence at that position. To determine the consensus residue at each position, a scoring scheme that scores absolute matches of a structural type as 5, mismatches as 3.75 and gap exchanges as 1 (Figure 1), is employed. Although score assignments were chosen non-empiri- cally, they were optimized on analyzing a large variety of alignments that varied in the number of representatives and the employed scheme was effective in differentiating strictly structurally conserved positions from indel regions. The scoring scheme was applied to score all chema of CUSP algorithm and the scoring scheme employed for identifying structurally conserved and unconserved blocks SSB and USB] igure 1 Schema of CUSP algorithm and the scoring scheme employed for identifying structurally conserved and f Sc e a o CUS a go t a t e sco g sc e e e p oye o e t y g st uctu a y co se ve a u co se ve b oc s [SS a US ] gu e Schema of CUSP algorithm and the scoring scheme employed for identifying structurally conserved and unconserved blocks [SSB and USB]. Steps 1–4 illustrate the steps involved in processing structure-based alignments of an example domain superfamily. Scoring schemes that capture structural type exchanges at each position in the alignment (repre- sented as X1 and X2 exchanges for comparisons of each pair) are first applied to each position. Consecutive positions with high scores are merged to identify structurally conserved blocks and distinguish them from indels. An average score is associ- ated with each such block and used to annotate the alignment to distinguish indel regions (USB) from 'core' regions (SSB). CUSP: Detection of conserved units of structure in protein structural alignments g Starting from PASS2 alignments [4], CUSP maps DSSP assignments of secondary structure[13] to the alignment Page 2 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/8/28 Validation of the algorithm and scoring schemes f g g The scoring scheme that we have employed was arrived at after examining domain superfamily alignments that var- ied in the number of representative members in the align- ment. Although it is well appreciated that different approaches produce quite different alignments[16], struc- tural alignments of ten superfamilies, derived independ- ently using other alignment methods such as CE[17] and CDD[18], were also tested with the CUSP scoring scheme. Primarily, we wanted to determine if the applied scores were robust in identifying structurally conserved features in related domains. The number of structurally equivalent positions reported by either method was obtained and compared with the number of 'core' conserved residues identified by the CUSP scoring scheme when applied to domain superfamily alignments from PASS2. In each of the superfamilies considered, the CUSP scoring scheme was robust in capturing strictly conserved features. Specif- ically, inherent biases in the superfamily, for instance, the conservation of a minimum of 4 helices in the cyto- chrome C superfamily, could be captured independent of the alignment method and to that extent the schemes employed are predictive and can describe the strictly con- served features of a superfamily. The scoring scheme that we have employed was arrived at after examining domain superfamily alignments that var- ied in the number of representative members in the align- ment. Although it is well appreciated that different approaches produce quite different alignments[16], struc- tural alignments of ten superfamilies, derived independ- ently using other alignment methods such as CE[17] and CDD[18], were also tested with the CUSP scoring scheme. http://www.biomedcentral.com/1472-6807/8/28 BMC Structural Biology 2008, 8:28 sizes were calculated for every member using standard for- mula and averaged for the entire superfamily. The degree of length variation for every member from the mean domain size of its superfamily was calculated by express- ing as a ratio the length difference of each member to its mean domain size. exchanges between observed structural types at each posi- tion (Figure 1: described as X1–X2 exchanges) and an average score (Figure 1: Sc[i]) and a consensus structural type was assigned to every alignment position. Next, con- secutive alignment positions of identical structural types were merged to form a structural block and a Block score, an average of the sum of scores at each position, was asso- ciated with every block (Figure 1: Block_score[i] [j]). Thus, a block represents a consensus unit that is conserved in all members in the alignment. exchanges between observed structural types at each posi- tion (Figure 1: described as X1–X2 exchanges) and an average score (Figure 1: Sc[i]) and a consensus structural type was assigned to every alignment position. Next, con- secutive alignment positions of identical structural types were merged to form a structural block and a Block score, an average of the sum of scores at each position, was asso- ciated with every block (Figure 1: Block_score[i] [j]). Thus, a block represents a consensus unit that is conserved in all members in the alignment. Length difference = − ∗ \| \| li M M 100 Length difference = − ∗ \| \| li M M 100 where li = length of a protein superfamily member In a similar manner, average solvent accessibility scores were also associated with each structural block (not shown in the figure). Since the score is averaged over each position in the alignment, the block score is indicative of the extent of conservation of each structural type (H, C, E or -) in each block. Each structural block was associated with the tags 'Poor' (block score < 3), medium (block score 3–4.5) and high (block score 4.5–5.0). Finally, a consensus structural alignment is derived for the protein superfamily that not only delineates the structurally con- served blocks (SSB: H, E or C) from structurally uncon- served blocks (USB: , -) but also annotates such regions based on block scores as 'high, medium or poor' to indi- cate degree of conservation. Results and Discussion Results and Discussion Extent of length variation in protein domain superfamilies Length variations in protein domains are universal and observed in all protein classes. Figure 2a shows that domain superfamilies from all classes show long length variations (> 50 residue standard deviation from the aver- age domain size). ~20% of protein superfamilies from the α/β class exhibit > 50 residue deviations in domain length. ~70% of the superfamily members in all classes show < 50 residue deviation (Figure 2a). The extent of length variation in different multi-membered super- families from all classes ranges from 5 to > 45% of the mean domain size (Figure 2b) and has been used to dis- M = Mean domain size of each superfamily The distribution of length differences of the ~2500 pro- teins in the 353 superfamilies was plotted. Superfamilies in which a majority of the members (> 75%) show > 30% length variation were considered as "length-deviant" superfamilies and others with less than 10% variation in length from the mean domain size were considered as "length-rigid" superfamilies. The extent of deviation and the number of members that distributed in various length ranges were both employed in the distinction of super- families as length-rigid and length-deviant (also see Addi- tional information Section I: S1–S5, Figures 2 and S1, and Results later). http://www.biomedcentral.com/1472-6807/8/28 In the example, highly conserved structural blocks (H, E and C) identified by high block scores (> 4.5), are indicated in maroon, dark blue and dark green respectively. Conserved blocks that show 'medium' conservation are also indicated (red (helix), cyan (strand) and light green (coil)). The remaining regions are treated as USB. Page 3 of 14 (page number not for citation purposes) Page 3 of 14 (page number not for citation purposes) Page 3 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/8/28 Application of CUSP on diverse folds and functional implications of indels Structurally conserved features of several domain super- families, by careful examinations of multiple alignments, have been studied in detail in the past and are available in literature. We have applied CUSP to a few classical domain superfamilies such as globin, ferritin and cyto- chrome C domain superfamilies (see Additional informa- tion) to determine CUSPs performance in identifying core regions and in distinguishing indel regions in these well characterized folds. In addition, other structure alignment methods were also applied to such folds. The functional and structural implications of indel regions detected by CUSP were also examined. Length variation in protein superfamilies Mean domain sizes for each superfamily were determined by averaging over the lengths of individual members. Standard deviations in length from the mean domain Page 4 of 14 (page number not for citation purposes) Page 4 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/8/28 BMC Structural Biology 2008, 8:28 a) Distribution of length variation (described by mean standard deviation) in 353 domain superfamily members of Alpha, Beta, Alpha/Beta (AorB) and Alpha +Beta (AplusB) classes Figure 2 a) Distribution of length variation (described by mean standard deviation) in 353 domain superfamily mem- bers of Alpha, Beta, Alpha/Beta (AorB) and Alpha +Beta (AplusB) classes. b) Class specific distribution of the extent of length variation (expressed as a ratio of mean domain size) of all superfamily members. ! " # $ % & ! ' ( ) * + , + - . * + , / , . * + - / - . * / , 0 , . * / - 0 - . * 0 , 1 , . * 0 - 1 - . * 1 , , . * 1 - - . * , (a) (b) 2 3 4 3 5 3 6 3 7 3 3 8 9 : ; < = > ? @ A B A < 9 C D E F G H I J K L M J 3 7 3 N 3 O 3 P 3 Q 3 3 R Q 3 Q 3 R 7 3 3 S 7 3 3 T : @ U < = V > W V @ ? A 8 X Y Z [ \ ] ^ _ ` Y Z a Y b a a \ c d Y ` d ] Z _ F e f K F H G g h K i M I L f (a) 2 3 4 3 5 3 6 3 7 3 3 8 9 : ; < = > ? Length variation in protein superfamilies @ A B A < 9 C D E F G H I J K L M J 3 7 3 N 3 O 3 P 3 Q 3 3 R Q 3 Q 3 R 7 3 3 S 7 3 3 T : @ U < = V > W V @ ? A 8 X Y Z [ \ ] ^ _ ` Y Z a Y b a a \ c d Y ` d ] Z _ F e f K F H G g h K i M I L f (a) ! " # $ % & ! ' ( ) * + , + - . * + , / , . * + - / - . * / , 0 , . * / - 0 - . * 0 , 1 , . * 0 - 1 - . * 1 , , . * 1 - - . * , (b) * + , + - . * + , / , . * + - / - . * / , 0 , . * / - 0 - . * 0 , 1 , . * 0 - 1 - . * 1 , , . * 1 - - . * , * + , + - . * + , / , . * + - / - . * / , 0 , . * / - 0 - . * 0 , 1 , . * 0 - 1 - . * 1 , , . * 1 - - . * , (b) a) Distribution of length variation (described by mean standard deviation) in 353 domain superfamily members of Alpha, Beta, Alpha/Beta (AorB) and Alpha Beta (AplusB) classes Figure 2 a) Distribution of length variation (described by mean standard deviation) in 353 domain superfamily mem- bers of Alpha, Beta, Alpha/Beta (AorB) and Alpha +Beta (AplusB) classes. b) Class specific distribution of the extent of length variation (expressed as a ratio of mean domain size) of all superfamily members. Length variation in protein superfamilies Highly populated domain superfamilies (> 10 numbers). S.No Class No_members Average domain size Sequence Identity Description Page 6 of 14 (page number not for citation purposes) Length variation in protein superfamilies tinguish length-rigid from length deviant domain super- families (Table 1). These trends are also observed on consideration of the 64 length-deviant domain super- families alone. For 50 of the length-deviant domain superfamilies, we observe that < 20% of the members show < 5 residue variation (Figure S1b). superfamilies, we observe that < 20% of the members show < 5 residue variation (Figure S1b). superfamilies, we observe that < 20% of the members show < 5 residue variation (Figure S1b). Page 5 of 14 (page number not for citation purposes) Page 5 of 14 (page number not for citation purposes) Page 5 of 14 (page number not for citation purposes) BMC Structural Biology 2008, 8:28 http://www.biomedcentral.com/1472-6807/8/28 http://www.biomedcentral.com/1472-6807/8/28 BMC Structural Biology 2008, 8:28 Table 1: List of length-rigid and length-deviant domain superfamilies. This list is shown only for helix-rich class. Please look into Additional Tables 1 and 2 for full list. Highly populated domain superfamilies (> 10 numbers). a) List of 'Length-rigid superfamilies' (> 4 members) Table 1: List of length-rigid and length-deviant domain superfamilies. This list is shown only for helix-rich class. Please look into Additional Tables 1 and 2 for full list. Highly populated domain superfamilies (> 10 numbers). a) List of 'Length-rigid superfamilies' (> 4 members). S.No Class No_members Average domain size Sequence Identity Description (%) 1 α 8 417 21 Cytochrome P450 2 α 6 323 14 Terpenoid synthases 3 α 8 250 25 Nuclear receptor ligand-binding domain 4 α 5 204 23 DNA-glycosylase 5 α 5 114 26 Calponin-homology domain, CH-domain b) List of 'Length-deviant superfamilies' (> 4 members). 6 α 22 101 24 Cytochrome c 7 α 32 64 26 Homeodomain-like 8 α 48 88 21 Winged helix" DNA-binding domain 9 α 6 92 32 C-terminal effector domain of bipartite response regulator 10 α 5 90 27 Putative DNA-binding domain 11 α 12 88 29 Histone-fold 12 α 12 259 17 Ferritin-like 13 α 22 142 18 4-helical cytokines 14 α 35 125 23 EF-hand 15 α 5 75 33 Met repressor-like 16 α 6 76 37 IHF-like DNA-binding proteins 17 α 6 191 22 6-phosphogluconate dehydrogenase C-terminal domain-like 18 α 6 308 18 Terpenoid cylases/Protein prenyltransferases 19 α 9 369 17 ARM repeat 20 α 9 202 21 TPR-like able 1: List of length-rigid and length-deviant domain superfamilies. This list is shown only for helix-rich clas dditional Tables 1 and 2 for full list. Structural characteristics and lengths of 'indels' ; < @ ; < A ; ; < B C D E F C G H G I C H J K L M N O P O Q R N O P P R N O Q ; < A ; < S ; < T ; < U ; < V W X Y Z [ Y X \ V W X Y ] ^ _ ` a b V W X Y Z [ Y X \ ] ^ _ ` a b V W X Y Z [ Y X \ ] ^ _ ` a b c Y d e ` f [ ^ ` c W _ f c d Y g h f i K H j F k J C M F l m n o p m o n q r m s t u v w x q r r p r q v v u v y P (c) z { \| } { \| ~ { \| { \| { { \| { \| { \| { \| { \| { \| { \| z { \| ¡ ¢ £ ¤ ¤ ¥ ¦ § ¦ ¨ © ¥ ¦ § § © ¥ ¦ ¨ ª § « ¬ ® ¯ ¬ ° ± ® ¬ ² ³ ² ´ ¬ ³ µ ¶ · ² ´ ¸ ¹ ¯ ¬ ² ± · ¶ ³ º ® » ¯ ¹ ³ º ¶ ! " # $ % & ' & ( & ) * ) + & * , ' - . # / # . Structural characteristics and lengths of 'indels' Page 6 of 14 (page number not for citation purposes) Page 6 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/8/28 BMC Structural Biology 2008, 8:28 ecific distribution of the type of structure observed in indel regions pecific distribution of the type of structure observed in indel regions. b) Class specific distribu Distribution of indel lengths of various structural types [α-helix, β-strand, coils] in indel regions. ! " # $ % & ' & ( & ) * ) + & * , ' - . # / # . 0 1 2 3 3 4 5 6 7 8 9 (a) (b) : ; < = ; < > ; < ? Structural characteristics and lengths of 'indels' We have examined the nature and lengths of secondary structures that appear in indels. Here, we find that regular secondary structures such as helices and strands are observed in indels, in addition to coils (Figure 3a). In fact, a class-specific trend emerges in the present analysis (Fig- ures 3a and 4) and we find that additional length between related proteins is accrued in ~50% of protein super- families of the α-β (α/β or α+β) class as α-helices. Exami- nation of protein structures of representative 'giant' and 'dwarf' members of length-deviant protein superfamilies confirm these trends. 56% of protein superfamilies from the α-class such as the Cytochrome C (Figure 4a) show additional coils in indels while β-class proteins introduce either additional β-strands/coils (Figure 4b). The giant and dwarf members of the Actin-like ATPase and Lys- ozyme superfamilies (Figure 4c and 4d) accommodate up to two-fold variation in length primarily as additional hel- ices and coils. On consideration of the 64 length-deviant superfamilies alone, similar trends are obtained and heli- ces and coils are both highly favored in indels (Figure S1c). For 70% of the highly populated length-deviant domain superfamilies (Table 1), nearly 40% of indels are coils (Figure S1d). Manual examination of the locations of these additional structures shows that such indels can Structural modifications, it is observed, can form exten- sions of pre-existing structures or insert as new structural elements in the middle of domains. Such insertions although not contiguous in sequence may lie close to each other in structure and even form sub-domain like struc- tures. Alternately, they may accrue as additional regular (α-helix, β-strand) and irregular structures (coils) at the N and C terminal ends (Table S2). Since CUSP delineates protein alignments into structurally conserved regions and unconserved regions, it would be useful to identify if a selection principle is operational in identifying where structural modifications, because of additional lengths, can occur in related protein domains. Extent of length variation accommodated in SSB and USB 80% of length variations in length-deviant superfamilies from all classes are observed in USB regions with some superfamilies from the α/β class accommodating a wider range of length variation (Additional information, Sec- tion I: S2 – S4). Truncated structural elements account for 10% of these length differences (Additional Figure S1a). Structural characteristics and lengths of 'indels' 0 1 2 3 3 4 5 6 7 8 9 (a) (b) z { \| } { \| ~ { \| { \| { { \| { \| { \| { \| { \| { \| { \| z { \| ¡ ¢ £ ¤ ¤ ¥ ¦ § ¦ ¨ © ¥ ¦ § § © ¥ ¦ ¨ ª § « ¬ ® ¯ ¬ ° ± ® ¬ ² ³ ² ´ ¬ ³ µ ¶ · ² ´ ¸ ¹ ¯ ¬ ² ± · ¶ ³ º ® » ¯ ¹ ³ º ¶ (b) : ; < = ; < > ; < ? ; < @ ; < A ; ; < B C D E F C G H G I C H J K L M N O P O Q R N O P P R N O Q ; < A ; < S ; < T ; < U ; < V W X Y Z [ Y X \ V W X Y ] ^ _ ` a b V W X Y Z [ Y X \ ] ^ _ ` a b V W X Y Z [ Y X \ ] ^ _ ` a b c Y d e ` f [ ^ ` c W _ f c d Y g h f i K H j F k J C M F l m n o p m o n q r m s t u v w x q r r p r q v v u v y P (c) a) Class specific distribution of the type of structure observed in indel regions Figure 3 a) Class specific distribution of the type of structure observed in indel regions. b) Class specific distribution of indel lengths. c) Distribution of indel lengths of various structural types [α-helix, β-strand, coils] in indel regions. Structural characteristics and lengths of 'indels' Page 7 of 14 (page number not for citation purposes) Page 7 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/8/28 BMC Structural Biology 2008, 8:28 ength adjustments in length-deviant superfamilies from the four major classes igure 4 ength adjustments in length-deviant superfamilies from the four major classes. Panels' I-IV depict 'dwarf and giant' epresentative members (left and right respectively) of a deviant superfamily from alpha, beta, alpha/beta and alpha +beta class. epresentative members are indicated with PDB id and domain length. CUSP reported structurally conserved regions (SSB), hose lengths and structural type are retained across all domain superfamily members (in brown), are distinguished from nconserved regions/indels (USB in green) (a) Cytochrome C superfamily 'giant' members are 56% more likely to adjust extra Length adjustments in length-deviant superfamilies from the four major classes Figure 4 Length adjustments in length-deviant superfamilies from the four major classes. Panels' I-IV depict 'dwarf and giant' representative members (left and right respectively) of a deviant superfamily from alpha, beta, alpha/beta and alpha +beta class. Representative members are indicated with PDB id and domain length. CUSP reported structurally conserved regions (SSB), whose lengths and structural type are retained across all domain superfamily members (in brown), are distinguished from unconserved regions/indels (USB, in green). (a) Cytochrome C superfamily 'giant' members are 56% more likely to adjust extra ength as coils and short length helices.(b) Viral proteins from β-class have acquired additional strands and coils in indel regions. Up to two-fold length variations are seen as additional coils and helices in (c) Actin-like ATPase and (d) Lysozyme-like domain superfamilies. Solvent accessibility in conserved structural blocks are < 5 residues (Figure 3b). Medium-sized indels of between 5–10 residues are noticed in 20% of all indels in the dataset. Only 6% of all indels are found to be > 15 res- idues in length. Similar trends were also observed in ear- lier analysis on homologous superfamilies [5,6] although on smaller and different datasets. are < 5 residues (Figure 3b). Medium-sized indels of between 5–10 residues are noticed in 20% of all indels in the dataset. Only 6% of all indels are found to be > 15 res- idues in length. Similar trends were also observed in ear- lier analysis on homologous superfamilies [5,6] although on smaller and different datasets. y The conservation of a biochemical property such as sol- vent accessibility in regions annotated as SSB and USB was analyzed (see Additional information, S5) to determine if such regions behaved distinctly from each other. Addi- tional Figure S2a shows that in Beta class superfamilies, structurally unconserved regions (USB) arising from indels or structural replacements are usually exposed to the solvent. Amongst these, in structurally conserved regions (SSB), β-strands show a distinct preference for avoiding solvent while coils and α-helices are partially/ well- exposed to the solvent. Likewise, Additional Figure S2b shows the distribution of the average PSA scores in different types of structurally conserved blocks in all classes. (Additional Figures S3–S5 show trends in these parameters for other classes). 45% of the additional α-helices in indels of helix-rich length-deviant superfamilies are shorter than 5 residues (Figure 3c). A majority of the α-helices appearing in USB regions of β and α-β protein superfamilies are < 5 residues although in all superfamilies, longer α-helices (between 5 and 15 residues) are also observed (~20%). ~70% of indels appearing as β-strands are short length (< 5 resi- dues) and this may relate to the cost involved in satisfying the inherent nature of β-strands to form sheets. Additional strands of longer length (> 10 residues) are observed in fewer than 5% of all length-deviant β-rich superfamilies. Such strands in indels could be extensions of pre-existing strands or occur as shorter length β-hairpins and, there- fore, strands longer than 15 residues are not noticed in indel regions. Application of CUSP algorithm in the identification of structural scaffolds The CUSP algorithm examines structure-derived align- ments to delineate structurally conserved regions from structurally variable regions in protein domain super- families. Thus, applications of the algorithm on domain superfamily alignments are well capable of identifying 'core' regions, common to all members, from 'variant' regions. In order to verify this, we have examined the scores assigned to various conserved blocks identified by the program on some well characterized folds such as the Globins, Ferritins and Cytochrome C (see Additional information). Each of these folds is known to show con- siderable variations in length that are accommodated as indels. We observe that percentage variation in terms of the total number of α-helices, coils and β-strands is more in length- deviant superfamilies than in length-rigid superfamilies (see Additional information, Section I: S1, and Tables S4 and S5). In some of the length-deviant superfamilies (Additional Tables S2 and S5), the number of additional structures is large enough to form domain like structures. Manual examination of the structural alignments of the giant and dwarf domains of length-deviant domain super- families shows that in all classes, the accretion of single, long secondary structures is less common and instead many short length indels are arranged to form super sec- ondary structural motifs (Table S2). Thus, isolated or sol- vent-exposed extra secondary structures are avoided and additional units confer structural or functional support in each domain superfamily. In order to address if these trends are observed after including immediate sequence homologues of these superfamiles, we consulted the pre- curated results from GenDis database [11] for the top-five length-deviant and length-rigid superfamilies belonging to the four major structural classes. For each superfamily, between 250 to 800 sequence homologues were consid- ered for assessing trends in length variation. We find sim- ilar trends of length variation in the superfamilies distinguished as "length-rigid" and "length-deviant" using structural homologues alone, even on the inclusion of sequence homologues in these superfamilies (data not shown). This suggests that superfamilies identified as length-deviant/rigid are likely to remain so even with the availability of more structures. Length adj Figure 4 Length adjustments in length deviant superfamilies from the four major classes Figure 4 Length adjustments in length-deviant superfamilies from the four major classes. Panels' I-IV depict 'dwarf and giant' representative members (left and right respectively) of a deviant superfamily from alpha, beta, alpha/beta and alpha +beta class. Representative members are indicated with PDB id and domain length. CUSP reported structurally conserved regions (SSB), whose lengths and structural type are retained across all domain superfamily members (in brown), are distinguished from unconserved regions/indels (USB, in green). (a) Cytochrome C superfamily 'giant' members are 56% more likely to adjust extra length as coils and short length helices.(b) Viral proteins from β-class have acquired additional strands and coils in indel regions. Up to two-fold length variations are seen as additional coils and helices in (c) Actin-like ATPase and (d) Lysozyme-like domain superfamilies. Length adjustments in length deviant superfamilies from the four major classes Figure 4 Length adjustments in length-deviant superfamilies from the four major classes. Panels' I-IV depict 'dwarf and giant' representative members (left and right respectively) of a deviant superfamily from alpha, beta, alpha/beta and alpha +beta class. Representative members are indicated with PDB id and domain length. CUSP reported structurally conserved regions (SSB), whose lengths and structural type are retained across all domain superfamily members (in brown), are distinguished from unconserved regions/indels (USB, in green). (a) Cytochrome C superfamily 'giant' members are 56% more likely to adjust extra length as coils and short length helices.(b) Viral proteins from β-class have acquired additional strands and coils in indel regions. Up to two-fold length variations are seen as additional coils and helices in (c) Actin-like ATPase and (d) Lysozyme-like domain superfamilies. and lysozyme (Figures 4c and 4d), such indels are long and sub-domain like. either act as extensions of previous secondary structures or occur as insertions in the middle of domains. Some of these insertions extend from the N and C terminal domains and in superfamilies such as the SAM domain The length distributions of such indels in different classes also show interesting trends. We find that 60% of indels Page 8 of 14 (page number not for citation purposes) Page 8 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/8/28 http://www.biomedcentral.com/1472-6807/8/28 BMC Structural Biology 2008, 8:28 Page 9 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/8/28 http://www.biomedcentral.com/1472-6807/8/28 BMC Structural Biology 2008, 8:28 dues as structurally conserved while CDD reports 84 residues as strictly conserved. such well conserved regions and recognizes the four core helices. In fact, these scores and structurally conserved blocks are also identified by using alignments involving different members and methods. For the same super- family, we have examined the alignments generated by CE [17] and CDD[18]. Irrespective of the number of sequences and the alignment algorithm applied to align the sequences, CUSP is seen to detect the structural scaf- fold involving the core A, B, G, H helices to a high accu- racy. Figure 5a and Table S3 show that in the globin domain superfamily, CUSP reports 107 residues as struc- turally equivalent, CE derived alignments treat 119 resi- Globins The three-dimensional structures of globins are known, from crystallographic analyses, to be very similar. In an earlier analysis of the conserved features of this fold involving 226 sequences, it has been shown that the globin family of proteins differ greatly in their amino acid sequences and conserve only two residues in all sequences. Residue identities of some pairs of sequences are even as low as 16% [19]. Structure-guided alignments generated for these proteins have shown that although individual chains vary in size between 132 and 157 resi- dues, only 102 residue sites are common to all globins due to many deletions and insertions. These sites form six separate regions that lie in the core conserved helices. Insertions and deletions between these regions involve separations of different lengths in different sequences. Other detailed reviews on the phylogenies and differences between constituent members universally agree on the conservation of a core fold constituted by the A, B G and H helices [20]. Functional variety is attributed to differ- ences in the remaining structural elements. As shown in Figure 5a, the CUSP algorithm attributes high scores to Page 9 of 14 (page number not for citation purposes) Page 9 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/8/28 Role of indels in structural and functional diversity f f y Indels, irrespective of their location, seem to confer a structural or functional variation to the domain super- family in which they occur (Table S2). For instance, the members of the SH3 domain superfamily (Src Homol- ogy) show up to two-fold length variation. This is a family of molecular modules that is conserved amongst diverse proteins which function in protein-protein interactions for intracellular signal transduction. These interactions are effected through the recognition of a short proline-rich sequence, that adopts a left-handed polyproline type II helical conformation, embedded in proteins [22]. A giant member of this domain superfamily, MIA (Melanoma inhibitory activity protein), differs from the typical mem- bers in structural and functional aspects. In contrast to the typical members that are intracellular and modular, MIA is a single domain, extracellular protein. Here, additional lengths are not only seen as N and C terminal extensions that result in wider and larger barrels (Figure 6a–c) but also as additional sheets and 310 helices in the middle of the structure. Length differences extend to the RT loop and 60s–70s loops that flank the ligand binding region of SH3 domain. A superposition of four domains in this super- family shows incremental additions to the termini and acquisition of additional structures in the middle of the domain such as in the RT and 60s–70s loops (Figure 6d). These indels mediate functional differences from the typ- ical domain members and involve in the ability to recog- nize ligands other than conventional polyproline helices. Thus, in the SH3 domain superfamily, structural add-ons tune a conventional scaffold to meet new requirements of location, structure and function. We have presented a method, CUSP, which processes pro- tein structure-driven alignments to identify conserved structural units, common to all related proteins. In doing so, regions that allow variations to accumulate and confer uniqueness to each protein, annotated as USB, are also identified for every superfamily. The scoring schemes were arrived at after examining alignments derived independ- ently from other approaches such as CE and CDD. In 8 of the 10 superfamilies examined, CUSP detects > 60% of the conserved residues reported by other alignment meth- ods. For the two superfamilies which show < 45% cover- age, the large difference in the number of structural entries examined may be responsible for the difference in per- formance. Ferritin like superfamily An iron-coordinating glutamic or aspartic acid is located in the first helix and there is an EXXH (single-letter code for amino acids) motif in the second, but there are no other obvious sequence homologies. CUSP when applied to structure-based alignments for the domain superfamily detects the consecutive helices that strictly co- ordinate Fe (Figure 5b). These conserved helices, in fact, typify the conserved scaffold of the domain superfamily and are also detected from independently derived CE alignments of Ferritin domains. As seen in Table S3, both CE and CUSP agree well on the number of structurally equivalent residues for the domain superfamily. Large dif- ference in size that occurs as additional helices and several loops are found to be associated with the number of inter- acting domains in the giant member MMO which is far more than ruberythrin. This difference could account for the acquisition of extra structural elements that can inter- act with different domains. Ferritin like superfamily This superfamily of the alpha-rich fold includes members that are di-iron carboxylate proteins. The average domain size of the superfamily is 250 residues and includes small domains such as ruberythrin (1dvba1, 147 residues) and giant domains such as methane monoxygenase hydroxy- lase/MMO (1mtyd,512 residues). The two domains cata- lyze dioxygen-dependent oxidation-hydroxylation reactions[21]. All members are characterized by the pres- a Structurally conserved regions identified in the globin fold (in pink) by CUSP on independently derived alignments from PASS2 and CE (left and right respectively) Figure 5 a Structurally conserved regions identified in the globin fold (in pink) by CUSP on independently derived align- ments from PASS2 and CE (left and right respectively).b Dwarf and giant domains in the Ferritin superfamily ([1dvba1 (1–147)] and [1mtyd- (15–526)], left and right respectively) show a common conserved core of 4 helices (in brown) surround- ing a central Fe atom. Additional lengths in methane monoxygenase hydroxylase, the giant domain, (in green) participate in domain interactions. a Structurally conserved regions identified in the globin fold (in pink) by CUSP on independently derived alignments from PASS2 and CE (left and right respectively) Figure 5 a Structurally conserved regions identified in the globin fold (in pink) by CUSP on independently derived align- ments from PASS2 and CE (left and right respectively).b Dwarf and giant domains in the Ferritin superfamily ([1dvba1 (1–147)] and [1mtyd- (15–526)], left and right respectively) show a common conserved core of 4 helices (in brown) surround- ing a central Fe atom. Additional lengths in methane monoxygenase hydroxylase, the giant domain, (in green) participate in domain interactions. y g g ( p ) y p y g ( g p y) g a Structurally conserved regions identified in the globin fold (in pink) by CUSP on independently derived align- ments from PASS2 and CE (left and right respectively).b Dwarf and giant domains in the Ferritin superfamily ([1dvba1 (1–147)] and [1mtyd- (15–526)], left and right respectively) show a common conserved core of 4 helices (in brown) surround- ing a central Fe atom. Additional lengths in methane monoxygenase hydroxylase, the giant domain, (in green) participate in domain interactions. Page 10 of 14 (page number not for citation purposes) Page 10 of 14 (page number not for citation purposes) BMC Structural Biology 2008, 8:28 http://www.biomedcentral.com/1472-6807/8/28 http://www.biomedcentral.com/1472-6807/8/28 ence of a duplicated motif consisting of two consecutive helices. Conclusion ence of a duplicated motif consisting of two consecutive helices. An iron-coordinating glutamic or aspartic acid is located in the first helix and there is an EXXH (single-letter code for amino acids) motif in the second, but there are no other obvious sequence homologies. CUSP when applied to structure-based alignments for the domain superfamily detects the consecutive helices that strictly co- ordinate Fe (Figure 5b). These conserved helices, in fact, typify the conserved scaffold of the domain superfamily and are also detected from independently derived CE alignments of Ferritin domains. As seen in Table S3, both CE and CUSP agree well on the number of structurally equivalent residues for the domain superfamily. Large dif- ference in size that occurs as additional helices and several loops are found to be associated with the number of inter- acting domains in the giant member MMO which is far more than ruberythrin. This difference could account for the acquisition of extra structural elements that can inter- act with different domains. In our analysis, we have estimated the extent of length var- iation in protein superfamilies and employed it as a meas- ure of structural variation between homologous proteins. Numerous measures have been used to quantify protein structural similarity and these include RMSD, SSAP, con- tact maps, DALI and VAST scores [23-26]. We are inter- ested in the tolerance of folds to large variations in length and have, therefore, employed standard deviation and mean length variation to determine this. Proteins of simi- lar lengths may still differ in the orientations of individual secondary structures and adopt different folds. To that extent, a simple scoring scheme that parses pre-derived structural alignments of known related proteins from the PASS2 database and quantifies the extent of length varia- tion in all protein superfamilies is used to empirically esti- mate trends emerging in the dataset. We have also performed the analysis on multi-membered domain superfamilies (> 3 members) for an empirical assessment of the data involving 353 domain superfamilies. Addi- tionally, trends obtained in the dataset are noticed on consideration of the most length-deviant or highly popu- lated domain superfamilies alone. Role of indels in structural and functional diversity While the alignments from CDD included very close sequence homologues, the structural representatives considered in the CE alignment included domain mem- bers of similar lengths and also include more sequence diverse members. A strict cut-off of 75% is employed to characterize structural types at each alignment position as H, E or C and this in fact, increases the stringency of the scores. These assignment and scores therefore, are repre- sentative and predictive of SSB assignments in all and new sequence relatives in the superfamily. Cut-off schemes similar to ours have been employed earlier in JOY repre- sentations of structural alignments[15] and in estimating equivalences of secondary structures (SSE) for deriving matrices[27]. We have also attempted a study of the domain contexts, associations of length deviant domains and their func- tional consequences (Table S2, and manuscript under Page 11 of 14 (page number not for citation purposes) Page 11 of 14 (page number not for citation purposes) BMC Structural Biology 2008, 8:28 http://www.biomedcentral.com/1472-6807/8/28 Giant and dwarf domains of the SH3 domain like superfamily (a) [1i1ja(1–106)] and (b) [1gcqa-(158–213)]) show additional structures near the ligand-binding site Figure 6 Giant and dwarf domains of the SH3 domain like superfamily (a) [1i1ja(1–106)] and (b) [1gcqa-(158–213)]) show additional structures near the ligand-binding site. Structural superposition of the domain superfamily members (c) shows an appreciable conservation of the core structures (in yellow). (d) Structview representation of the alignment of different domain members of the protein superfamily shows a well conserved core involving β-strands and indels acquiring secondary structure in the giant domain. Page 12 of 14 (page number not for citation purposes) Giant and structures Figure 6 Giant and dwarf domains of the SH3 domain like superfamily (a) [1i1ja(1 106)] and (b) [1gcqa (158 213)]) show additional structures near the ligand binding site Figure 6 Giant and dwarf domains of the SH3 domain like superfamily (a) [1i1ja(1–106)] and (b) [1gcqa-(158–213)]) show additional structures near the ligand-binding site. Structural superposition of the domain superfamily members (c) shows an appreciable conservation of the core structures (in yellow). (d) Structview representation of the alignment of different domain members of the protein superfamily shows a well conserved core involving β-strands and indels acquiring secondary structure in the giant domain. ples of different superfamilies that exhibit class-specific nature in accommodating length variations. preparation). Reeves et. al., [6] have examined equivalent secondary structures between CATH superfamilies and suggest that such additional structural elements contrib- ute effectively to functional variety in the highly popu- lated superfamilies. We find that in all superfamilies examined, the structur- ally unconserved regions amongst related proteins do not all retain a uniform pattern in solvent accessibility. This coincides with the expectation that it is in such regions that variation in lengths between proteins is introduced. To preserve the core scaffold, which may be the driving force in limiting the number of folds, indel regions are more prone to structural changes and this may result in greater solvent exposure in some proteins or alter protein surfaces to modify interaction interfaces. β-strands show a universal preference for solvent avoidance and this reflects the preference of such strands to avoid isolations from the protein core and integrate into the structure as well- ordered sheets (Table S2). In proteins of the α-β class, coils show a clear preference for solvent exposure, more so Since the CUSP algorithm works with a scoring scheme to detect consensus trends in a majority of the superfamily members, the extent of conservation of each structural type in each block is annotated and it is possible, there- fore, to extract features that correlate with the extent of conservation of each structural type. An analysis of the nature of such USBs shows that additional lengths can either occur as extensions or insert in the middle of a pro- tein structure. A class-specific trend for the type of struc- ture adopted in indel regions has also emerged in the current analysis and each class prefers a specific type of structure (Figure 3, Figure S1 (b-d)). Authors' contributions in α + β class superfamilies where they are vital in segre- gating α and β units. Inferences on solvent exposure, in the present analysis, are limited to individual domains of the proteins and do not consider multi-domain contexts and oligomerisation states of the proteins. in α + β class superfamilies where they are vital in segre- gating α and β units. Inferences on solvent exposure, in the present analysis, are limited to individual domains of the proteins and do not consider multi-domain contexts and oligomerisation states of the proteins. SS coded the algorithm, performed the analysis on the PASS2 dataset and drafted the manuscript. BP performed an initial manual analysis on five PASS2 superfamilies, MKG coded the JAVA based graphical viewer, Structview. NS and BO participated in design and review of the man- uscript. RS conceived of the study, design, co-ordination and critically reviewed the manuscript. All authors read and approved the final manuscript. Based on the extent of length variation observed in differ- ent superfamilies, we have clustered all the superfamilies into length-rigid and length-deviant groups. Interestingly, length-rigid proteins are not as well-populated (as reflected in the number of members that are functionally diverse and in the number of families) as length-deviant proteins. While on the one hand, this does indicate that with the availability of more structures, trends in length- deviations could be affected in the identified rigid super- families, one may argue that such superfamilies are not preferred due to their strict length limitations and limited functional promiscuity (as reflected in the number of fam- ilies). Length-deviant proteins, on the other hand, are found to include superfolds such as the P-loop NTP hydrolases, Ferredoxin folds etc., that have already been shown to be well represented in many genomes. Additional file 1 CUSP: an algorithm to distinguish structurally conserved and uncon- served protein domain alignments and its application in the study of large length variations. The data provided represent the various analysis carried out to determine and describe the length variation in the dataset (Section I: S1–S5) and also contains an example of the functional implications of indels in Cytochrome C domain superfamily (Section II). Additional fig- ures and tables that support the data in the main text are also included. Click here for file [http://www.biomedcentral.com/content/supplementary/1472- 6807-8-28-S1.pdf] In many length-deviant protein superfamilies, despite large differences in length (over two fold in some cases), the core is often well preserved. The large additional lengths often do not involve the active site and in many cases they affect the oligomerization states and interacting surfaces of the protein (Ferritin like domain superfamily), introduce substrate-specificity (SH3 domains) and in some cases play an auto-regulatory role (Table S2). Since our analysis is derived from the PASS2 database of domain superfamilies, which in turn is guided by the domain definitions of SCOP, it is highly likely that severe length deviation, exhibited as additional domains, have escaped our attention. Giant and structures Figure 6 Figure 4 shows exam- Page 12 of 14 (page number not for citation purposes) Page 12 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/8/28 http://www.biomedcentral.com/1472-6807/8/28 http://www.biomedcentral.com/1472-6807/8/28 BMC Structural Biology 2008, 8:28 BMC Structural Biology 2008, 8:28 Acknowledgements R.S was an International Senior Research Fellow of the Wellcome Trust, U.K. S.S thanks the Council of Scientific and Industrial Research, India for PhD research fellowship. We gratefully acknowledge NCBS-TIFR for infra- structural support. References 1. Berman HM, Bhat TN, Bourne PE, Feng Z, Gilliland G, Weissig H, Westbrook J: The Protein Data Bank and the challenge of structural genomics. Nat Struct Biol 2000, 7(Suppl):957-959. g , ( pp ) 2. Wolf Y, Madej T, Babenko V, Shoemaker B, Panchenko AR: Long- term trends in evolution of indels in protein sequences. BMC Evol Biol 2007, 7:19. 3. Zhang J: Protein-length distributions for the three domains of life. Trends Genet 2000, 16(3):107-109. These interesting trends that we have obtained on the nature and type of indels in protein superfamilies from different classes could impact the area of comparative modeling in indel regions of newer superfamily members. We have obtained some distinct trends on indels that are class-specific, with information on typical lengths. Such information, we expect, will be useful in the choice of spe- cific structural types for newer relatives of protein super- families. Each superfamily shows a distinct trend in length variability and such information can be fed, by the assign- ment of variable gap penalties, into sequence alignment approaches to improve homology detection amongst members that vary considerably in length. We trust that such analyses would provide guiding principles during sequence searches, alignment and homology modeling of distant relationships. ( ) 4. 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https://openalex.org/W2625805967	https://europepmc.org/articles/pmc5472187?pdf=render	English	null	A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts	MBio	2,017	cc-by	13,001	RESEARCH ARTICLE crossm RESEARCH ARTICLE A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts Address correspondence to Neal A. DeLuca, ndeluca@pitt.edu. This article is a direct contribution from a Fellow of the American Academy of Microbiology. External solicited reviewers: Sandra Weller, University of Connecticut School of Medicine; Rozanne Sandri-Goldin, University of California, Irvine. Received 12 May 2017 Accepted 23 May 2017 Published 13 June 2017 Citation Fox HL, Dembowski JA, DeLuca NA. 2017. A herpesviral immediate early protein promotes transcription elongation of viral transcripts. mBio 8:e00745-17. https://doi.org/ 10.1128/mBio.00745-17. Editor Michael J. Imperiale, University of Michigan—Ann Arbor Copyright © 2017 Fox et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Neal A. DeLuca, ndeluca@pitt.edu. This article is a direct contribution from a Fellow of the American Academy of Microbiology. External solicited reviewers: Sandra Weller, University of Connecticut School of Medicine; Rozanne Sandri-Goldin, University of California, Irvine. Received 12 May 2017 Accepted 23 May 2017 Published 13 June 2017 IMPORTANCE HSV-1 interacts with many cellular proteins throughout productive infection. Here, we demonstrate the interaction of a viral protein, ICP22, with a sub- set of cellular proteins known to be involved in transcription elongation. We deter- mined that ICP22 is required to recruit the FACT complex and other transcription elongation factors to viral genomes and that in the absence of ICP22 viral transcrip- tion is globally reduced late in productive infection, due to an elongation defect. This insight deﬁnes a fundamental role of ICP22 in HSV-1 infection and elucidates the involvement of cellular factors in HSV-1 transcription. ® mbio.asm.org 1 A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts Hannah L. Fox, Jill A. Dembowski, Neal A. DeLuca Hannah L. Fox, Jill A. Dembowski, Neal A. DeLuca Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA Hannah L. Fox, Jill A. Dembowski, Neal A. DeLuca Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh Pennsylvania, USA Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA ABSTRACT Herpes simplex virus 1 (HSV-1) genes are transcribed by cellular RNA polymerase II (RNA Pol II). While four viral immediate early proteins (ICP4, ICP0, ICP27, and ICP22) function in some capacity in viral transcription, the mechanism by which ICP22 functions remains unclear. We observed that the FACT complex (com- prised of SSRP1 and Spt16) was relocalized in infected cells as a function of ICP22. ICP22 was also required for the association of FACT and the transcription elongation factors SPT5 and SPT6 with viral genomes. We further demonstrated that the FACT complex interacts with ICP22 throughout infection. We therefore hypothesized that ICP22 recruits cellular transcription elongation factors to viral genomes for efﬁcient transcription elongation of viral genes. We reevaluated the phenotype of an ICP22 mutant virus by determining the abundance of all viral mRNAs throughout infection by transcriptome sequencing (RNA-seq). The accumulation of almost all viral mRNAs late in infection was reduced compared to the wild type, regardless of kinetic class. Using chromatin immunoprecipitation sequencing (ChIP-seq), we mapped the loca- tion of RNA Pol II on viral genes and found that RNA Pol II levels on the bodies of viral genes were reduced in the ICP22 mutant compared to wild-type virus. In con- trast, the association of RNA Pol II with transcription start sites in the mutant was not reduced. Taken together, our results indicate that ICP22 plays a role in recruiting elongation factors like the FACT complex to the HSV-1 genome to allow for efﬁcient viral transcription elongation late in viral infection and ultimately infectious virion production. Received 12 May 2017 Accepted 23 May 2017 Published 13 June 2017 Citation Fox HL, Dembowski JA, DeLuca NA. 2017. A herpesviral immediate early protein promotes transcription elongation of viral transcripts. mBio 8:e00745-17. https://doi.org/ 10.1128/mBio.00745-17. Editor Michael J. Imperiale, University of Michigan—Ann Arbor Copyright © 2017 Fox et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. KEYWORDS RNA polymerases, herpes simplex virus, immediate early proteins, transcriptional regulation D uring productive infection, herpes simplex virus 1 (HSV-1) transcription is com- plexly regulated by both viral and cellular factors. Genes are transcribed by cellular RNA polymerase II (RNA Pol II) (1) in an ordered cascade with four classes: immediate early (), early (), leaky late (1), and true late (2) genes (2, 3). Four of the ﬁve immediate early proteins, ICP4, ICP0, ICP27, and ICP22, are involved in the regulation of viral transcription. ICP4 is a transcription factor that recruits cellular complexes, includ- ing TFIID and mediator, to viral genes to enhance transcription initiation and can also D ® mbio.asm.org 1 ® mbio.asm.org 1 May/June 2017 Volume 8 Issue 3 e00745-17 ® Fox et al. function to repress transcription of some viral genes (4–6). ICP0 is an E3 ubiquitin ligase (7) and plays a role in counteracting the repression of incoming viral genomes by host factors (8, 9) and abrogating the interferon response to infection (10, 11). ICP27 is involved in the nuclear export of viral mRNAs and has a role in recruiting RNA Pol II to viral genes (12–14). The immediate early protein ICP22 has been implicated in the promotion of late gene transcription, but the mechanism by which it functions is unknown (15–18). ICP22 is a 420-amino-acid protein that is extensively posttranslationally modiﬁed, including phosphorylation by the viral protein UL13 (19–21). ICP22 is required for efﬁcient productive infection in vivo and in ﬁbroblast cells such as MRC5 cells. ICP22 mutants are notably less restricted in Vero, HeLa, and HEp-2 cell lines (15, 17, 22, 23). Studies of ICP22 mutants in restrictive cells have shown that in the absence of ICP22 the expression of certain late genes is reduced (15, 17, 18, 24, 25). Additionally, virions produced by an ICP22 mutant virus are abnormally assembled, possibly due to reduced levels of the products of the late genes that encode virion assembly and structural proteins (26). ICP22 expression is associated with two effects on the cellular environment that may be related to its role in productive viral infection: the modiﬁcation of RNA Pol II and the formation of VICE (virus-induced chaperone-enriched) domains (27). In uninfected cells, the C-terminal domain (CTD) of RNA Pol II is sequentially phosphorylated during cellular gene transcription. KEYWORDS RNA polymerases, herpes simplex virus, immediate early proteins, transcriptional regulation These modiﬁcations follow a pattern in which hypophosphorylated RNA Pol II is recruited to promoters and is then phosphorylated on serine-5 (Ser-5) of the CTD around the start of transcription initiation. As RNA Pol II proceeds along the gene, serine-2 (Ser-2) of the CTD is phosphorylated and Ser-5 phosphorylation gradu- ally decreases toward the 3= end of the gene. Phosphorylation of Ser-2 of the CTD is typically associated with the ability of RNA Pol II to overcome promoter-proximal pausing and facilitate elongation (28, 29). During infection with HSV-1, the CTD of RNA Pol II is uniquely modiﬁed: RNA Pol II exhibiting both Ser-5 and Ser-2 phosphorylation of the CTD is rapidly decreased in the cell, and RNA Pol II exhibiting only Ser-5 phosphorylation of the CTD accumulates. The alteration of CTD modiﬁcations requires ICP22 and has been hypothesized to affect viral transcription in some way (25, 30–32). VICE domains have been shown to sequester host protein chaperones (33) and to accumulate nascent proteins during HSV-1 infection (34). Our laboratory recently developed a modiﬁed iPOND (isolation of proteins on nascent DNA) protocol to purify viral genomes and analyze proteins associated with these genomes during productive infection (35, 36). Many viral and cellular proteins were found to be associated with viral genomes, including ICP22 and a number of cellular proteins involved in transcription. Some transcription factors identiﬁed by this method have been previously shown to interact with ICP4, such as TFIID and mediator (37). The FACT transcription elongation complex was one of the most abundant protein complexes identiﬁed and was found to relocalize to viral replication compartments. The mechanism underlying this recruitment has not been determined (35, 36). y Preliminary experiments with viruses deﬁcient for one or more immediate early proteins suggested that ICP22 may be involved in the recruitment of the FACT complex to viral genomes. To elucidate the role of ICP22 in HSV-1 productive infection, we undertook to further characterize the phenotype of an ICP22 mutant. In this study, we puriﬁed wild-type (wt) and ICP22 mutant viral genomes from infected cells to deter- mine which proteins require ICP22 in order to associate with viral DNA. We found that the amounts of FACT complex subunits SSRP1 and Spt16 were substantially reduced on ICP22 mutant genomes. The FACT complex was originally identiﬁed for its role in transcription elongation through nucleosomes (38). May/June 2017 Volume 8 Issue 3 e00745-17 KEYWORDS RNA polymerases, herpes simplex virus, immediate early proteins, transcriptional regulation We therefore sought to character- ize the role of ICP22 in the recruitment of the FACT complex to viral genomes during productive HSV-1 infection and to investigate the transcriptional defects that occur in the absence of ICP22. We demonstrate that ICP22 physically interacts with the FACT complex and is essential for FACT complex and transcription elongation factor recruit- ment to viral DNA. Furthermore, in the absence of ICP22, RNA Pol II association with May/June 2017 Volume 8 Issue 3 e00745-17 mbio.asm.org 2 ® HSV ICP22 Promotes Elongation of Viral Transcripts FIG 1 Abundance of FACT complex proteins and localization of SSRP1 in infected cells. (A) MRC5 cells were infected with wild-type (KOS) virus at an MOI of 10. Cell lysates were collected at the indicated hours postinfection. Cell lysates were run on a 10% SDS-PAGE gel and probed with antibodies against Spt16, SSRP1, ICP4, and GAPDH. (B) Vero cells were infected with unlabeled KOS, or KOS prelabeled with 10 M EdC, at an MOI of 10. Cells infected with prelabeled KOS were ﬁxed at 3 hpi. Cells infected with unlabeled KOS were incubated in medium containing 10 M EdC starting at 4 hpi and then ﬁxed at 6 hpi. Fixed cells were stained with Hoechst stain (blue) and reacted with Alexa Fluor azide as described in Materials and Methods to visualize viral DNA (green). SSRP1 was detected by immunoﬂuorescence (red). FIG 1 Abundance of FACT complex proteins and localization of SSRP1 in infected cells. (A) MRC5 cells were infected with wild-type (KOS) virus at an MOI of 10. Cell lysates were collected at the indicated hours postinfection. Cell lysates were run on a 10% SDS-PAGE gel and probed with antibodies against Spt16, SSRP1, ICP4, and GAPDH. (B) Vero cells were infected with unlabeled KOS, or KOS prelabeled with 0 M EdC, at an MOI of 10. Cells infected with prelabeled KOS were ﬁxed at 3 hpi. Cells infected with unlabeled KOS were incubated in medium containing 10 M EdC starting at 4 hpi and then ﬁxed at 6 hpi. ixed cells were stained with Hoechst stain (blue) and reacted with Alexa Fluor azide as described in Materials and Methods to visualize viral DNA (green). SSRP1 was detected by immunoﬂuorescence (red). KEYWORDS RNA polymerases, herpes simplex virus, immediate early proteins, transcriptional regulation Ab d f FACT l i d l li i f SSRP i i f d ll (A) MRC ll FIG 1 Abundance of FACT complex proteins and localization of SSRP1 in infected cells. (A) MRC5 cells were infected with wild-type (KOS) virus at an MOI of 10. Cell lysates were collected at the indicated hours postinfection. Cell lysates were run on a 10% SDS-PAGE gel and probed with antibodies against Spt16, SSRP1, ICP4, and GAPDH. (B) Vero cells were infected with unlabeled KOS, or KOS prelabeled with 10 M EdC, at an MOI of 10. Cells infected with prelabeled KOS were ﬁxed at 3 hpi. Cells infected with unlabeled KOS were incubated in medium containing 10 M EdC starting at 4 hpi and then ﬁxed at 6 hpi. Fixed cells were stained with Hoechst stain (blue) and reacted with Alexa Fluor azide as described in Materials and Methods to visualize viral DNA (green). SSRP1 was detected by immunoﬂuorescence (red). gene bodies was reduced while the association with transcription start site regions of viral genes was not affected. Taken together, our results indicate that ICP22 plays a role in recruiting elongation factors, including the FACT complex, to viral genomes to allow for efﬁcient transcription elongation of viral genes. RESULTS Previously, our lab has reported that the FACT complex is abundant on HSV-1 genomes during viral replication (35). Additionally, SSRP1 is redistributed within the nucleus upon infection in KOS-infected cells compared to mock-infected cells, although Spt16 and SSRP1 protein levels are not altered by infection (35) (Fig. 1). In order to determine if any of the viral immediate early proteins were responsible for this redistribution and recruitment of the FACT complex to viral genomes, we used immu- noﬂuorescence to assay SSRP1 redistribution in cells infected with a panel of viruses deﬁcient for one or more immediate early proteins (see Table S1 in the supplemental material). We determined that SSRP1 was not redistributed upon infection with viruses deﬁcient for ICP22. We therefore hypothesized that ICP22 may play a role in recruiting the FACT complex to viral genomes and that, by deﬁning the relationship between ICP22 and the FACT complex, we could elucidate the role of ICP22 in productive infection. May/June 2017 Volume 8 Issue 3 e00745-17 Redistribution of FACT complex subunit SSRP1 requires ICP22 expression. Immu- noﬂuorescence was used to further characterize the redistribution of the FACT complex Redistribution of FACT complex subunit SSRP1 requires ICP22 expression. Immu- noﬂuorescence was used to further characterize the redistribution of the FACT complex mbio.asm.org 3 May/June 2017 Volume 8 Issue 3 e00745-17 ® Fox et al. FIG 2 Effect of ICP22 on the localization of SSRP1 in HSV-1-infected cells. Vero cells were infected with wild-type (KOS) or ICP22 mutant (n199) virus at an MOI of 10. (A) EdC (10 M) was added to the medium of infected cells from 4 to 6 hpi. The cells were then ﬁxed, stained with Hoechst stain (blue), and reacted with Alexa Fluor azide as described in Materials and Methods to visualize viral DNA (green). SSRP1 was detected by immunoﬂuorescence (red). Single cells shown are representative of cells in two independent replicates. (B) Infected cells were ﬁxed at 3 hpi and stained with Hoechst stain (blue). ICP4 (green) and SSRP1 (red) were detected by immunoﬂuorescence. Single cells shown are representative of cells in two independent replicates. FIG 2 Effect of ICP22 on the localization of SSRP1 in HSV-1-infected cells. Vero cells were infected with wild-type (KOS) or ICP22 mutant (n199) virus at an MOI of 10. (A) EdC (10 M) was added to the medium of infected cells from 4 to 6 hpi. The cells were then ﬁxed, stained with Hoechst stain (blue), and reacted with Alexa Fluor azide as described in Materials and Methods to visualize viral DNA (green). SSRP1 was detected by immunoﬂuorescence (red). Single cells shown are representative of cells in two independent replicates. (B) Infected cells were ﬁxed at 3 hpi and stained with Hoechst stain (blue). ICP4 (green) and SSRP1 (red) were detected by immunoﬂuorescence. Single cells shown are representative of cells in two independent replicates. relative to viral genomes as a function of ICP22. Vero cells were infected with either wild-type virus (KOS) or ICP22 mutant virus (n199), and cultures were incubated with the nucleoside analogue in the medium from 4 to 6 h postinfection (hpi). 5-Ethynyl- 2=-deoxycytidine (EdC) labeling of replicating viral genomes enables tagging of viral DNA with Alexa Fluor via click chemistry (35, 36). The cultures were ﬁxed at 6 hpi, labeled viral genomes were tagged with Alexa Fluor, and SSRP1 was probed via indirect immunoﬂuorescence. In mock-infected cells, SSRP1 was concentrated in the nucleolus (Fig. 2A), where it is known to play a role in RNA polymerase I-mediated transcription of rRNA (39). May/June 2017 Volume 8 Issue 3 e00745-17 Redistribution of FACT complex subunit SSRP1 requires ICP22 expression. Immu- noﬂuorescence was used to further characterize the redistribution of the FACT complex In KOS-infected cells, SSRP1 colocalized with viral genomes in replication compartments in addition to gathering in foci around viral replication compartments. In n199-infected cells, SSRP1 did not relocalize and was excluded from viral replication compartments. Because ICP22 is an immediate early protein, we also used immunoﬂuorescence to visualize the localization of SSRP1 early in infection. Vero cells were infected with KOS or n199, ﬁxed at 3 hpi, and probed with antibodies speciﬁc for ICP4 and SSRP1. ICP4 was used to represent the location of viral DNA because it has been shown to colocalize with viral genomes throughout infection (35). In cells infected with KOS, SSRP1 was redistributed into discrete foci in the nucleus by 3 hpi (Fig. 2B), and these foci were distinct from the majority of ICP4-containing foci. In cells infected with the ICP22 mutant n199, SSRP1 remained concentrated in the nucleolus, resembling mock- infected cells. We additionally observed that in cells infected with viral mutants that express ICP22 but do not express ICP4 or other combinations of immediate early proteins (Table S1), SSRP1 is redistributed similarly to SSRP1 in wild-type-infected cells. Redistribution of FACT complex subunit SSRP1 requires ICP22 expression. Immu- noﬂuorescence was used to further characterize the redistribution of the FACT complex mbio.asm.org 4 May/June 2017 Volume 8 Issue 3 e00745-17 ® HSV ICP22 Promotes Elongation of Viral Transcripts TABLE 1 Comparison of proteins copurifying with ICP22 mutant and wild-type viral a TABLE 1 Comparison of proteins copurifying with ICP22 mutant and wild-type viral genomesa Protein Spectral count by replicate and virus Replicate 1 Replicate 2 KOS n199 KOS n199 Viral proteins ICP4 689 696 834 916 ICP22 60 2 63 3 ICP8 592 539 972 910 UL9 199 218 255 341 UL30 213 229 456 507 UL42 369 255 411 320 Cellular repair proteins XRCC5 (KU80) 89 88 101 130 XRCC6 (KU70) 62 66 55 65 RNA polymerase II subunits PolrA 35 17 102 82 PolrB 19 6 47 42 Transcription elongation factors Spt16 57 4 78 7 SSRP1 43 0 29 0 Spt5 0 0 32 10 Spt6 12 0 41 2 Cdk9 5 9 6 11 Transcription initiation factors TBP (TFIID) 0 0 6 5 TAF1 (TFIID) 0 0 5 12 TAF6 (TFIID) 0 10 2 8 ERCC2 (TFIIH) 6 13 8 13 ERCC3 (TFIIH) 3 13 12 32 MED12 (mediator) 13 20 43 62 MED14 (mediator) 8 26 41 92 MED23 (mediator) 23 50 60 112 MED 24 (mediator) 15 23 25 52 aMRC5 cells were infected with wild-type (KOS) or an ICP22 mutant (n199) at an MOI of 10 and were incubated with EdC-containing medium or negative-control medium (no EdC) from 4 to 6 hpi. At 6 hpi, nuclei were harvested and the DNA was puriﬁed as described in Materials and Methods. Proteins eluted from genomes were analyzed by mass spectrometry. Values indicate spectral counts determined by mass spectrometry. aMRC5 cells were infected with wild-type (KOS) or an ICP22 mutant (n199) at an MOI of 10 and were incubated with EdC-containing medium or negative-control medium (no EdC) from 4 to 6 hpi. At 6 hpi, nuclei were harvested and the DNA was puriﬁed as described in Materials and Methods. Proteins eluted from genomes were analyzed by mass spectrometry. Values indicate spectral counts determined by mass spectrometry. The redistribution of SSRP1 in infected cells is therefore a speciﬁc function of ICP22. These data suggest that ICP22 affects the redistribution of SSRP1 in the cell throughout infection. ICP22 is necessary for the association of elongation factors with viral genomes. Redistribution of FACT complex subunit SSRP1 requires ICP22 expression. Immu- noﬂuorescence was used to further characterize the redistribution of the FACT complex In order to determine how ICP22 affects the association of the FACT complex and other viral and cellular factors with the viral genome, we puriﬁed replicating KOS and n199 viral DNA, along with associated proteins, from infected cells as previously described (35, 36). We infected MRC5 cells at a multiplicity of infection (MOI) of 10 PFU/cell with either n199 or KOS and incubated cells in the presence of EdC from 4 to 6 hpi to label replicating viral DNA. Infected cultures without EdC added to the medium served as a negative control. At 6 hpi, nuclei were harvested and EdC-labeled viral DNA was covalently tagged with biotin via click chemistry. Genomes were then puriﬁed on streptavidin beads, and associated proteins were identiﬁed by mass spectrometry. Many viral and cellular proteins were enriched on both the KOS and n199 viral genomes relative to unlabeled negative controls (Table S2). The association of most viral and cellular proteins with viral genomes was compa- rable between KOS and n199 as indicated by the similar recoveries of spectral counts (SpC) (Table 1). Levels of viral DNA binding proteins ICP4, UL42, and ICP8 were very mbio.asm.org 5 May/June 2017 Volume 8 Issue 3 e00745-17 ® Fox et al. similar between KOS and n199 EdC-labeled samples. Furthermore, the viral replication proteins UL9 (origin binding protein) and UL30 (DNA polymerase), as well as cellular DNA repair proteins, were present at similar levels on KOS and n199 genomes. Transcription initiation factors were found to be present at similar levels on KOS and n199 genomes or were slightly increased on n199 genomes. Interestingly, the levels of a subset of cellular transcription factors were selectively reduced on n199 genomes relative to KOS genomes. Most striking was the relative deﬁciency of a number of cellular transcription elongation factors. Speciﬁcally, there was a reduction in Spt5, Spt6, and FACT complex members Spt16 and SSRP1, as well as a modest reduction in the levels of RNA Pol II subunits. Both Spt16 and SSRP1 levels were reduced more than 20-fold, providing evidence that ICP22 plays a role in recruiting the FACT complex to replicating viral genomes. ICP22 interacts with FACT complex members SSRP1 and Spt16. Results thus far demonstrated that ICP22 is required for the relocalization of the FACT complex in infected cells and its association with viral genomes. May/June 2017 Volume 8 Issue 3 e00745-17 Redistribution of FACT complex subunit SSRP1 requires ICP22 expression. Immu- noﬂuorescence was used to further characterize the redistribution of the FACT complex In order to determine if these observations involve an interaction between ICP22 and FACT complex components, we constructed a virus expressing N-terminally FLAG-tagged ICP22 in a KOS background (ICP22-FLAG) to use in immunoﬂuorescence and coimmunoprecipitation experiments. ICP22 interacts with FACT complex members SSRP1 and Spt16. Results thus far demonstrated that ICP22 is required for the relocalization of the FACT complex in infected cells and its association with viral genomes. In order to determine if these observations involve an interaction between ICP22 and FACT complex components, we constructed a virus expressing N-terminally FLAG-tagged ICP22 in a KOS background (ICP22-FLAG) to use in immunoﬂuorescence and coimmunoprecipitation experiments. As in wt virus-infected cells, SSRP1 was relocalized from the pattern seen in mock-infected cells to discrete nuclear foci in ICP22-FLAG-infected cells (Fig. 3A). Furthermore, FLAG-tagged ICP22 colocalized with SSRP1. The number of SSRP1 foci in ICP22-FLAG-infected cells in Fig. 3A is fewer than the number of foci seen in Fig. 2B in wt virus-infected cells. While this may indicate that the relocalization is delayed in ICP22-FLAG-infected cells, we believe that this is most likely due to cell-to-cell variation. We therefore immunoprecipitated ICP22-FLAG to determine if FACT complex mem- bers physically interact with ICP22. We infected cells with ICP22-FLAG, lysed cells at 3 or 6 hpi, and immunoprecipitated ICP22-FLAG and associated proteins with anti-FLAG antibody-coated magnetic beads. Mock-, KOS-, and n199-infected cells were used as controls. Proteins were eluted from the beads with 3FLAG peptide and subjected to Western blot analysis. Both of the FACT complex subunits, SSRP1 and Spt16, copre- cipitated with ICP22. Treatment of puriﬁed complexes prior to immunoprecipitation with Benzonase, a nuclease that degrades all forms of DNA and RNA, did not disrupt the interactions between ICP22 and FACT complex members, indicating that the presence of nucleic acid in the sample did not contribute to coprecipitation (Fig. 3B). Taken together, these data indicate that ICP22 interacts with complex members either directly or as part of a complex. We therefore immunoprecipitated ICP22-FLAG to determine if FACT complex mem- bers physically interact with ICP22. We infected cells with ICP22-FLAG, lysed cells at 3 or 6 hpi, and immunoprecipitated ICP22-FLAG and associated proteins with anti-FLAG antibody-coated magnetic beads. Mock-, KOS-, and n199-infected cells were used as controls. Proteins were eluted from the beads with 3FLAG peptide and subjected to Western blot analysis. Both of the FACT complex subunits, SSRP1 and Spt16, copre- cipitated with ICP22. Redistribution of FACT complex subunit SSRP1 requires ICP22 expression. Immu- noﬂuorescence was used to further characterize the redistribution of the FACT complex (B) Isolation of FLAG-tagged ICP22 and associated proteins in infected cells. MRC5 cells were infected with ICP22-FLAG at an MOI of 10. Cells were harvested at 3 or 6 hpi. Lysates were treated with () or without (-) Benzonase and bound to anti-FLAG beads. Proteins bound to the beads were eluted with 3FLAG peptide and run on an SDS-PAGE gel. Gels were immunoblotted with anti-SSRP1, anti-Spt16, and anti-FLAG. FIG 3 Interaction of ICP22 and the FACT complex in infected cells. (A) Localization of SSRP1 and ICP22-FLAG in infected cells. Vero cells were infected with virus expressing FLAG-tagged ICP22 protein FIG 3 Interaction of ICP22 and the FACT complex in infected cells. (A) Localization of SSRP1 and ICP22-FLAG in infected cells. Vero cells were infected with virus expressing FLAG-tagged ICP22 protein (ICP22-FLAG) at an MOI of 10. Cells were ﬁxed at 3 and 6 hpi and stained with Hoechst stain (blue). ICP22-FLAG (green) and SSRP1 (red) were detected by immunoﬂuorescence. Single cells shown are representative of cells in two independent replicates. (B) Isolation of FLAG-tagged ICP22 and associated proteins in infected cells. MRC5 cells were infected with ICP22-FLAG at an MOI of 10. Cells were harvested at 3 or 6 hpi. Lysates were treated with () or without (-) Benzonase and bound to anti-FLAG beads. Proteins bound to the beads were eluted with 3FLAG peptide and run on an SDS-PAGE gel. Gels were immunoblotted with anti-SSRP1, anti-Spt16, and anti-FLAG. abundance throughout infection to identify transcriptional defects with n199. Tran- scriptome sequencing (RNA-seq) was used to measure viral transcript levels at different times after infection with wt (KOS) or ICP22 mutant (n199) virus. RNA-seq data indicated that although late gene mRNA accumulation was indeed impaired in n199 as reported, the expression of viral genes in other classes was also affected (Fig. 4B). Reduced gene expression appears to depend more on the time postinfection than on the kinetic class of a particular gene. Speciﬁcally, the expression of viral genes that are increasingly expressed after 6 hpi in KOS was reduced in n199 compared to KOS, regardless of kinetic class. These genes include those previously identiﬁed to be less expressed in ICP22 mutant-infected cells, such as RL1, which codes for ICP0; US11 (18); UL48, which codes for VP16; UL44, which codes for gC; and UL36 (25). Redistribution of FACT complex subunit SSRP1 requires ICP22 expression. Immu- noﬂuorescence was used to further characterize the redistribution of the FACT complex Treatment of puriﬁed complexes prior to immunoprecipitation with Benzonase, a nuclease that degrades all forms of DNA and RNA, did not disrupt the interactions between ICP22 and FACT complex members, indicating that the presence of nucleic acid in the sample did not contribute to coprecipitation (Fig. 3B). Taken together, these data indicate that ICP22 interacts with complex members either directly or as part of a complex. Viral mRNA and genome abundance in ICP22 mutant-infected cells. To further investigate the signiﬁcance of the interaction between the FACT complex and ICP22, we reevaluated the gene expression and DNA replication phenotypes of n199 (40) using more sensitive approaches. It has previously been reported that the rate of viral DNA replication of ICP22 mutant virus is similar to that of wild-type virus based on hybridization assays (17, 25, 40). To obtain a more quantitative account of viral DNA replication in the absence of ICP22, we used quantitative PCR to measure the number of viral genomes over time. We carried out infections in MRC5 cells, which are known to be restrictive to ICP22 mutant virus. One million cells were infected at an MOI of 5, and DNA was harvested throughout the productive viral replication cycle. DNA repli- cation rates did not differ signiﬁcantly between KOS and n199, indicating that ICP22 likely does not play a signiﬁcant role in viral DNA replication (Fig. 4A). We also assessed infectious virus production throughout infection using plaque assays. We observed an almost 20-fold reduction in infectious virus produced from n199-infected cells com- pared to KOS-infected cells (Fig. 4A). These data suggest that although the n199 genome efﬁciently replicates, it has some defect or defects that prevent infectious virion production. Previously, a reduction in late gene expression has been presumed to contribute to the reduced infectivity associated with n199. We therefore quantiﬁed viral mRNA mbio.asm.org 6 May/June 2017 Volume 8 Issue 3 e00745-17 ® HSV ICP22 Promotes Elongation of Viral Transcripts FIG 3 Interaction of ICP22 and the FACT complex in infected cells. (A) Localization of SSRP1 and ICP22-FLAG in infected cells. Vero cells were infected with virus expressing FLAG-tagged ICP22 protein (ICP22-FLAG) at an MOI of 10. Cells were ﬁxed at 3 and 6 hpi and stained with Hoechst stain (blue). ICP22-FLAG (green) and SSRP1 (red) were detected by immunoﬂuorescence. Single cells shown are representative of cells in two independent replicates. Redistribution of FACT complex subunit SSRP1 requires ICP22 expression. Immu- noﬂuorescence was used to further characterize the redistribution of the FACT complex We also observed decreased expression of genes such as genes UL9, UL42, UL50, and UL52; 1 genes US6 and UL19; and 2 genes UL45, UL35, and UL38, among others. Elongation of RNA polymerase transcription is impaired in an ICP22 mutant. In ICP22 mutant-infected cells, there was a global reduction in the abundance of all viral mbio.asm.org 7 May/June 2017 Volume 8 Issue 3 e00745-17 ® Fox et al. FIG 4 Replication and gene expression of an ICP22 mutant. (A) DNA replication and viral replication of ICP22 mutant n199 compared wild-type KOS. MRC5 cells were infected and assayed at 4, 6, 8, 12, and 24 hpi. DNA replication was determined by quantitative PCR (genome Points are averages from 2 experiments; error bars represent standard deviations. Viral replication was determined by plaque assay (PFU). Poin are averages from 2 experiments; error bars represent standard deviations. (B) RNA-seq analysis of select genes in all kinetic classes in ICP mutant (n199) compared to wild type (KOS). MRC5 cells were infected at an MOI of 5 and assayed at 2, 4, 6, 8, 12, and 16 hpi. Viral transcrip were quantiﬁed by RNA-seq as described in Materials and Methods. May/June 2017 Volume 8 Issue 3 e00745 17 FIG 4 Replication and gene expression of an ICP22 mutant. (A) DNA replication and viral replication of ICP22 mutant n199 compared to wild-type KOS. MRC5 cells were infected and assayed at 4, 6, 8, 12, and 24 hpi. DNA replication was determined by quantitative PCR (genomes). Points are averages from 2 experiments; error bars represent standard deviations. Viral replication was determined by plaque assay (PFU). Points are averages from 2 experiments; error bars represent standard deviations. (B) RNA-seq analysis of select genes in all kinetic classes in ICP22 mutant (n199) compared to wild type (KOS). MRC5 cells were infected at an MOI of 5 and assayed at 2, 4, 6, 8, 12, and 16 hpi. Viral transcripts were quantiﬁed by RNA-seq as described in Materials and Methods. FIG 4 Replication and gene expression of an ICP22 mutant. (A) DNA replication and viral replication of ICP22 mutant n199 compared to wild-type KOS. MRC5 cells were infected and assayed at 4, 6, 8, 12, and 24 hpi. DNA replication was determined by quantitative PCR (genomes). Points are averages from 2 experiments; error bars represent standard deviations. Viral replication was determined by plaque assay (PFU). May/June 2017 Volume 8 Issue 3 e00745-17 Redistribution of FACT complex subunit SSRP1 requires ICP22 expression. Immu- noﬂuorescence was used to further characterize the redistribution of the FACT complex Both 8WG16 and 4H8 have been used in previous ChIP-seq studies to determine RNA Pol II levels on individual genes and have been found to detect similar patterns (41). Therefore, by using both 8WG16 and 4H8, we are able to detect all forms of RNA Pol II that have been identiﬁed to be present in KOS- and n199-infected cells (25). Immunoprecipitated DNA was sequenced to determine the relative abundance of RNA Pol II and ICP4 across the viral genome. Consistent with results from the DNA replication assay (Fig. 4A), the total numbers of reads that map to the viral genome in the input samples were similar between KOS and n199 (Fig. S1A). Furthermore, con- sistent with mass spectrometry analysis (Table 1), the numbers of reads that map to the viral genome in ICP4 ChIP samples were similar between KOS and n199 and the numbers of reads that map to the viral genome in the RNA Pol II ChIP samples were somewhat reduced for n199 relative to KOS (Fig. S1A). The reads mapping across the viral genome for ICP4 and RNA Pol II are shown in Fig. S1B. The distribution of ICP4 across the viral genome was unaffected by mutation of ICP22. The details of ICP4 binding to the viral genome will be published elsewhere. At this level of resolution, the difference between the RNA Pol II binding proﬁles of n199 and KOS is quite subtle. The peaks in the n199 samples appear to be better deﬁned with a possible reduction in read numbers between the peaks compared to the KOS samples. 8WG16 and 4H8 antibodies produced read patterns similar to one another and reveal the same differences in binding patterns between KOS and n199. A higher-resolution representation of Fig. S1B in the region between UL42 and UL48 is shown in Fig. 5A. These genes are all expressed at 6 hpi in KOS-infected cells and represent members of the early through true late classes of genes. The numbers of reads in the RNA Pol II ChIP at or near transcription start sites were similar between KOS and n199. However, the numbers of reads mapping beyond the start site, in the message bodies, appeared to be signiﬁcantly reduced in n199 (Fig. 5A). Redistribution of FACT complex subunit SSRP1 requires ICP22 expression. Immu- noﬂuorescence was used to further characterize the redistribution of the FACT complex Points are averages from 2 experiments; error bars represent standard deviations. (B) RNA-seq analysis of select genes in all kinetic classes in ICP22 mutant (n199) compared to wild type (KOS). MRC5 cells were infected at an MOI of 5 and assayed at 2, 4, 6, 8, 12, and 16 hpi. Viral transcripts were quantiﬁed by RNA-seq as described in Materials and Methods. May/June 2017 Volume 8 Issue 3 e00745-17 mbio.asm.org ® HSV ICP22 Promotes Elongation of Viral Transcripts transcripts late in infection (Fig. 4B). Furthermore, the main difference in the recruit- ment of cellular proteins to the n199 genome relative to KOS appears to be limited to factors involved in transcription elongation (Table 1). In order to determine if the reduced abundance of viral transcripts in the absence of ICP22 could be due to a defect in transcription elongation, we mapped the distribution of RNA Pol II on KOS and n199 genomes by chromatin immunoprecipitation sequencing (ChIP-seq) at 6 hpi, a critical time for the observed phenotypes. MRC5 cells were infected with either KOS or n199 and cross-linked at 6 hpi to capture the state of the genome at that time, and parallel immunoprecipitations were performed with antibodies speciﬁc for RNA Pol II and ICP4. p p p p In wt HSV-1-infected cells, a unique form of RNA Pol II (IIi), in which the CTD is phosphorylated on Ser-5, accumulates. Additionally, reduction in the unphosphory- lated form (IIa) and even greater reduction in the hyperphosphorylated form (IIo), in which the CTD is phosphorylated on both Ser-5 and Ser-2, are observed (30, 31). In ICP22 mutant virus-infected cells, the IIi form does not accumulate and there is greater abundance of the IIa form than in KOS-infected cells (25). We performed parallel immunoprecipitations with the two RNA Pol II antibodies 8WG16 and 4H8. 8WG16 recognizes both the IIa and the HSV-speciﬁc IIi forms of RNA Pol II, both of which are present in HSV-1-infected cells (30). 4H8 recognizes Ser-5-modiﬁed RNA Pol II and should therefore recognize IIi. Additionally, 4H8 recognizes RNA Pol II with both Ser-5 and Ser-2 phosphorylated because Ser-2 phosphorylation does not inhibit binding of the antibody to its Ser-5 epitope and should therefore recognize IIo (29). Although 8WG16 is more speciﬁc for IIa than 4H8, 4H8 can also recognize IIa. May/June 2017 Volume 8 Issue 3 e00745-17 Redistribution of FACT complex subunit SSRP1 requires ICP22 expression. Immu- noﬂuorescence was used to further characterize the redistribution of the FACT complex In order to compare the relative abundance of Pol II and ICP4 on the start sites and message bodies of n199 and KOS, we calculated the ratio of normalized n199 to KOS reads for individual gene mRNA start sites and gene bodies of select genes represen- tative of different kinetic classes for both ICP4 and RNA Pol II (Fig. 5B). For the calculations, we used the data from the 4H8 antibody as this antibody should detect the forms of RNA Pol II involved in the transcription of viral genes in KOS- and mbio.asm.org 9 May/June 2017 Volume 8 Issue 3 e00745-17 ® Fox et al. FIG 5 ChIP analysis of ICP4 and RNA Pol II on ICP22 mutant versus wt (KOS) genomes. MRC5 cells were infected with wild type (KOS) or ICP22 mutant (n199) at an MOI of 10. The infected cells were harvested and processed for ChIP-seq as described in Materials and Methods. (A) Mapped reads of the region between UL42 and UL48 on the genome for ChIP of ICP4, RNA Pol II (4H8), or RNA Pol II (8WG16) in n199- or KOS-infected cells. The maximums on the y axis are to the top left of each panel. Experiments were repeated twice, and the ﬁgure represents one data set. (B) Normalized ratio of n199 over KOS reads for start sites or gene bodies of representative IE, E, and L genes. The values used were averages from two experiments using the 4H8 antibody. FIG 5 ChIP analysis of ICP4 and RNA Pol II on ICP22 mutant versus wt (KOS) genomes. MRC5 cells were infected with wild type (KOS) or ICP22 mutant (n199) at an MOI of 10. The infected cells were harvested and processed for ChIP-seq as described in Materials and Methods. (A) Mapped reads of the region between UL42 and UL48 on the genome for ChIP of ICP4, RNA Pol II (4H8), or RNA Pol II (8WG16) in n199- or KOS-infected cells. The maximums on the y axis are to the top left of each panel. Experiments were repeated twice, and the ﬁgure represents one data set. (B) Normalized ratio of n199 over KOS reads for start sites or gene bodies of representative IE, E, and L genes. The values used were averages from two experiments using the 4H8 antibody. FIG 5 ChIP analysis of ICP4 and RNA Pol II on ICP22 mutant versus wt (KOS) genomes. DISCUSSION HSV-1 genes are transcribed by cellular RNA Pol II (1). However, the Pol II machinery of the cell is perturbed or augmented by viral gene products, resulting in the selective and efﬁcient expression of viral genes. For example, the immediate early protein ICP4 plays an important role in the regulation of transcription initiation. ICP4 binds to viral DNA and interacts with crucial cellular proteins involved in transcription initiation such as transcription factors, TFIID, and mediator (37, 42), facilitating their recruitment to the viral genome. In this study, we describe ﬁndings that shed light on how the immediate early protein ICP22 affects RNA Pol II transcription of viral genes, thus promoting the production of viral progeny. Speciﬁcally, ICP22 interacts with the FACT complex and is responsible for the relocation of FACT in the nucleus and its recruitment to the viral genome along with two other transcription elongation factors, Spt5 and Spt6. In addition, while ICP22 was not required for wt levels of RNA Pol II on the transcription start sites of viral genes, the amount of RNA Pol II in the bodies of viral genes was signiﬁcantly reduced in the absence of ICP22. These observations suggest that ICP22 promotes the elongation of RNA Pol II transcription of viral genes late after infection, likely through the recruitment of transcription elongation factors. The FACT complex is a histone chaperone that is known to dissociate H2A-H2B dimers from nucleosomes to allow access to DNA by elongating RNA Pol II and other factors (43, 44). The FACT complex is involved in cellular transcription elongation in addition to other processes in the cell such as DNA replication (45, 46). In addition to displacing nucleosomes by dissociating H2A-H2B dimers, the FACT complex can also destabilize nucleosomes globally, contributing to the maintenance of a loose chroma- tin structure (47). The FACT complex may therefore contribute to the establishment and maintenance of the loosely associated chromatin structure observed on HSV-1 ge- nomes during viral DNA replication (48). Furthermore, the FACT complex has been found to be involved in the latent replication of Kaposi’s sarcoma-associated herpes- virus and in the efﬁcient transcription of human cytomegalovirus immediate early genes (49, 50). In addition to the FACT complex, we discovered a substantial decrease in the amount of Spt6 and Spt5 recruited to viral genomes in the absence of ICP22 (Table 1). DISCUSSION All of these proteins have established roles in cellular transcription elongation and are known to interact with RNA Pol II and one another (51–54). If these factors are not recruited to paused RNA Pol II, transcription arrest can occur (28). Previously, it was determined that late gene transcription, as well as the transcrip- tion of the immediate early gene that codes for ICP0, is attenuated in ICP22 mutants (15, 17, 18, 26). However, the connection between the genes affected was not eluci- dated. It has been suggested that a common sequence may be responsible for uniting those genes affected by ICP22 (27). We used RNA-seq to characterize the transcription of viral genes in the ICP22 mutant and observed a global decrease in viral mRNA abundance beginning at 4 to 6 hpi, after the onset of viral DNA replication. Late genes are affected more drastically and noticeably because they are typically upregulated 4 to 6 hpi. Immediate early and early genes that are typically expressed at consistent or rising levels at 4 to 6 hpi, including ICP0, are impacted by the absence of ICP22 in a manner similar to late genes (Fig. 4B). Consistent with previous observations (17, 25, 40), we did not ﬁnd a defect in viral DNA replication in the n199 background (Fig. 4A). The observation that transcription elongation factors are reduced on ICP22 mutant genomes suggests that transcription elongation may be responsible for the inefﬁcient viral gene expression in ICP22 mutants. This hypothesis is further supported by the ChIP-seq data demonstrating a speciﬁc reduction in the amount of RNA Pol II on the bodies of viral genes in the ICP22 mutant compared to wild-type virus, whereas that amount of RNA Pol II on their transcription start sites was relatively unaffected (Fig. 5). The forms of RNA Pol II in HSV-infected cells are relevant to these observations. In uninfected cells, unphosphorylated RNA Pol II (IIa) is recruited to promoters, where it is phosphorylated on Ser-5 at the start of transcription initiation. Ser-2 phosphorylation of paused RNA Pol II results in the hyperphosphorylated form of RNA Pol II (IIo) (55). This Previously, it was determined that late gene transcription, as well as the transcrip- tion of the immediate early gene that codes for ICP0, is attenuated in ICP22 mutants (15, 17, 18, 26). However, the connection between the genes affected was not eluci- dated. Redistribution of FACT complex subunit SSRP1 requires ICP22 expression. Immu- noﬂuorescence was used to further characterize the redistribution of the FACT complex MRC5 cells were infected with wild type (KOS) or ICP22 mutant (n199) at an MOI of 10. The infected cells were harvested and processed for ChIP-seq as described in Materials and Methods. (A) Mapped reads of the region between UL42 and UL48 on the genome for ChIP of ICP4, RNA Pol II (4H8), or RNA Pol II (8WG16) in n199- or KOS-infected cells. The maximums on the y axis are to the top left of each panel. Experiments were repeated twice, and the ﬁgure represents one data set. (B) Normalized ratio of n199 over KOS reads for start sites or gene bodies of representative IE, E, and L genes. The values used were averages from two experiments using the 4H8 antibody. n199-infected cells, including Ser-5-phosphorylated RNA Pol II and RNA Pol II phos- phorylated on both Ser-5 and Ser-2 (IIo). An example of the analysis is shown in Fig. S2. In the ICP4 immunoprecipitations, n199-over-KOS read ratios were consistently close to 1 for both gene start sites and gene bodies. On the other hand, the n199-over-KOS read ratios for the RNA Pol II immunoprecipitations were reduced on n199 viral gene bodies compared to KOS, particularly for genes whose mRNA levels were reduced later in infection in the ICP22 mutant (Fig. 4B). The number of RNA Pol II reads on transcription start sites was not reduced in n199 relative to KOS, with more reads on the n199 start sites in some cases (Fig. 5B). This observation supports a model in which transcription elongation is reduced in the absence of ICP22; however, transcription initiation is unaffected. mbio.asm.org 10 May/June 2017 Volume 8 Issue 3 e00745-17 ® HSV ICP22 Promotes Elongation of Viral Transcripts DISCUSSION It has been suggested that a common sequence may be responsible for uniting those genes affected by ICP22 (27). We used RNA-seq to characterize the transcription of viral genes in the ICP22 mutant and observed a global decrease in viral mRNA abundance beginning at 4 to 6 hpi, after the onset of viral DNA replication. Late genes are affected more drastically and noticeably because they are typically upregulated 4 to 6 hpi. Immediate early and early genes that are typically expressed at consistent or rising levels at 4 to 6 hpi, including ICP0, are impacted by the absence of ICP22 in a manner similar to late genes (Fig. 4B). Consistent with previous observations (17, 25, 40), we did not ﬁnd a defect in viral DNA replication in the n199 background (Fig. 4A). The observation that transcription elongation factors are reduced on ICP22 mutant genomes suggests that transcription elongation may be responsible for the inefﬁcient viral gene expression in ICP22 mutants. This hypothesis is further supported by the ChIP-seq data demonstrating a speciﬁc reduction in the amount of RNA Pol II on the bodies of viral genes in the ICP22 mutant compared to wild-type virus, whereas that amount of RNA Pol II on their transcription start sites was relatively unaffected (Fig. 5). The forms of RNA Pol II in HSV-infected cells are relevant to these observations. In uninfected cells, unphosphorylated RNA Pol II (IIa) is recruited to promoters, where it is phosphorylated on Ser-5 at the start of transcription initiation. Ser-2 phosphorylation of paused RNA Pol II results in the hyperphosphorylated form of RNA Pol II (IIo) (55). This May/June 2017 Volume 8 Issue 3 e00745-17 mbio.asm.org 11 ® Fox et al. form of RNA Pol II is closely associated with actively elongating cellular transcripts, and elongation factors such as Spt6 are known to bind to Ser-2-phosphorylated forms (56, 57). However, Ser-2-phosphorylated forms of Pol II are greatly depleted in wild-type and ICP22 mutant virus-infected cells (25, 30–32, 58). This raises the question of how transcription elongation occurs in infected cells with altered Pol II CTD phosphorylation. The formation of the IIo form of RNA Pol II requires the action of cdk9. However, there is a lack of consensus on the role of phosphorylation of Ser-2 of the CTD by cdk9 in HSV-1 gene transcription. MATERIALS AND METHODS Cells and viruses. Experiments were performed using MRC5 (human fetal lung) or Vero (African green monkey kidney) cells obtained from and propagated as recommended by ATCC. The viruses used in this study include wild-type KOS, 22/n199 (n199), and ICP22-FLAG. n199 is derived from wild-type KOS and contains a linker carrying stop codons in the US1 gene as has been previously described (40). ICP22-FLAG was generated using red-mediated recombination in a bacterial artiﬁcial chromosome (BAC) containing KOS DNA (64–66). A FLAG tag was inserted after the start codon of US1. Modiﬁed BAC constructs were then transfected into Vero cells using Lipofectamine 2000 transfection reagent (Life Technologies, Inc.). Virus produced was harvested, plaque puriﬁed, and sequenced to verify FLAG tag insertion. Additional viruses used include d109, d106, d92, and d99 (67); d95, d96, and DMP (68); d120 (6); and 5dl1.2 (69). Immunoblotting for protein abundance. MRC5 cells were infected at an MOI of 10 by incubation with KOS virus in Tricine-buffered saline (TBS) for 1 h before inoculum was removed and cells were rinsed with TBS. Cells were then incubated at 37°C in Dulbecco’s modiﬁed Eagle’s medium (DMEM) containing 2% fetal bovine serum (FBS) for the indicated amount of time. Samples were rinsed with ice-cold TBS plus 0.1 mM tosyl-L-lysyl-chloromethane hydrochloride (TLCK) before being scraped into 2 SDS-PAGE sample buffer. Samples were boiled for 10 min before analysis. Samples were run on a 10% precast gel (Bio-Rad), transferred to a polyvinylidene ﬂuoride membrane (Amersham), and probed with the following primary antibodies: rabbit anti-Spt16 H300 (Santa Cruz; 1:500), mouse anti-SSRP1 10D1 (BioLegend; 1:500), anti-ICP4 58S (1:500), and anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) Ambion AM4300. After incubation with anti-rabbit–horseradish peroxidase (HRP) or anti-mouse–HRP secondary antibody (Promega), bands were visualized using ECL Prime Western blot detection reagent (Amersham). Imaging of viral DNA and immunoﬂuorescence. Vero cells (2 105) were plated on coverslips in 12-well dishes. Cells were infected at an MOI of 10 by incubation with virus in TBS for 1 h before inoculum was removed and cells were rinsed with TBS. Cells were then incubated at 37°C in Dulbecco’s modiﬁed Eagle’s medium (DMEM) containing 2% fetal bovine serum (FBS) for the indicated amount of time. Labeling of replicating genomes was carried out as described previously (35). Brieﬂy, EdC (10 M) was added to the medium of infected cells at 4 hpi and ﬁxed at 6 hpi. DISCUSSION It has been postulated that ICP22 interacts with or recruits cdk9 in order to phosphorylate Ser-2 of the CTD (59, 60). There is evidence that ICP22 physically associates with cdk9 (59, 60) and that inhibition of kinases such as cdk9 can lead to decreased transcription during HSV-1 infection (61). Others have observed a rapid loss of RNA Pol II Ser-2 phosphorylation when ICP22 is expressed in cells either transiently or at later times during infection, indicating that Ser-2 phosphorylation by cdk9 may not be necessary for efﬁcient viral transcription (24, 25, 31, 32). Moreover, it has been proposed that ICP22 interacts with cdk9 in order to inhibit it and prevent Ser-2 phosphorylation (62, 63). Supporting this is the observation that cellular tran- scripts are repressed during infection with HSV-1. Notably, this reduction in cellular transcription is enhanced late in infection (58). It may be that cdk9 and Ser-2 phos- phorylation is more important early in infection and that late after infection ICP22 is required to sustain transcription elongation. In addition, persisting small amounts of Ser-2 phosphorylation in Vero cells may be a possible explanation for the reduced requirement of ICP22 in Vero cells relative to human ﬁbroblasts. The possible temporal involvement of cdk9 in HSV infection and the basis for the differential requirement for ICP22 in different cell types remain to be tested. We propose that ICP22 facilitates the recruitment of Spt5, Spt6, and the FACT complex to transcribing RNA Pol II independently of the form of Pol II involved in elongation in uninfected cells. Evidence exists that ICP22 interacts with RNA Pol II (61). The recruitment of the elongation factors through these interactions would then facilitate the elongation of viral transcripts, possibly by ensuring a looser chromatin structure on viral gene bodies. The precise nature of these interactions, the role of chromatin, and the implications for cellular transcription are under investigation. May/June 2017 Volume 8 Issue 3 e00745-17 MATERIALS AND METHODS EdC-labeled DNA was then visualized by conjugating EdC to Alexa Fluor 488 azide using the Click-iT Alexa Fluor 488 imaging kit (Thermo Fisher). The immunoﬂuorescence assay was carried out as described previously (35) with the following May/June 2017 Volume 8 Issue 3 e00745-17 mbio.asm.org 12 ® HSV ICP22 Promotes Elongation of Viral Transcripts primary antibodies: rabbit anti-ICP4 N15 (1:500), mouse anti-SSRP1 10D1 (BioLegend; 1:200), and mouse anti-FLAG M2 (Sigma; 1:5,000). Secondary antibodies used included goat anti-rabbit conjugated to Alexa Fluor 488 (Invitrogen; 1:500), goat anti-mouse conjugated to Alexa Fluor 594 (Invitrogen; 1:500), goat anti-mouse IgG2b conjugated to Alexa Fluor 594 (Invitrogen; 1:500), and goat anti-mouse IgG1 conju- gated to Alexa Fluor 488 (Invitrogen; 1:500). Images were obtained using an Olympus FluoView FV1000 confocal microscope. Isolation of viral genomes and associated proteins. Viral DNA isolation was carried out as described previously (36) with the following modiﬁcations. MRC5 cells were infected with KOS or n199 at an MOI of 10 and incubated at 37°C in DMEM containing 2% FBS for 4 h, at which time 10 M EdC was added to the medium. Infections carried out in the absence of EdC were used as negative controls. At 6 hpi, nuclei were harvested and processed as described above. Genome-associated proteins were analyzed by mass spectrometry as described previously (35). Mass spectrometry was carried out by MSbioworks, LLC, Ann Arbor, MI. Coimmunoprecipitation and immunoblotting. MRC5 cells were grown to conﬂuence in a 150-mm plate. Cells were infected at an MOI of 10 by incubation with indicated virus or mock infection in TBS for 1 h before the inoculum was removed and cells were rinsed with TBS. Cells were then incubated at 37°C in DMEM containing 2% FBS for the indicated amount of time. After incubation, cells were rinsed with ice-cold TBS plus 0.1 mM tosyl-L-lysyl-chloromethane hydrochloride (TLCK). Cells were then lysed at 4°C for 30 min in 1.5 ml lysis buffer (50 mM Tris-HCl [pH 7.4], 300 mM NaCl, 1% Triton X-100, 1 mM EDTA, Roche protease inhibitor cocktail). Lysates were then centrifuged at 1.8 103 g for 10 min at 4°C, and supernatants were ﬁltered through a 100-m cell strainer (Corning). Indicated samples were incubated in 10 l Benzonase (Novagen) at this step for 10 min. MATERIALS AND METHODS Samples were then combined 1:1 with binding buffer (50 mM Tris-HCl [pH 7.4], 1% Triton X-100, 1 mM EDTA, Roche protease inhibitor cocktail) and added to anti-FLAG M2 magnetic beads (Sigma). Immunoprecipitation was carried out according to the manufacturer’s protocol with a few modiﬁcations. Brieﬂy, samples were bound to the anti-FLAG beads overnight and following a series of washes with Tris-buffered saline (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, Roche protease inhibitor cocktail). Bound proteins were eluted using the recommended concen- tration of 3FLAG peptide (Sigma). Samples were combined 1:1 with 2 SDS-PAGE sample buffer and boiled for 10 min before analysis. Samples were run on a 7.5% precast gel (Bio-Rad), transferred to a polyvinylidene ﬂuoride membrane (Amersham), and probed with the following primary antibodies: rabbit anti-Spt16 H300 (Santa Cruz; 1:500), mouse anti-SSRP1 10D1 (BioLegend; 1:500), and mouse anti-FLAG M2 (Sigma; 1:500). After incubation with anti-rabbit–HRP or anti-mouse–HRP secondary antibody (Promega), bands were visualized using ECL Prime Western blot detection reagent (Amersham). Viral DNA quantiﬁcation and growth curve MRC5 cells (1 106) in 35 mm plates were infected Viral DNA quantiﬁcation and growth curve. MRC5 cells (1 106) in 35-mm plates were infected with n199 or KOS at an MOI of 5. Cells were infected by incubation with indicated virus or mock infected in TBS for 1 h before removing inoculum and rinsing cells three times with warm TBS. Cells were then incubated at 37°C in DMEM containing 2% FBS for the indicated amount of time. For DNA quantiﬁcation, cells were lysed in 200 l DNA lysis buffer (0.5% SDS, 100 mM NaCl, 400 g/ml proteinase K), incubated at 37°C 4 h to overnight, incubated at 65°C for 30 min, and diluted 1:1,000 in 1 TE (100 mM Tris, 10 mM EDTA). Quantitative real-time PCR was carried out as described previously (70). Genome quantity was determined based on the ampliﬁcation of the HSV-1 TK gene using the following primers: TkdsF1 (5=-ACC CGC TTA ACA GCG TCA ACA-3=) and TkdsR1 (5=-CCA AAG AGG TGC GGG AGT TT-3=). Standard curves were established using serially diluted puriﬁed KOS genomic DNA. To assess infectious virus production, cells were harvested by scraping into incubation medium. Cells were then lysed by freeze-thaw and sonication, following which cells were pelleted and supernatant was isolated. Supernatant was serially diluted and used to infect Vero cell monolayers in 6-well plates. May/June 2017 Volume 8 Issue 3 e00745-17 REFERENCES during herpes simplex virus infection by the C3HC4 viral protein ICP0. J Gen Virol 75:1223–1233. https://doi.org/10.1099/0022-1317-75-6-1223. during herpes simplex virus infection by the C3HC4 viral protein ICP0. J Gen Virol 75:1223–1233. https://doi.org/10.1099/0022-1317-75-6-1223. 1. Alwine JC, Steinhart WL, Hill CW. 1974. Transcription of herpes simplex type 1 DNA in nuclei isolated from infected HEp-2 and KB cells. Virology 60:302–307. https://doi.org/10.1016/0042-6822(74)90390-0. 1. Alwine JC, Steinhart WL, Hill CW. 1974. Transcription of herpes simplex type 1 DNA in nuclei isolated from infected HEp-2 and KB cells. Virology 60:302–307. https://doi.org/10.1016/0042-6822(74)90390-0. 9. Gu H, Liang Y, Mandel G, Roizman B. 2005. Components of the REST/ CoREST/histone deacetylase repressor complex are disrupted, modiﬁed, and translocated in HSV-1-infected cells. Proc Natl Acad Sci U S A 102:7571–7576. https://doi.org/10.1073/pnas.0502658102. 2. Honess RW, Roizman B. 1974. Regulation of herpesvirus macromolecular synthesis. I. Cascade regulation of the synthesis of three groups of viral proteins. J Virol 14:8–19. 2. Honess RW, Roizman B. 1974. Regulation of herpesvirus macromolecular synthesis. I. Cascade regulation of the synthesis of three groups of viral proteins. J Virol 14:8–19. 10. Eidson KM, Hobbs WE, Manning BJ, Carlson P, DeLuca NA. 2002. Expres- sion of herpes simplex virus ICP0 inhibits the induction of interferon- stimulated genes by viral infection. J Virol 76:2180–2191. https://doi .org/10.1128/jvi.76.5.2180-2191.2002. 3. Honess RW, Roizman B. 1975. Regulation of herpesvirus macromolecular synthesis: sequential transition of polypeptide synthesis requires func- tional viral polypeptides. Proc Natl Acad Sci U S A 72:1276–1280. https:// doi.org/10.1073/pnas.72.4.1276. 3. Honess RW, Roizman B. 1975. Regulation of herpesvirus macromolecular synthesis: sequential transition of polypeptide synthesis requires func- tional viral polypeptides. Proc Natl Acad Sci U S A 72:1276–1280. https:// doi.org/10.1073/pnas.72.4.1276. 11. Lin R, Noyce RS, Collins SE, Everett RD, Mossman KL. 2004. The herpes simplex virus ICP0 RING ﬁnger domain inhibits IRF3- and IRF7-mediated activation of interferon-stimulated genes. J Virol 78:1675–1684. https:// doi.org/10.1128/JVI.78.4.1675-1684.2004. 4. Dixon RA, Schaffer PA. 1980. Fine-structure mapping and functional analysis of temperature-sensitive mutants in the gene encoding the herpes simplex virus type 1 immediate early protein VP175. J Virol 36:189–203. 12. Sandri-Goldin RM. 1998. ICP27 mediates HSV RNA export by shuttling through a leucine-rich nuclear export signal and binding viral intronless RNAs through an RGG motif. Genes Dev 12:868–879. https://doi.org/10 .1101/gad.12.6.868. 5. DeLuca NA, McCarthy AM, Schaffer PA. 1985. Isolation and characteriza- tion of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4. J Virol 56:558–570. 6. MATERIALS AND METHODS The beads were then resuspended in IP elution buffer (1.0% sodium dodecyl sulfate, 0.1 M NaHCO3) and incubated at 65°C for 2 h, after which time the beads were removed and incubation of the supernatant was continued overnight at 65°C to reverse cross-links. The samples were then extracted with phenol-chloroform-isoamyl alcohol (25:24:1) twice and with chloroform-isoamyl alcohol (24:1) once and then puriﬁed using Qiagen PCR cleanup columns. The quantity of DNA in each sample was quantiﬁed using a Qubit 2.0 ﬂuorometer (Invitrogen). Ten nanograms of each sample was used to create sequencing libraries using the NEBNext Ultra II DNA library preparation kit (New England Biolabs). Individual samples each with unit barcodes were quantiﬁed using an Agilent DNA 7500 chip and a 2100 Bioanalyzer. Pooled multiplexed samples were sequenced at the Tufts University genomics facility. The demultiplexed sequence read ﬁles were mapped to the HSV strain KOS genome and analyzed using CLC Genomics Workbench. SUPPLEMENTAL MATERIAL Supplemental material for this article may be found at https://doi.org/10.1128/mBio 00745-17. Supplemental material for this article may be found at https://doi.org/10.1128/mBio 00 .00745-17. FIG S1, TIF ﬁle, 12.1 MB. FIG S2, TIF ﬁle, 12.2 MB. TABLE S1, PDF ﬁle, 0.1 MB. TABLE S2, XLSX ﬁle, 0.2 MB. ACKNOWLEDGMENTS We acknowledge Francis Sivrich and Sarah Dremel for technical assistance and helpful discussions. We also thank Richard Jones, at MSbioworks (Ann Arbor, MI), for his expertise in mass spectrometry. None of the authors have a conﬂict of interest of any kind with this subject of this study. This work was supported by the NIH grants R01AI030612, T32AI049820, and F31AI126632. The funders had no role in study design, data collection and interpreta- tion, or the decision to submit the work for publication. MATERIALS AND METHODS After infection, cells were incubated at 37°C in overlay medium (DMEM, 10 g/liter methylcellulose, 2% FBS). After 3 days, the medium was removed and cells were stained with 10 mg/ml crystal violet in 50% ethanol. PFU was determined as an average from two duplicate wells. RNA-seq. MRC5 cells were seeded into 60-mm dishes at a density of 2 106 cells per dish, infected with n199 or KOS at an MOI of 10 PFU/cell, and incubated at 37°C. At the appropriate time postinfection, RNA was isolated using the Ambion RNaqueous-4 PCR kit using the included protocol. The isolated total RNA was quantiﬁed using an Agilent eukaryotic RNA Nano kit and a 2100 Bioanalyzer. One microgram of total RNA from each sample was used to create sequencing libraries using the TruSeq RNA sample preparation kit (Illumina). Individual samples each with unit barcodes were quantiﬁed using an Agilent DNA 7500 chip and a 2100 Bioanalyzer. Pooled multiplexed samples were sequenced at the Tufts University genomics facility. The demultiplexed sequence read ﬁles were mapped to the HSV strain KOS genome and quantiﬁed using CLC Genomics Workbench. ChIP-seq. MRC5 cells were seeded into 600-cm2 square tissue culture dishes at a density of 7 107 cells per dish, infected with n199 or KOS at an MOI of 10 PFU/cell, overlaid with 100 ml medium, and incubated at 37°C. At 6 hpi, 5 ml of 20% formaldehyde was added to the culture medium. The cultures were incubated at room temperature for 5 min, followed by the addition of 5 ml of 2.5 M glycine. All subsequent procedures were performed at 4°C unless otherwise stated. The cultures were then washed with TBS and scraped into 50 ml of FLB {5 mM PIPES [piperazine-N,N=-bis(2-ethanesulfonic acid)], pH 8.0, 85 mM KCl, 0.5% Igepal CA-630 [US Biological], Roche protease inhibitor cocktail}. The cells were then pelleted by low-speed centrifugation, resuspended in 5 ml FLB, and pelleted again. The cell pellet was resuspended in 1.2 ml RIPA buffer (1 phosphate-buffered saline [PBS], 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, Roche protease inhibitor cocktail) and sonicated for 6 intervals of 30 s each on ice with a Sonics Vibra-Cell VCX 130 sonicator equipped with a 3-mm microprobe. The sonicated material was centrifuged at 1.8 103 g for 15 min. DNA was isolated from 50 l of the supernatant as a total mbio.asm.org 13 ® Fox et al. input control. MATERIALS AND METHODS The remainder was divided equally to use in immunoprecipitations using the ICP4 and RNA Pol II monoclonal antibodies, 58S and either 8WG16 (Abcam; ab817) or 4H8 (Abcam, Inc.; ab5408), respectively. Twenty-ﬁve microliters of monoclonal antibody was bound to 50 l of Dynabeads M280 sheep anti-mouse IgG beads (Invitrogen) in 5% bovine serum albumin (BSA) in PBS overnight. The antibody-coated beads were extensively washed with 5% BSA in PBS and then added to the reserved sonicated extracts, which were incubated overnight. The immunoprecipitation mixtures were washed ﬁve times with LiCl wash buffer (100 mM Tris-HCl buffer, pH 7.5, 500 mM LiCl, 1.0% Igepal CA-630, 1.0% sodium deoxycholate) and once with TE. The beads were then resuspended in IP elution buffer (1.0% sodium dodecyl sulfate, 0.1 M NaHCO3) and incubated at 65°C for 2 h, after which time the beads were removed and incubation of the supernatant was continued overnight at 65°C to reverse cross-links. The samples were then extracted with phenol-chloroform-isoamyl alcohol (25:24:1) twice and with chloroform-isoamyl alcohol (24:1) once and then puriﬁed using Qiagen PCR cleanup columns. The quantity of DNA in each sample was quantiﬁed using a Qubit 2.0 ﬂuorometer (Invitrogen). Ten nanograms of each sample was used to create sequencing libraries using the NEBNext Ultra II DNA library preparation kit (New England Biolabs). Individual samples each with unit barcodes were quantiﬁed using an Agilent DNA 7500 chip and a 2100 Bioanalyzer. Pooled multiplexed samples were sequenced at the Tufts University genomics facility. The demultiplexed sequence read ﬁles were mapped to the HSV strain KOS genome and analyzed using CLC Genomics Workbench. input control. The remainder was divided equally to use in immunoprecipitations using the ICP4 and RNA Pol II monoclonal antibodies, 58S and either 8WG16 (Abcam; ab817) or 4H8 (Abcam, Inc.; ab5408), respectively. Twenty-ﬁve microliters of monoclonal antibody was bound to 50 l of Dynabeads M280 sheep anti-mouse IgG beads (Invitrogen) in 5% bovine serum albumin (BSA) in PBS overnight. 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https://openalex.org/W4389134276	https://www.researchsquare.com/article/rs-3625118/latest.pdf	English	null	Potential the relationship between clinical use metformin and frailty index:a Mendelian randomization study	Research Square (Research Square)	2,023	cc-by	6,760	Potential the relationship between clinical use metformin and frailty index:a Mendelian randomization study Potential the relationship between clinical use metformin and frailty index:a Mendelian randomization study Deqiang Jiao Guizhou University of traditional Chinese Medicine Jiang Liang Deqiang Jiao Guizhou University of traditional Chinese Medicine Jiang Liang Guizhou University of traditional Chinese Medicine 1 Introduction In recent years, the challenge of an aging global population has become increasingly significant. The United Nations’ 2022 World population Outlook report (https://population.un.org) and the 2023 World Social report [1] indicate that the global population aged over 65 was 761 million in 2021 and is projected to reach 1.6 billion by 2050, with countries like China and Japan experiencing the most notable increases. Consequently, frailty in the elderly has emerged as a major research focus. Frailty is a condition characterized by increased vulnerability and reduced physiological function. The Frailty Index (FI), which assesses frailty based on 70 clinical aspects such as falls, disability, disease, and mortality risk[2],is an important tool for evaluating this condition. Clinical studies have demonstrated that as the FI accumulates, there is a heightened risk of serious adverse events like fall [3], fracture [4], and depression [5]. Moreover, the FI is closely linked to mortality predictions [6]. Research in China shows that FI increases with age and is significantly associated with all-cause mortality and specific-cause mortality, including heart disease, stroke, cancer, chronic obstructive pulmonary disease, and diabetes. However, for those dying from these specific causes, the FI's relation to age is not evident [7]. The accumulation of the FI can result from various factors, including age, chronic diseases, trauma, malnutrition, and long-term medication use. For instance, the use of high-dose chemotherapy drugs like methotrexate can impact metabolism [8] [9]. Metformin, a long-standing weight loss drug widely used in diabetes treatment, has sparked debate over its relationship with the increase in frailty index. A cross-sectional study of type 2 diabetic veterans aged 65 and older indicated an association between metformin exposure and a reduced risk of frailty among community-dwelling veterans [10]. This is attributed to metformin's protective role in musculoskeletal aging-related diseases [11]. However, other clinical studies have found that patients with muscular dystrophy on metformin exhibit a significantly higher frailty index compared to non-muscular hypoglycemic patients [12], and hypoglycemic drugs like metformin may increase myopenia risk by reducing dietary protein intake [13]. Furthermore, metformin is known to cause morphological changes in myotubes, making them long and thin [14], and to atrophy gastrocnemius muscle fibers in WT mice, accompanied by reduced serum myoglobin levels [15]. Clinical studies have shown that while metformin does not directly interfere with protein synthesis, it can disrupt mitochondrial respiratory function and counteract muscle gains from exercise [16] [17] [18]. Research Article Keywords: metformin, frailty index, causal analysis, Mendelian randomization Posted Date: November 29th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3625118/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Keywords: metformin, frailty index, causal analysis, Mendelian randomization Page 1/16 Abstract Background: Observational studies present conflicting evidence regarding the association between metformin use and the risk of an increase in the Frailty Index. Some studies suggest a significant increase, while others indicate the opposite. Objective: This study aims to explore the causal relationship between metformin use and an increased risk of the Fraility Index . Methods: We conducted a two-sample Mendelian randomization (MR) analysis using various methods: inverse-variance weighted (IVW), weighted median, MR-Egger regression, simple mode, and weighted mode. Publicly available summary statistics datasets from genome-wide association studies (GWAS) meta-analyses were utilized. These datasets focused on metformin use in individuals of European descent (n = 462933) as the exposure variable, and a GWAS on doctor-diagnosed frailty index increase in individuals from the UK Biobank (n =175226) as the outcome variable. Additionally, we validated the outcomes using data from another group of metformin patients. Results: We identified 61 single nucleotide polymorphisms (SNPs) associated with metformin use at genome-wide significance as instrumental variables (P<1e-5; linkage disequilibrium r2 <0.001). The IVW (b = 2.646518, SE = 0.3763135, P = 2.03E-12), MR-Egger regression (b = 1.986775, SE = 1.4747723, P = 1.83E-01), simple mode (b = 3.450373, SE = 1.0903862, P = 2.44E-03), weighted median (b = 2.388585, SE = 0.4366406, P =4.49E-08), and Weighted mode (b = 3.08067, SE = 0.9913658, P =2.88E-03) methods all indicated a potential causal relationship between metformin use and the frailty index. Cochran’s Q test and funnel plot analysis showed no evidence of heterogeneity or asymmetry, suggesting no directional pleiotropy. Similar trends were observed in both forward and reverse validation within an additional 49 datasets. Conclusion: Our MR analysis findings suggest a potential causal relationship between metformin use and an increase risk of frailty index. 1 Introduction Additionally, metformin use may lead to adverse effects such as hypoglycemia, nausea, vomiting, digestive tract issues, lactic acidosis, all potentially contributing to an increased frailty index. Thus, the correlation between metformin and frailty index requires further investigation. Mendelian randomization experiments, which analyze the correlation between large sample exposure factors and specific outcomes based on existing research data, are a current popular research method. Our aim is to determine whether there is an objective causal relationship between metformin use and the increase in frailty index through this method, thereby enhancing clinical practice by promoting strengths, mitigating frailty, and reducing adverse outcomes. 2 Materials and methods Page 2/16 Page 2/16 2.3 Evaluation of the causal relationship between metformi 2.3 Evaluation of the causal relationship between metformin use and frailty index We primarily utilized the Inverse variance weighted (IVW) method to meta-analyze the Wald ratio estimates of included SNPs and the Weighted Median estimator to assess the causal relationship between metformin and the frailty index[24]. The Weighted Median method involves ranking the effects of individual SNPs by weight, taking the median, and then calculating causality, requiring at least 50% of the instrumental variables to be valid[25]. Additionally, the MR-Egger regression method complements the Weighted Median method, evaluating causal relationships through weighted coefficient linear regression slopes between exposure and outcome variables, with the intercept providing an estimate of average horizontal pleiotropic effects across genetic variants[26]. Simple Mode increases the stability of MR pleiotropy, whileWeighted Mode enhances the sensitivity of pattern estimation bandwidth selection[27] 2.4 Sensitivity analysis Heterogeneity of SNP was assessed using I2 statistics and Cochran's Q-statistics, with P < 0.05 indicating heterogeneity. The MR-Egger regression method's intercept terms evaluated the possibility of horizontal pleiotropy[28]. The 'Leave-one-out' method was employed to identify SNPs with a potentially greater influence on outcomes, where OR > 1 suggests a negative impact of exposure factors on outcomes[29]. Finally, the reliability of these findings to the overall results of both groups was evaluated. 2.1 Data sources and selection In this study, data were obtained from the MR Base database (https://www.ebi.ac.uk/gwas) and Bio bank (http://www.nealelab.is/uk-biobank). The exposure variable was GWAS analysis of metformin use in European individuals (n = 462933), and the outcome variable was GWAS of the frailty index (n = 175226). In the validation group, exposure and outcome variables were interchanged. Detailed information is presented in Table 1. Table 1 Data set details for exposure variables and outcome variables ID Exposure/Outcome PubmedID Sample size First author Consortium Year SNP size population ukb-b−14609 metformin NA 462933 Ben Elsworth MRC-IEU 2018 9851867 European ebi-a- GCST90020053 Frailty index 34431594 175226 Atkins JL NA 2021 7589717 - 2 2 Selection of instrumental variables Table 1 Data set details for exposure variables and outcome variables 2.2 Selection of instrumental variables Instrumental variables were selected based on pre-established criteria, showing a direct relationship with exposure variables but not with outcomes. Both the research and validation groups selected appropriate instrumental variables to investigate the causal relationship between exposure variables and outcomes. The research group identified 61 instrumental variables, and the validation group 49, all showing significant correlations with exposure variables (P < 1e-5) and no linkage imbalance (LD) among SNPs (r2 < 0.001). F-statistics were calculated, confirming that all SNPs met the criterion of F-statistics > 10 for MR Analysis[19]. The Phenoscanner tool (http://www.phenoscanner.medschl.cam.ac.uk) was used to eliminate confounding factors related to exposure variables. For the frailty index scale [20][21], 26 confounding factors were considered; in the validation group, six confounding factors associated with metformin use were identified, including hyperglycemia, insulin resistance, body fat, obesity, diabetes, and body weight[22][23]. Moreover, the final F-statistics for all SNPs inboth groups were greater than 10, indicating a strong impact of these instrumental variables on exposure variables compared to other confounding factors[30]. 3.1 Detailed SNP Information Table 2. Specific SNP information for the research and validation groups, including the location of each SNP allele, effect allele (EA), effect allele frequency (EAF), and correlation indices with metformin and frailty index, comprising beta value, SE value, and P value. In the research group, 61 SNPs, such as rs10842675, rs10860210, and rs10938398, were significantly correlated with metformin and frailty index. In the validation group, 49 SNP including rs10246100, rs10838115, and rs10932334 showed significant correlation with the frailty index. The P values of IVs in both the research group (rs9273268, rs9630985, rs1781719, rs2598503, rs6508338, rs67422659, rs113404564, rs58643433, rs6819189, rs2723063, rs924150, rs614730, rs190795754, rs1473781, rs12233366, rs12505194, rs80193967, rs149266061) and validation group (rs10838115, rs2896595, rs17740759, rs12367100, rs555911977, rs374943348, rs62414191, rs116421847, rs71460103, rs77233221, rs6443450, rs33970119, rs4146140) were all less than 0.05, suggesting potential correlation or validity between these SNP and the research group (frailty index) and validation group (metformin use). Page 3/16 Page 3/16 Moreover, the final F-statistics for all SNPs inboth groups were greater than 10, indicating a strong impact of these instrumental variables on exposure variables compared to other confounding factors[30]. 3.3 Sensitivity analysis Sensitivity analysis revealed that the MR regression intercepts for the research and validation groups were estimated at 0.0013 (SE = 0.0028,P = 0.645) and 0.000137 (SE = 0.00027,P = 0.617), respectively. This suggests an absence of genetic polymorphism in both groups. The funnel charts and MR-Egger regression tests indicated no asymmetry, implying no horizontal pleiotropy in either group. Heterogeneity tests showed that the Heterogeneity P_values of MR-Egger and IVW analyses for both groups were less than 0.05, indicating no heterogeneity of IVs (Table 4, Fig. 4). The 'leave-one-out' analysis demonstrated that all SNPs in both groups significantly positively affected the causal relationship between metformin use and the increase in the frailty index. This suggests a strong causal link between metformin use and an increase in the frailty index (Fig. 5). 3.1 Detailed SNP Information Page 4/16 Table 2 Table 2 SNP details table for research group and validation group SNPs Effect allele Metformin Frailty index F- statistics β SE P β SE P rs10842675 rs10860210 rs10938398 rs10985855 rs11156444 rs11157386 rs113404564 rs114147823 rs11528477 rs115837176 rs12233366 rs12296994 rs12505194 rs12829351 rs12866578 rs13382799 rs1405630 rs147338695 rs1473781 rs149266061 rs1613599 rs16903339 rs1781719 rs190795754 rs1999899 rs2268967 rs2598503 rs2723063 rs28479795 rs35096620 rs3857298 rs41288297 rs4287436 rs4752792 rs497520 rs55824575 585944 A A A G T C A A T A T A A T A A T C A T T G T T T A T G T T T T C A A A T 0.00157438 −0.00151648 0.00171941 −0.00319468 −0.00146316 0.00440582 −0.00241301 0.00369338 0.00263404 0.00422147 0.00237137 −0.00166862 0.00155237 −0.00165185 0.00207289 0.00310716 −0.00155223 0.00290934 0.00176504 −0.00357312 0.00150312 0.00189756 −0.00144035 −0.00349277 0.00219733 0.00173304 −0.00164954 −0.00164977 0.00192583 0.00190352 −0.00160784 0.00337147 0.00174613 0.00209176 −0.00149358 −0.00370875 0 00180232 0.000338649 0.00033786 0.000327314 0.000630171 0.000326415 0.000954179 0.000541629 0.000780248 0.00056878 0.00085182 0.000475929 0.000369227 0.000346739 0.000358906 0.000389933 0.000662073 0.000328344 0.000649618 0.000341292 0.000781939 0.000325588 0.000376512 0.000324191 0.000698322 0.000483643 0.000386824 0.000335253 0.000327859 0.000390232 0.000369333 0.00033496 0.000760839 0.000332859 0.00032437 0.00032714 0.000744804 0 000383473 3.30E−06 7.20E−06 1.50E−07 4.00E−07 7.40E−06 3.90E−06 8.40E−06 2.20E−06 3.60E−06 7.20E−07 6.30E−07 6.20E−06 7.60E−06 4.20E−06 1.10E−07 2.70E−06 2.30E−06 7.50E−06 2.30E−07 4.90E−06 3.90E−06 4.70E−07 8.90E−06 5.70E−07 5.50E−06 7.50E−06 8.60E−07 4.90E−07 8.00E−07 2.60E−07 1.60E−06 9.40E−06 1.60E−07 1.10E−10 5.00E−06 6.40E−07 2 60E 06 −0.0011 0.0027 0.0048 −0.0122 −0.0029 0.0114 −0.0148 0.0066 −0.0032 0.0144 0.011 −0.0014 0.0073 −0.0017 0.005 −0.0071 −0.0015 7.00E−04 0.0082 −0.0158 2.00E−04 9.00E−04 −0.0104 −0.0169 −0.0015 0.0071 −0.0106 −0.0082 0.0042 0.007 −0.0029 0.0118 0.0048 −0.003 −0.0063 −0.0115 0 0062 0.0035 0.0035 0.0033 0.0064 0.0033 0.0097 0.0055 0.0079 0.0058 0.0085 0.0048 0.0038 0.0035 0.0037 0.004 0.0068 0.0033 0.0067 0.0035 0.008 0.0033 0.0038 0.0033 0.0071 0.005 0.004 0.0034 0.0034 0.004 0.0038 0.0035 0.0077 0.0034 0.0033 0.0033 0.0075 0 0039 0.7451 0.4423 0.149 0.05648 0.3819 0.2393 0.007165 0.4005 0.5831 0.09117 0.02353 0.7023 0.03891 0.6355 0.2084 0.2978 0.6583 0.915 0.01886 0.0479 0.942 0.8151 0.001629 0.01812 0.7596 0.07163 0.00186 0.01421 0.2915 0.06393 0.4025 0.128 0.1567 0.3632 0.05836 0.1248 0 1135 2.16E + 01 2.01E + 01 2.76E + 01 2.57E + 01 2.01E + 01 2.13E + 01 1.98E + 01 2.24E + 01 2.14E + 01 2.46E + 01 2.48E + 01 2.04E + 01 2.00E + 01 2.12E + 01 2.83E + 01 2.20E + 01 2.23E + 01 2.01E + 01 2.67E + 01 2.09E + 01 2.13E + 01 2.54E + 01 1.97E + 01 2.50E + 01 2 06E + Page 5/16 SNPs Effect allele Metformin Frailty index F- statistics β SE P β SE P rs58643433 rs614730 rs62414233 rs62421936 rs6508338 rs6694989 rs67232546 rs67422659 rs6819189 rs6941355 rs7178030 rs72631105 rs73188924 rs7376543 rs75932023 rs7651465 rs769136 rs80193967 rs8021298 rs898181 rs924150 rs9273268 rs927742 rs9630985 rs10246100 rs10838115 rs10932334 rs11599901 rs116421847 rs116954057 rs11790299 rs11951033 rs12367100 rs12523352 rs12739184 rs13258335 rs145529723 rs1528892 rs17144411 rs17740759 G A A T T G T G A G G A A G C A C G A C C C T C A T T A T T T T T T A A A T T A −0.00299678 0.00202433 −0.00175042 −0.0027678 0.00147424 0.00170082 0.00227404 0.00200498 −0.00153078 0.00152279 −0.0019526 0.00193826 0.00217376 −0.00232207 −0.00399972 −0.00146865 0.00174999 0.00162449 0.00158746 0.00197255 −0.00149514 0.00227428 −0.00150016 0.00154149 −0.000111677 0.000892704 −5.55E−05 0.000551252 −0.00202315 0.000825423 0.00041061 0.000486359 0.000909858 −0.000627867 −0.00016333 9.60E−05 −0.000428217 −4.46E−05 −0.000134485 −0.0016513 0.000656343 0.000392058 0.000330244 0.000571629 0.000326796 0.00034934 0.000398149 0.000447871 0.000324553 0.000326323 0.000440597 0.000416544 0.000389733 0.00038888 0.000901619 0.000328659 0.000394521 0.000365908 0.000343581 0.000388137 0.000333176 0.00041352 0.000328328 0.000342942 0.000327278 0.00032348 0.000323681 0.00040457 0.000872712 0.000829994 0.000431437 0.000342139 0.000353389 0.000325428 0.000574011 0.000393193 0.000829401 0.000436384 0.000414803 0.000637542 5.00E−06 2.40E−07 1.20E−07 1.30E−06 6.40E−06 1.10E−06 1.10E−08 7.60E−06 2.40E−06 3.10E−06 9.30E−06 3.30E−06 2.40E−08 2.40E−09 9.20E−06 7.90E−06 9.20E−06 9.00E−06 3.80E−06 3.70E−07 7.20E−06 3.80E−08 4.90E−06 7.00E−06 0.730000235 0.005800027 0.860000097 0.170000031 0.02 0.320000016 0.340000065 0.160000006 0.01 0.053999533 0.780000714 0.809999965 0.610000232 0.919999942 0.749999545 0.009599973 −0.0177 0.0096 −0.0037 −0.0083 0.0097 0.0058 −0.0033 0.0131 −0.0084 0.0054 0.0023 0.0022 −0.0055 −0.0051 1.00E−04 0.0031 0.0068 0.0077 0.0035 −8.00E−04 −0.0082 0.0264 5.00E−04 0.0131 0.015 0.0146 0.0169 0.0189 −0.0437 −0.0409 0.0202 −0.0181 0.0163 −0.0161 −0.0264 0.0188 0.0419 0.0198 0.0217 0.0312 0.0068 0.004 0.0034 0.0058 0.0033 0.0036 0.0041 0.0046 0.0033 0.0033 0.0045 0.0043 0.004 0.004 0.0092 0.0034 0.004 0.0037 0.0035 0.004 0.0034 0.0042 0.0033 0.0035 0.0033 0.0033 0.0033 0.0041 0.009 0.0084 0.0044 0.0035 0.0036 0.0033 0.0059 0.004 0.0084 0.0044 0.0042 0.0065 0.00907 0.01679 0.2764 0.1523 0.003513 0.1011 0.4157 0.004237 0.01139 0.1027 0.6154 0.607 0.1659 0.2043 0.9891 0.3636 0.09422 0.03953 0.3252 0.8448 0.0166 3.91E−10 0.871 0.0001966 7.18E−06 9.79E−06 2.80E−07 4.34E−06 1.07E−06 1.02E−06 4.36E−06 2.32E−07 6.27E−06 1.23E−06 7.33E−06 2.96E−06 6.86E−07 7.75E−06 3.19E−07 1.92E−06 2.01E + 01 2.42E + 01 2.53E + 01 2.44E + 01 2.66E + 01 2.30E + 01 1.96E + 01 2.75E + 01 4.16E + 01 2.08E + 01 2.48E + 01 2.21E + 01 2.08E + 01 2.67E + 01 2.81E + 01 2.34E + 01 2.04E + 01 2.37E + 01 3.26E + 01 2.00E + 01 2.22E + 01 2.18E + 01 1.96E + 01 2.17E + 01 3.11E + 01 3.57E + 01 1.97E + 01 Page 6/16 Page 6/16 Group SNPs Effect allele Metformin Frailty index F- statistics β SE P β SE P rs2250490 rs28453001 rs2896595 rs33970119 rs34211526 rs34223426 rs34651062 rs35096827 rs374943348 rs3814049 rs3908480 rs4146140 rs4733108 rs555911977 rs56299474 rs60451143 rs60570631 rs61802920 rs62414191 rs6443450 rs6793973 rs6986533 rs71460103 rs72651389 rs72793147 rs72809317 rs73092909 rs77233221 rs8008059 rs9356282 rs9673104 rs998141 T A A A A A T T A T A T T T A T C T A C T A T C A T A T A T T A 0.000439559 −0.000350733 −0.00113081 0.00130367 0.000339958 −0.000385446 0.000586934 0.000357628 0.000939471 −0.000273413 0.000709547 −0.000655608 0.000639442 0.00111547 −7.36E−05 −0.000630775 0.000446807 0.000449457 0.000940735 −0.000658857 −0.000551314 −0.000138074 −0.000920304 0.000239207 0.000276074 0.000104906 −0.000484205 0.00200965 0.000655872 −0.000177449 5.02E−05 0.000554689 0.000363938 0.000324108 0.000430298 0.000662098 0.000457737 0.000390983 0.000713796 0.000540728 0.000381127 0.000425691 0.000523105 0.000334911 0.000352569 0.000451542 0.000426604 0.000677054 0.000336189 0.000539788 0.000383281 0.000324213 0.000428177 0.000343172 0.000408355 0.000359212 0.00055949 0.000324834 0.000522845 0.000950364 0.000695005 0.000383098 0.000403199 0.00037029 0.230000087 0.27999998 0.008600031 0.049000442 0.460000178 0.320000016 0.410000135 0.509999793 0.014000063 0.519999589 0.170000031 0.05 0.070000316 0.0129999 0.860000097 0.349999964 0.180000205 0.410000135 0.014000063 0.042000069 0.200000002 0.689999856 0.023999931 0.509999793 0.620000443 0.749999545 0.349999964 0.034000085 0.349999964 0.640000038 0.89999998 0.129999895 0.0167 −0.0167 −0.0206 0.0333 −0.0209 −0.0177 0.037 −0.03 0.0274 0.0211 0.0258 −0.0198 0.0167 0.034 0.0241 −0.0316 0.0154 0.0252 0.0192 −0.0155 0.0198 −0.0167 −0.0224 0.0164 −0.0258 −0.015 −0.0266 0.0447 0.0332 0.0213 −0.0185 0.018 0.0037 0.0033 0.0044 0.0068 0.0047 0.004 0.0073 0.0056 0.0039 0.0044 0.0054 0.0034 0.0036 0.0046 0.0044 0.0069 0.0034 0.0055 0.0039 0.0033 0.0044 0.0035 0.0042 0.0037 0.0057 0.0033 0.0053 0.0098 0.0072 0.0039 0.0041 0.0038 7.26E−06 4.40E−07 2.78E−06 9.55E−07 7.58E−06 8.98E−06 3.42E−07 8.58E−08 3.23E−12 1.55E−06 1.47E−06 6.83E−09 3.46E−06 2.10E−13 3.94E−08 4.75E−06 7.08E−06 4.36E−06 9.01E−07 2.98E−06 6.66E−06 1.90E−06 6.87E−08 7.70E−06 6.75E−06 6.80E−06 5.38E−07 4.80E−06 3.85E−06 5.29E−08 6.61E−06 1.92E−06 2.00E + 01 1.97E + 01 1.97E + 01 2.13E + 01 2.58E + 01 2.01E + 01 3.02E + 01 2.09E + 01 2.02E + 01 2.07E + 01 1.96E + 01 2.62E + 01 2.12E + 01 2.36E + 01 2.37E + 01 2.11E + 01 2.67E + 01 2.05E + 01 2.38E + 01 2.00E + 01 2 21E + Page 7/16 Group SNPs Effect allele Metformin Frailty index F- statistics β SE P β SE P 2.04E + 01 2.56E + 01 2.19E + 01 2.40E + 01 1.98E + 01 1.96E + 01 2.57E + 01 2.87E + 01 4.94E + 01 2.30E + 01 2.28E + 01 3.39E + 01 2.15E + 01 5.46E + 01 3.00E + 01 2.10E + 01 2.05E + 01 2.10E + 01 2.42E + 01 2.21E + 01 2.02E + 01 2.28E + 01 Page 8/16 3.2 Mendelian randomized results Table 3. Statistical analysis of Mendelian randomization results for both groups. The Odds Ratio (OR) values for both groups were greater than 1, indicating a positive relationship between metformin use and frailty index. However, the P_value for MR-Egger regression and Simple mode in the research group was > 0.05, lacking statistical significance. Results are visually represented in the forest maps and scatter plots of Figs. 2 and 3. Figure 2 shows that values of All-MR Egger and ALL-IVW for both groups were greater than 0, with 61 SNPs in the research group exceeding 0, two of which (rs9273268, rs9630985) were notably prominent. Only 17 SNP values in the validation group were less than 0. The scatter plots indicate an upward trend in the MR regression lines for both groups, supporting a potential causal relationship between metformin use and frailty index. Table 3 Mendelian randomized results of research group and validation group Group MR method nSNP β SE OR(95%CI) P_value Research group Validation group MR Egger Weighted median Inverse variance weighted Simple mode Weighted mode MR Egger Weighted median Inverse variance weighted Simple mode Weighted mode 61 61 61 61 61 49 49 49 49 49 1.986775 2.388585 2.646518 3.450373 3.08067 0.008835345 0.018089828 0.015166749 0.027099445 0.028828057 1.4747723 0.4366406 0.3763135 1.0903862 0.9913658 0.013067005 0.004332668 0.003561006 0.011902705 0.012158369 7.292 (0.405,131.279) 10.898 (4.631,25.646) 14.105 (6.746 ,29.491) 31.512 (3.718,267.075) 21.773 (3.119,151.980) 1.009 (0.983,1.035) 1.018 (1.010,1.027) 1.015 (1.008,1.022) 1.027 (1.004,1.052) 1.029 (1.005,1.054) 1.83E−01 4.49E−08 2.03E−12 2.44E−03 2.88E−03 5.02E−01 2.98E−05 2.05E−05 2.73E−02 2.18E−02 .3 Sensitivity analysis 4 Discussion Page 9/16 Page 9/16 The Frailty index is currently employed to evaluate patient quality of life by assessing weakness. Numerous factors influence the Frailty Index, including age, underlying diseases, and nutritional status. However, long-term intake of certain medications is also a significant factor[8][31]. Metformin, a drug with a long history, is widely used for weight loss, hypoglycemia, and even anti-aging purposes, although it is associated with many side effects. Yet, the impact of metformin on increasing the Frailty Index remains a topic of debate. Several randomized controlled trials have yielded conflicting results, suggesting metformin has no effect on mitochondrial respiratory function, the release of reactive oxygen species, or oxidative stress in skeletal muscle[32][33]. These findings might relate to the age of the subjects, the small sample size, and the duration of intervention in clinical trials. Traditional epidemiological studies, often limited by confounding factors, struggle to establish causality. Therefore, this study aims to explore the relationship between metformin use and an increase in the Frailty Index through a two- sample Mendelian randomization study based on GWAS data. In this study, a two-sample Mendelian randomization method was utilized to investigate the potential causal relationship between metformin use and an increased risk of the Frailty Index. For the research group, 61 SNPs significantly related to metformin use were selected as instrumental variables. The GWAS data on the rising frailty index were analyzed using the inverse variance weighted (IVW) method, weighted median estimator, MR-Egger regression, Simple mode, and Weighted mode. A joint analysis was conducted using a dataset with a metformin sample size of 462933 and a single frailty index dataset. In the validation group, the exposure and outcome data sets were exchanged, and 49 SNPs were analyzed using the same methods. Ultimately, we observed a potential causal relationship between metformin use and an increased risk of the Frailty Index. The same conclusion was reached using the dataset involving metformin, suggesting that metformin use may promote an increase in the Frailty Index, which is a cautionary finding for the long-term use of metformin. The specific reason for a metformin-induced increase in the frailty index remain unclear. Previous studies have suggested several possible factors: 1) lactic acidosis[34], 2) induced hepatotoxicity such as acute hepatitis[35], 3) hypoglycemia[36], 4) gastrointestinal adverse reactions including nausea, vomiting, and diarrhea[37], and 5) potential myasthenia. However, the link between metformin and muscular dystrophy remains unconfirmed. 5 Deficiency This study faced limitations due to the lack of public baseline data for the exposure and outcome groups, restricting the depth of causal analysis. This limitation impedes the use of existing data for a more detailed exploration of causality. Regarding the exposure group's data, it is important to consider that some elderly individuals might use metformin for various reasons without medical consultation, potentially affecting the accuracy of data analysis due to undisclosed medication status. Additionally, the ethnicity of the frailty index dataset in this study is unknown, and the metformin dataset is exclusively European. Ethnic differences may therefore influence the study's results. Table 4 Sensitivity Analysis of MR. I2 = (Q − df)/Q Group MR method Cochran Q- statistic Heterogeneity P_value Q_df I2 Research group Validation group MR Egger Inverse variance weighted MR Egger Inverse variance weighted 116.1337 116.5555 75.06229 75.46784 1.32E−05 1.70E−05 0.005747497 0.006893891 59 60 47 48 0.4920 0.4852 0.3739 0.3640 Declarations Conflict of interest All authors have no conflicts of interest. On behalf of all authors, the corresponding author states that there is no conflict of interest. Author Contribution All authors have no conflicts of interest. On behalf of all authors, the corresponding author states that there is no conflict of interest. 4 Discussion While the primary goal of aging and long-term drug use is to prevent cardiovascular events, it is undeniable that some patients experience weakness due to the progressive worsening of primary diabetes over time. Yet, reports from clinical randomized controlled trials do not fully account for weakness as a consequence of primary diseases. Our two Mendelian randomization trials’ results diverge from those of previous clinical trials, suggesting the need for clinical studies in younger and middle-aged patients who require metformin to assess whether the drug could increase their frailty index. References 1. Wilmoth J R, Bas D, Mukherjee S, et al. World Social Report 2023: Leaving No One Behind in an Ageing World[M]. UN, 2023. 1. Wilmoth J R, Bas D, Mukherjee S, et al. World Social Report 2023: Leaving No One Behind in an Agei 1. Wilmoth J R, Bas D, Mukherjee S, et al. World Social Report 2023: Leaving No One Behind in an Ageing World[M]. UN, 2023. 2. Ryan J, Espinoza S, Ernst ME, et al. Validation of a Deficit-Accumulation Frailty Index in the ASPirin in Reducing Events in the Elderly Study and Its Predictive Capacity for Disability-Free Survival. J Gerontol A Biol Sci Med Sci. 2022;77(1):19-26. doi:10.1093/gerona/glab225 3 Taguchi CK Menezes PL Melo ACS et al Frailty syndrome and risks for falling in the elderly community Síndrome da fragilidade e riscos 2. Ryan J, Espinoza S, Ernst ME, et al. Validation of a Deficit-Accumulation Frailty Index in the ASPirin in Reducing Events in the Elderly Study and Its Predictive Capacity for Disability-Free Survival. J Gerontol A Biol Sci Med Sci. 2022;77(1):19-26. doi:10.1093/gerona/glab225 3. Taguchi CK, Menezes PL, Melo ACS, et al. 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Genetic Causal Association between Iron Status and Osteoarthritis: A Two-Sample Mendelian Randomization. Nutrients. 2022;14(18):3683. Published 2022 Sep 6. doi:10.3390/nu14183683 27. Xu J, Zhang S, Tian Y, et al. Genetic Causal Association between Iron Status and Osteoarthritis: A Two-Sample Mendelian Randomization. Nutrients. 2022;14(18):3683. Published 2022 Sep 6. doi:10.3390/nu14183683 28. References Eur Rev Med Pharmacol Sci. 2013;17 Suppl 1:45-49. 35. Mian A, Bani Fawwaz BA, Singh G, et al. Metformin-Induced Acute Hepatitis. Cureus. 2023;15(5):e38908. Published 2023 May 11. doi:10.7759/cureus.38908 35. Mian A, Bani Fawwaz BA, Singh G, et al. Metformin-Induced Acute Hepatitis. Cureus. 2023;15(5):e38908. Published 2023 May 11. doi:10.7759/cureus.38908 36. Mansouri D, Khayat E, Khayat M, et al. Self-Monitoring of Blood Glucose and Hypoglycemia Association During Fasting in Ramadan Among Patients with Diabetes. Diabetes Metab Syndr Obes. 2020;13:1035-1041. Published 2020 Apr 5. doi:10.2147/DMSO.S234675 36. Mansouri D, Khayat E, Khayat M, et al. Self-Monitoring of Blood Glucose and Hypoglycemia Association During Fasting in Ramadan Among Patients with Diabetes. Diabetes Metab Syndr Obes. 2020;13:1035-1041. Published 2020 Apr 5. doi:10.2147/DMSO.S234675 37. Wen Q, Hu M, Lai M, et al. Effect of acupuncture and metformin on insulin sensitivity in women with polycystic ovary syndrome and insulin resistance: a three-armed randomized controlled trial. Hum Reprod. 2022;37(3):542-552. doi:10.1093/humrep/deab272 37. Wen Q, Hu M, Lai M, et al. Effect of acupuncture and metformin on insulin sensitivity in women with polycystic ovary syndrome and insu resistance: a three-armed randomized controlled trial. Hum Reprod. 2022;37(3):542-552. doi:10.1093/humrep/deab272 37. Wen Q, Hu M, Lai M, et al. Effect of acupuncture and metformin on insulin sensitivity in women with polycystic ovary syndrome and insulin resistance: a three-armed randomized controlled trial. Hum Reprod. 2022;37(3):542-552. doi:10.1093/humrep/deab272 Figures Page 12/16 Page 12/16 Figure 1 Figure 1 Overview of the study's design roadmap. Overview of the study's design roadmap. Figure 2 Page 13/16 Forest map illustrating the association between metformin and the frailty index in both the research and validation groups. The black dotdenotes the logarithmic ratio (Odds Ratio, OR) of each single nucleotide polymorphism (SNP) per standard deviation (SD) increase between metformin and the frailty index. The red dot indicates the combined causal estimates for all SNPs in each group when used as single instrumental variables (IVs), using the inverse variance weighted (IVW) and MR-Egger methods. The horizontal line represents the 95% confidence interval range. Forest map illustrating the association between metformin and the frailty index in both the research and validation groups. The black dotdenotes the logarithmic ratio (Odds Ratio, OR) of each single nucleotide polymorphism (SNP) per standard deviation (SD) increase between metformin and the frailty index. The red dot indicates the combined causal estimates for all SNPs in each group when used as single instrumental variables (IVs), using the inverse variance weighted (IVW) and MR-Egger methods. The horizontal line represents the 95% confidence interval range. Forest map illustrating the association between metformin and the frailty index in both the research and validation groups. The black dotdenotes the logarithmic ratio (Odds Ratio, OR) of each single nucleotide polymorphism (SNP) per standard deviation (SD) increase between metformin and the frailty index. The red dot indicates the combined causal estimates for all SNPs in each group when used as single instrumental variables (IVs), using the inverse variance weighted (IVW) and MR-Egger methods. The horizontal line represents the 95% confidence interval range. Figure 3 Figure 3 SNP scatter diagram showing the relationship between metformin and the frailty index in both the research and validation groups. This diagram displays the effect of SNP on metformin and the frailty index (X-axis, unit SD) and the relationship between SNP-frailty index and SNP-metformin (Y-axis, OR) through the 95% confidence interval. The regression slope of the line is represented using the inverse variance weighted (IVW) method, the weighted median, and the total causal estimates from MR-Egger, Simple Mode, and Weighted Mode methods. SNP scatter diagram showing the relationship between metformin and the frailty index in both the research and validation groups. This diagram displays the effect of SNP on metformin and the frailty index (X-axis, unit SD) and the relationship between SNP-frailty index and SNP-metformin (Y-axis, OR) through the 95% confidence interval. The regression slope of the line is represented using the inverse variance weighted (IVW) method, the weighted median, and the total causal estimates from MR-Egger, Simple Mode, and Weighted Mode methods. Page 14/16 Page 14/16 Figure 4 Funnel diagrams for the research and validation groups, used to evaluate heterogeneity and horizontal pleiotropy. The light blue line is the inverse variance weighted (IVW) estimate, while the dark blue line indicates the MR-Egger estimate. Funnel diagrams for the research and validation groups, used to evaluate heterogeneity and horizontal pleiotropy. The light blue line is the inverse variance weighted (IVW) estimate, while the dark blue line indicates the MR-Egger estimate. Funnel diagrams for the research and validation groups, used to evaluate heterogeneity and horizontal pleiotropy. The light blue line is the nverse variance weighted (IVW) estimate, while the dark blue line indicates the MR-Egger estimate. Figure 5 Page 15/16 The research group and validation group removed a SNP related to metformin and frailty index respectively. The black dot is the result of inverse variance weighted analysis of all SNPs, and the red dot is the estimation from the IVW analysis of all SNPs. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. attachments.zip Page 16/16
https://openalex.org/W3128209380	https://biblio.ugent.be/publication/8692039/file/8692041	English	null	Multivariate Correlation Measures Reveal Structure and Strength of Brain–Body Physiological Networks at Rest and During Mental Stress	Frontiers in neuroscience	2,021	cc-by	18,066	ORIGINAL RESEARCH published: 04 February 2021 doi: 10.3389/fnins.2020.602584 ORIGINAL RESEARCH published: 04 February 2021 doi: 10.3389/fnins.2020.602584 Keywords: network physiology, brain–heart connection, cardiovascular oscillations, EEG waves, physiological stress, time series analysis, wearable devices Multivariate Correlation Measures Reveal Structure and Strength of Brain–Body Physiological Networks at Rest and During Mental Stress Riccardo Pernice1, Yuri Antonacci2, Matteo Zanetti3, Alessandro Busacca1, Daniele Marinazzo4, Luca Faes1* and Giandomenico Nollo3 Riccardo Pernice1, Yuri Antonacci2, Matteo Zanetti3, Alessandro Busacca1, Daniele Marinazzo4, Luca Faes1* and Giandomenico Nollo3 1 Department of Engineering, University of Palermo, Palermo, Italy, 2 Department of Physics and Chemistry “Emilio Segrè,” University of Palermo, Palermo, Italy, 3 Department of Industrial Engineering, University of Trento, Trento, Italy, 4 Department of Data Analysis, Ghent University, Ghent, Belgium In this work, we extend to the multivariate case the classical correlation analysis used in the ﬁeld of network physiology to probe dynamic interactions between organ systems in the human body. To this end, we deﬁne different correlation-based measures of the multivariate interaction (MI) within and between the brain and body subnetworks of the human physiological network, represented, respectively, by the time series of δ, θ, α, and β electroencephalographic (EEG) wave amplitudes, and of heart rate, respiration amplitude, and pulse arrival time (PAT) variability (η, ρ, π). MI is computed: (i) considering all variables in the two subnetworks to evaluate overall brain–body interactions; (ii) focusing on a single target variable and dissecting its global interaction with all other variables into contributions arising from the same subnetwork and from the other subnetwork; and (iii) considering two variables conditioned to all the others to infer the network topology. The framework is applied to the time series measured from the EEG, electrocardiographic (ECG), respiration, and blood volume pulse (BVP) signals recorded synchronously via wearable sensors in a group of healthy subjects monitored at rest and during mental arithmetic and sustained attention tasks. We ﬁnd that the human physiological network is highly connected, with predominance of the links internal of each subnetwork (mainly η−ρ and δ−θ, θ−α, α−β), but also statistically signiﬁcant interactions between the two subnetworks (mainly η−β and η−δ). MI values are often spatially heterogeneous across the scalp and are modulated by the physiological state, as indicated by the decrease of cardiorespiratory interactions during sustained attention and by the increase of brain–heart interactions and of brain–brain interactions at the frontal scalp regions during mental arithmetic. These ﬁndings illustrate the complex and multi-faceted structure of interactions manifested within and between different physiological systems and subsystems across different levels of mental stress. Edited by: Edited by: Plamen Ch. Ivanov, Boston University, United States Reviewed by: Andy Schumann, University Hospital Jena, Germany Georgios D. Mitsis, McGill University, Canada Reviewed by: Andy Schumann, University Hospital Jena, Germany Georgios D. Mitsis, McGill University, Canada Correspondence: Luca Faes luca.faes@unipa.it Correspondence: Luca Faes luca.faes@unipa.it Specialty section: This article was submitted to Autonomic Neuroscience, a section of the journal Frontiers in Neuroscience Specialty section: This article was submitted to Autonomic Neuroscience, a section of the journal Frontiers in Neuroscience Received: 03 September 2020 Accepted: 16 December 2020 Published: 04 February 2021 INTRODUCTION The latter has been widely employed for the study of brain connectivity, where the coupling between two time series is often assessed removing indirect eﬀects from other multiple series through the use of partial correlation matrices (Marrelec et al., 2006; Oliver et al., 2019). More sophisticated analysis techniques have been proposed for the study of dynamic brain– heart and brain–body interactions, e.g., information-theoretic- based measures able to assess the information produced by each physiological system and transferred to the other connected systems starting from their output time series, which exploit, for example, Granger Causality or penalized regression (we refer the reader to Faes et al., 2014; Duggento et al., 2016; Greco et al., 2019; Zanetti et al., 2019a; Antonacci et al., 2020 for further details) or diﬀerent approaches like the one calculating the maximal information coeﬃcient (Valenza et al., 2016). However, correlation-based measures have the advantage of being simple, computationally eﬃcient, and usable also for short data sequences. These advantages are highly desirable in the ﬁeld of network physiology where often only short stationary sequences can be obtained in the challenging analysis conditions where physiological states change transiently with time (Ivanov et al., 2016; Valente et al., 2018). Moreover, the availability of eﬃcient estimators favors their implementation in non-invasive IoT applications using wearable sensors and providing real-time evaluations (Baig et al., 2017; Baker et al., 2017; Pernice et al., 2019c; Vinciguerra et al., 2019). Among the variety of organ system interactions, brain– heart interactions play an important role since they underlie the activity of the autonomic nervous system (ANS) and the central nervous system (CNS), which are strictly interconnected through anatomical and functional links and inﬂuence each other continuously (Thayer et al., 2012; Beissner et al., 2013; Silvani et al., 2016). Eﬀects of such interactions have also practical importance, as, for instance, cerebral diseases like ischemic stroke and transient ischemic attacks can be due to cardiac arrhythmias such as atrial ﬁbrillation (Marini et al., 2005; Buchwald et al., 2016). On the other hand, the heartbeat dynamics are typically aﬀected by the ANS response to emotional stress, arousal, and physical activity (Dimsdale, 2008; Silvani et al., 2016). INTRODUCTION e.g., including muscular and ocular activities (Ivanov et al., 2017; Boonstra et al., 2019). Network physiology is a novel research ﬁeld describing the human organism as an integrated network in which nodes correspond to the organs and edges map organ interactions (Bashan et al., 2012; Bartsch et al., 2015; Ivanov et al., 2016). Since the human physiological network is highly dynamic, the strength of the interactions among organs changes over time across diﬀerent physiological states as a response to cognitive or homeostatic control mechanisms (e.g.: rest or stress; emotion elicitation; consciousness or unconsciousness; wake, sleep, sleep stages), or due to pathological conditions (Jänig, 2008; Bashan et al., 2012; Waterhouse, 2013; Valenza et al., 2016; Zanetti et al., 2019b). The continuous and dynamic interaction among organs is fundamental for maintaining the individual in good health; a failure in such interaction mechanisms could provoke diseases related to organ dysfunctions or, in the worst case, even the collapse of the whole organism (Ivanov et al., 2016). Therefore, taking into account the human body as a whole and investigating the interactions among multiple organs can provide additional information to that obtained focusing on each physiological system individually (Bartsch et al., 2015). This can now also be easily achieved in non-clinical conditions thanks to the widespread adoption of wearable sensors and systems allowing the non-invasive synchronous acquisition of multiple signals from diﬀerent physiological districts (Heikenfeld et al., 2018; Jovanov, 2019; Pernice et al., 2019c; Vinciguerra et al., 2019). In this context, a main challenge that has emerged in the last years is the development of proper time series analysis techniques capable of suitably quantifying the interactions among diﬀerent physiological systems starting from the output signals measured from the diﬀerent organs. The pioneering works in the emerging ﬁeld of network physiology have used simple cross-correlation measures, showing that they can be a reliable tool to quantify brain–body and brain–brain interactions across diﬀerent sleep states (Bashan et al., 2012; Lin et al., 2020). In fact, cross- correlation is a well-established tool that has been widely used in many ﬁelds of biomedical signal processing, e.g., for assessing the connection between pairs of brain areas in functional magnetic resonance imaging (fMRI) (Cao and Worsley, 1999; Li et al., 2009). Crucially, this approach has also been extended to take into account one or more control variables through the so- called partial correlation (Marrelec et al., 2006; Wang et al., 2016). Citation: Pernice R, Antonacci Y, Zanetti M, Busacca A, Marinazzo D, Faes L and Nollo G (2021) Multivariate Correlation Measures Reveal Structure and Strength of Brain–Body Physiological Networks at Rest and During Mental Stress. Front. Neurosci. 14:602584. doi: 10.3389/fnins.2020.602584 February 2021 \| Volume 14 \| Article 602584 1 Frontiers in Neuroscience \| www.frontiersin.org Multivariate Correlation and Brain–Body Networks Pernice et al. Frontiers in Neuroscience \| www.frontiersin.org INTRODUCTION In particular, it has been shown that both mental load and physiological stress produce repeatable variations not only in the brain activity (Gevins et al., 1998; Berka et al., 2007; Al-shargie et al., 2018), but also in the dynamic control of the cardiovascular function and heart rate variability (HRV) (Petrowski et al., 2017; Kim et al., 2018; Pernice et al., 2018, 2019a); these eﬀects can be of clinical relevance as they can ultimately increase the risk of heart attacks and stroke (Steptoe and Kivimäki, 2013; Al-Shargie et al., 2016). Moreover, besides the interplay between brain and heart, the network of interactions sub-serving the regulation of the homeostatic function encompasses other physiological rhythms, such as the respiratory drive (Pfurtscheller et al., 2019; Javorka et al., 2020), the cardiovascular and baroreﬂex functions (Krohova et al., 2019, 2020; Ringwood and Bagnall- Hare, 2020), and other less studied but signiﬁcant vital signs, In the present study, the correlation-based approach to the study of physiological interactions is extended to the multivariate case, providing a formalism and a set of measures for quantifying how blocks of time series are correlated, how the correlation between a “target” time series and multiple “source” series can be dissected into meaningful contributions, and how a multivariate implementation of the concept of partial correlation allows to infer the topology of networks of physiological interactions. Speciﬁcally, extending our preliminary analyses carried out in Pernice et al. (2019d), we measure the overall brain–body interactions as the multivariate correlation between the time series representative of the diﬀerent brain rhythms [δ, θ, α, and β electroencephalographic (EEG) power] and the time series of February 2021 \| Volume 14 \| Article 602584 Frontiers in Neuroscience \| www.frontiersin.org Frontiers in Neuroscience \| www.frontiersin.org 2 Multivariate Correlation and Brain–Body Networks Pernice et al. (iii) A mental arithmetic test (MENTAL) lasting 7 min during which the volunteer had to carry out the maximum possible number of three-digit sums and subtractions. (iii) A mental arithmetic test (MENTAL) lasting 7 min during which the volunteer had to carry out the maximum possible number of three-digit sums and subtractions. heart rate, respiratory, and pulse arrival time (PAT) variability. Then, for each target time series from one of the two physiological subnetworks (brain or body), we compute interaction measures explaining how the multivariate correlation between the target and the other series arises from within- and between-subnetwork interactions, or from pairwise interactions. Time Series Extraction Data processing was carried out oﬄine employing MATLAB R2019b (MathWorks, Natick, MA, United States). To allow the analysis of brain–body interactions, the acquired physiological signals were processed extracting synchronous time series representative of the dynamical activity of the body and brain intended as separate physiological districts (sub-networks). ECG recordings were ﬁrst preprocessed to correct for artifacts and to remove baseline wander and high-frequency noise, respectively, using a high-pass ﬁlter (half power frequency of 1 Hz) and a low-pass ﬁlter (half power frequency of 20 Hz); zero-phase ﬁltering was adopted to avoid group delays. Afterward, a template matching algorithm (Dobbs et al., 1984; Speranza et al., 1993; Oweis and Al-Tabbaa, 2014; Zanetti et al., 2019a) was employed to extract the R peaks and thus obtain R-R interval (RRI) time series (variable η). R peaks detection was carried out ﬁnding the local maxima of the cross-correlation between a template of the QRS complex and the ECG, applying a threshold on the cross- correlation, and ﬁnally locating the time of the R peak at the time of the maximum value of the aligned template (Dobbs et al., 1984; Oweis and Al-Tabbaa, 2014). Tachograms were visually inspected to assess the accurate detection of R peaks, or otherwise to correct for missing and ectopic beats (Zanetti et al., 2019a). The breathing signal was sampled at the same time instants of the R peaks in the ECG to obtain the respiratory time series (variable ϱ). To assess the dynamical activity of the cardiovascular system, the PAT time series (variable π) was extracted as the sequence of consecutive time intervals between the ECG R peak and the maximum derivative of the BVP signal for each given cardiac cycle (Gao et al., 2016). As regards the brain district, the power spectral density (PSD) was calculated for each EEG signal using a 2-s sliding window with 50% overlap. In each window, the spectral power in the frequency bands 0.5–3, 3–8, 8–12, and 12–25 Hz (respectively, δ, θ, α, and β) was measured through integration of the spectral proﬁle within each band, extracting brain time series which resulted sampled at 1 Hz. The procedure was repeated for the signals recorded from all electrodes, to extract the spatial distribution of the EEG band-power time series. INTRODUCTION Our analysis is performed in a group of young healthy subjects monitored at rest and during diﬀerent levels of mental stress, mapping the interaction measures across the scalp EEG electrodes to evidence possible regional eﬀects, and assessing the statistical signiﬁcance of the proposed measures to reconstruct the topology of brain and body interactions in the diﬀerent physiological states. The three above-described conditions actually correspond to an increasing level of stress, since a sustained attention task produces higher mental involvement than a fully relaxed state, still not being as stressful as carrying out fast and continuous arithmetic calculations (Zanetti et al., 2019a). The experiment was approved by the Ethics Committees of the University of Trento. All volunteers participating in this study provided written informed consent. Further details on the measurement protocol employed for this study can be found in Zanetti et al. (2019a). Hardware Used for Data Acquisition U q Data used in this study were acquired using non-invasive wearable sensors (Zanetti et al., 2019b). In detail, the signals consisted of electrocardiographic (ECG), EEG, respiratory, and blood volume pulse (BVP) waveforms recorded using diﬀerent devices. A sensorized t-shirt provided by Smartex (Prato, Italy) was employed for acquiring both the ECG (lead II, sampling frequency of 250 Hz) and the breath signal (sampling rate of 25 Hz). The E4 wristband provided by Empatica (Milano, Italy) with a photoplethysmographic (PPG) sensor has been used for BVP signal (sampling rate of 64 Hz). Finally, for EEG data, the EPOC PLUS wireless headset provided by Emotiv (San Francisco, CA, United States) has been employed, recording 14 signals from electrodes positioned on the scalp according to a reduced version of the 10-20 international placement system (see Figure 1A). All the data were acquired synchronously and sent wirelessly via Bluetooth to a personal computer for the subsequent post-processing and analyses. Particular care has been paid to ensure the correct positioning of the wearable devices on the body. Moreover, an appositely designed method for ensuring synchronization of the diﬀerent acquired biosignals has been employed, based on the linear warping of the time axis with respect to the Smartex signal taken as a reference. We refer the reader to Zanetti et al. (2019b) for further details and the complete synchronization procedure. Time Series Extraction Maps were generated through interpolation, over a 100 × 100 grid, of the values of EEG band-power time series using the MATLAB built- in biharmonic spline method. The interpolation was used only for visualization purposes, while all the analyses were carried Measurement Protocol Eighteen young healthy volunteers (13 males, ﬁve females; age range: 20–30 years) were monitored during a measurement protocol consisting of three experimental conditions corresponding to diﬀerent levels of mental stress (Zanetti et al., 2019a): (i) A resting condition (REST), lasting 12 min and consisting in watching a video showing landscapes with relaxing background music; (ii) A sustained attention task (GAME) lasting 12 min and consisting in playing a serious game, i.e., following a cursor on the screen while trying to avoid some obstacles; (ii) A sustained attention task (GAME) lasting 12 min and consisting in playing a serious game, i.e., following a cursor on the screen while trying to avoid some obstacles; February 2021 \| Volume 14 \| Article 602584 Frontiers in Neuroscience \| www.frontiersin.org 3 Multivariate Correlation and Brain–Body Networks Pernice et al. FIGURE 1 \| Schematic representation of the data acquisition and analysis steps. (A) Graphical representation of the positioning of the 14 EEG electrodes over the scalp. (B) Physiological systems and variables considered in this work: cardiac variable η (R–R interval of the ECG), respiratory variable ϱ (respiration amplitude), and cardiovascular variable π (pulse arrival time) for the body subnetwork X; amplitude of the δ, θ, α, and β EEG waves for the scalp subnetwork Y; the two subnetworks form the overall physiological network Z. (C) Venn diagrams depicting the multivariate interaction measures used in this work: on the left, multivariate brain–body interaction, quantifying the variability shared by the two subnetworks X and Y (light green area); in the middle, “direct” interaction between two individual variables (here, the cardiac and respiratory variables η and ϱ), when all other variables are considered (orange area); on the right, decomposition of the interaction between one target variable (here, the cardiac variable η) and all other variables (green + blue areas) as the sum of the interactions internal to the target subnetwork (here, the body subnetwork X; green area) and the interactions exclusive of the other subnetwork (here, the brain subnetwork Y; blue area). FIGURE 1 \| Schematic representation of the data acquisition and analysis steps. (A) Graphical representation of the positioning of the 14 EEG electrodes over the scalp. Measurement Protocol (B) Physiological systems and variables considered in this work: cardiac variable η (R–R interval of the ECG), respiratory variable ϱ (respiration amplitude), and cardiovascular variable π (pulse arrival time) for the body subnetwork X; amplitude of the δ, θ, α, and β EEG waves for the scalp subnetwork Y; the two subnetworks form the overall physiological network Z. (C) Venn diagrams depicting the multivariate interaction measures used in this work: on the left, multivariate brain–body interaction, quantifying the variability shared by the two subnetworks X and Y (light green area); in the middle, “direct” interaction between two individual variables (here, the cardiac and respiratory variables η and ϱ), when all other variables are considered (orange area); on the right, decomposition of the interaction between one target variable (here, the cardiac variable η) and all other variables (green + blue areas) as the sum of the interactions internal to the target subnetwork (here, the body subnetwork X; green area) and the interactions exclusive of the other subnetwork (here, the brain subnetwork Y; blue area). out on the acquired data. The brain time series extracted in this way were synchronous with those obtained resampling at 1 Hz the three cardiovascular time series using spline interpolation (Zanetti et al., 2019a). The rate of 1 Hz, which sets a time scale for the analysis which is compatible with the spectrum of heart rhythms, has already been used in previous studies in the ﬁeld of network physiology for analyzing the time series from diﬀerent body locations (Bashan et al., 2012; Bartsch et al., 2015). The uniformity of the ﬁnal sampling rate and the synchronization of the signals acquired from the diﬀerent devices, carried out according to the procedure described in Section “Hardware Used for Data Acquisition,” permitted to obtain synchronous time series for all the physiological districts. Each time series consisted of 300 samples (corresponding to 5 min of signal recording) and particular care was taken to avoid transient phenomena during the diﬀerent conditions. This has been accomplished starting the considered time window 3 min after the beginning of the REST phase, and from 1 to 2 min after the start of a MENTAL or GAME condition (not more to avoid habituation of the volunteer to the more stressful condition). All time series were checked for a restricted form of weak sense stationarity using the algorithm proposed in Magagnin et al. Measurement Protocol (2011), which randomly extracts a given number of sub-windows from each time series and assesses the steadiness of mean and variance across the sub-windows. out on the acquired data. The brain time series extracted in this way were synchronous with those obtained resampling at 1 Hz the three cardiovascular time series using spline interpolation (Zanetti et al., 2019a). The rate of 1 Hz, which sets a time scale for the analysis which is compatible with the spectrum of heart rhythms, has already been used in previous studies in the ﬁeld of network physiology for analyzing the time series from diﬀerent body locations (Bashan et al., 2012; Bartsch et al., 2015). The uniformity of the ﬁnal sampling rate and the synchronization of the signals acquired from the diﬀerent devices, carried out according to the procedure described in Section “Hardware Used for Data Acquisition,” permitted to obtain synchronous time series for all the physiological districts. Each time series consisted of 300 samples (corresponding to 5 min of signal recording) and particular care was taken to avoid transient phenomena during the diﬀerent conditions. This has been accomplished starting the considered time window 3 min after the beginning of the REST phase, and from 1 to 2 min after the start of a MENTAL or GAME condition (not more to avoid habituation of the volunteer to the more stressful condition). All time series were checked for a restricted form of weak sense stationarity using the algorithm proposed in Magagnin et al. (2011), which randomly extracts a given number of sub-windows from each time series and assesses the steadiness of mean and variance across the sub-windows. In the following, we will denote X as the body subnetwork, consisting of the η, ϱ, and π variables, while Y denotes the brain subnetwork (scalp areas), consisting of the δ, θ, α, and β variables. We are aware that recent studies have highlighted that particular care should be assumed making inferences about brain regions when using EEG signals acquired on the scalp (Lai et al., 2018; Van de Steen et al., 2019), and will discuss this issue in Section “Discussion.” Figure 1 schematically depicts the approach followed in this study, with the time series analyzed (Figure 1B) and the measures of multivariate interaction (MI) (Figure 1C) which are presented in detail in the next subsection. Measurement Protocol In the following, we will denote X as the body subnetwork, consisting of the η, ϱ, and π variables, while Y denotes the brain subnetwork (scalp areas), consisting of the δ, θ, α, and β variables. We are aware that recent studies have highlighted that particular care should be assumed making inferences about brain regions when using EEG signals acquired on the scalp (Lai et al., 2018; Van de Steen et al., 2019), and will discuss this issue in Section “Discussion.” Figure 1 schematically depicts the approach followed in this study, with the time series analyzed (Figure 1B) and the measures of multivariate interaction (MI) (Figure 1C) which are presented in detail in the next subsection. Frontiers in Neuroscience \| www.frontiersin.org Multivariate Interaction Analysis 1 is the most commonly known expression for the squared correlation, it can also be formulated in terms of the determinant of the covariance matrix of the vector variable concatenating x and y, W = [xy], as R (X; Y) ≡−ln 1 −ρ2(X; Y) = ln \|6X\| 6X\|Y . (6) (6) ρ2(x; y) = 1 − 6[xy] 6x6y , The MI measure deﬁned in Eq. 6 is null when X and Y are uncorrelated and, contrary to the squared correlation, it tends to inﬁnity when X and Y are completely correlated. Also, we note that the MI can be expressed as the diﬀerence between two terms related to the covariance structure of the vector variables as: 6[xy] = 6W = E h (W −mW)T (W −mW) i , (2) (2) or in terms of the residuals of a simple linear regression model of the type x = ay + b + u, where a and b are the regression coeﬃcients and u is the prediction error, as R (X; Y) = V (X) −V (X \| Y), (7) (7) where V (X) = ln \|6X\| is a logarithmic form of the so-called generalized variance of X and V (X \| Y) = ln 6X\|Y is the logarithmic generalized partial variance of X given Y (Barrett et al., 2010), quantifying, respectively, the overall variability within X and the part of such variability that remains after regressing X on Y. Eq. 7 is depicted graphically in the Venn diagram of Figure 1C (left). The MI measure deﬁned in Eqs 6 and 7 is motivated by its link to information-theoretic quantities when the variables are jointly Gaussian (see Appendix), and because it oﬀers the possibility to decompose in a meaningful way the variability shared between group of variables, as seen in the following. ρ2(x; y) = 1 −6x\|y 6x , 6x\|y = 6u, (3) (3) in which 6x\|y is the so-called partial variance, i.e., the variance of the error of the regression of x on y. The derivation of Eqs 2 and 3 is reported in the Appendix. In the present work, we extend the above measures to the multivariate case, considering the random vectors X and Y that collect the variables of the so-called body subnetwork composed by the cardiac, respiratory, and cardiovascular processes, and the variables of the brain subnetwork composed by the EEG power- band processes. Multivariate Interaction Analysis In this work, the time series measuring the output values of the diﬀerent physiological systems introduced in the previous section are interpreted as consecutive observations of random variables mapping the system states. A typical approach used in network physiology to study the interactions between two physiological February 2021 \| Volume 14 \| Article 602584 4 Multivariate Correlation and Brain–Body Networks Pernice et al. variables x and y is to quantify their linear correlation (Bashan et al., 2012; Lin et al., 2020). The most common measure is the squared Pearson’s correlation coeﬃcient, deﬁned as: variables x and y is to quantify their linear correlation (Bashan et al., 2012; Lin et al., 2020). The most common measure is the squared Pearson’s correlation coeﬃcient, deﬁned as: and deﬁning the so-called partial covariance of X given Y as 6X\|Y ≡6X −6X;Y6−1 Y 6Y;X being 6X;Y and 6Y;X the cross- covariance matrices (Barnett et al., 2009), it can be shown (see Appendix) that the multivariate correlation can be formulated in analogy to Eq. 3 as ρ2(x; y) ≡ 62 x;y 6x6y (1) analogy to Eq. 3 as (1) ρ2(X; Y) = 1 − 6X\|Y \|6X\| , 6X\|Y = 6U. (5) (5) where 6x = E[(x −mx)2] and 6y = E[(y −my)2] are the variance of x and y being mx = E[x] and my = E[y] their mean values, and 6x;y = E[(x −mx)(y −my)] is their covariance (E represents the expectation operator). The squared correlation is a symmetric normalized measure of linear dependence between x and y, i.e., ρ2 x; y = ρ2 y; x , which ranges from 0 to 1 moving from the absence of correlation to full correlation. From Eq. 5, it is clear that the squared multivariate correlation is related to the covariance matrix of the prediction error of a multivariate linear regression. Moreover, it is symmetric (ρ2(X; Y) = ρ2(Y; X)) and ranges from 0 to 1, indicating, respectively, uncorrelation (obtained when A = 0) and full linear dependence (obtained when U = 0) between X and Y. Here, we further deﬁne a logarithmic version of the multivariate correlation between X and Y, which we denote as MI: While Eq. Frontiers in Neuroscience \| www.frontiersin.org Multivariate Interaction Analysis With the notation introduced above, the body and brain variables are the P-dimensional vector X = [η ρ π] and the Q-dimensional vector Y = [δ θ α β] (P = 3, Q = 4), which are further grouped in the vector describing the state of the whole physiological network, Z = [XY] = [Z1 · · · ZM] (M = P + Q = 7). Then, denoting as 6X = E (X −mX)T(X −mX) , 6Y = E (Y −mY)T(Y −mY) and 6Z = E (Z −mZ)T(Z −mZ) the P × P, Q × Q, and M × M covariance matrices of X, Y, and Z, we deﬁne the multivariate correlation between X and Y extending Eq. 2 as follows: Next, to quantify how a single physiological process is linked to the others, we derive measures of the MI between a scalar variable and a vector variable. To this end, let us consider a “target” scalar variable in the body subnetwork, xi ∈X, and denote as Xi = X\xi the remaining variables in X (i = 1,...,P); similarly, a target variable yj ∈Y can be chosen in the brain subnetwork, separating it from the other variables Yj = Y\yj (j = 1,...,Q). Then, the interaction between the target variable and all other variables in the network is deﬁned as: R(xi; Xi, Y) = V (xi) −V(xi\|Xi, Y), R yj; Yj, X = V yj −V yj Yj, X , (8) ρ2(X; Y) ≡1 − 6[XY] \|6X\| \|6Y\|, 6[XY] = 6Z. (4) (4) (8) where the generalized variances and partial variances are V (xi) = ln 6xi, V yj = ln 6yj, and V(xi\|Xi, Y) = ln 6xi\|Xi,Y, V(yj\|Yj, X) = ln 6yj;Yj,X. For example, for the cardiac variable xi = η, such that Xi = [ϱπ], This deﬁnition also has a straightforward interpretation in terms of linear regression. Indeed, considering the regression X = YA + B + U, where A and B are parameter vectors of dimension Q × P and 1 × P, and U is an 1 × P vector of residuals, February 2021 \| Volume 14 \| Article 602584 Frontiers in Neuroscience \| www.frontiersin.org Multivariate Correlation and Brain–Body Networks Pernice et al. of the generalized partial variances in Eq. 7 and of the partial variances in Eqs 8–11. Multivariate Interaction Analysis where RSSR and RSSF are the residual sum of squares of the reduced and full regression (leading to compute the ﬁrst and second V (· \| ·) terms, respectively), pR and pF are the number of coeﬃcients used in the reduced and full regression, and N is the time series length. The interaction measure is considered statistically signiﬁcant if F is larger than the critical value of the Fisher distribution with (pF −pR, N −pF) degrees of freedom at the signiﬁcance level α0.05. When it is necessary to solve only one linear regression problem, i.e., for the computation of R yj; Yj , R y; Yj, X , R xi; Xi , and R xi; Xi, Y , the RSSR reduces to the variance of the predicted variable, 6X. Lastly, for the computation of R (X; Y) in which X and Y are both multivariate, RSSR is the generalized variance of X, \|6X\|, and RSSR is the generalized partial variance of X given Y, \|6 X\|Y\|. R xi; Y Xi = V xi\|Xi −V xi Xi, Y , R yj; X Yj = V yj\|Yj −V yj Yj, X ; (10) (10) a graphical example with xi = η is in Figure 1C (right, blue). We note that Eqs 9 and 10 achieve a decomposition of Eq. 8, i.e., R(xi; Xi, Y) = R xi; Xi + R xi; Y Xi and R(yj; Yj, X) = R yj; Yj + R yj; X Yj . For instance, Figure 1C (right) depicts how the extent of common variability shared between the cardiac variable and all other physiological variables, R(η; π, ρ, Y), can be expanded as the sum of the variability the cardiac variable shares with the two other variables of the body subnetwork, R(η; π, ρ), and the variability that it shares with the brain subnetwork but not with the body subnetwork, R(η; Y\|π, ρ). As regards the statistical analysis, the deviation from homogeneity of the spatial distribution of each interaction measure was assessed using the non-parametric Kruskal–Wallis test, which was also used to assess the statistical signiﬁcance of the diﬀerence across conditions (REST, MENTAL, and GAME) of the median of the distribution of the measure computed over the 18 subjects, followed in this case by post hoc Dunn–Šidák test with correction for multiple comparisons (Šidák, 1967; Sawilowsky, 2007) to assess pairwise diﬀerences (REST vs. Multivariate Interaction Analysis we have R (η; ϱ, π,δ, θ, α,β) = ln(6η) −ln(6η\|ϱπδθαβ) [Figure 1C (right), blue + green]. In a similar way, the interaction between a target variable of a given subnetwork (brain or body) and the remaining variables in the same subnetwork is quantiﬁed as After computation of each interaction measure, its statistical signiﬁcance was tested, individually for each computation, by using a parametric Fisher statistic (Brandt and Williams, 2006) under the null hypothesis that the coeﬃcients of the considered linear relationship are all zero (Montalto et al., 2014; Siggiridou and Kugiumtzis, 2015). In all those cases in which it is necessary to solve two diﬀerent linear regression problems with scalar predicted variable, i.e., for the computation of R(xi; Y\|Xi), R(yj; X\|Yj) and R(zi; zj\|Z\{zi, zj}), the test statistic is: R xi; Xi = V (xi) −V xi Xi , R yj; Yj = V yj −V yj Yj ; (9) (9) a graphical example with xi = η is in Figure 1C (right, green). Moreover, conditional interactions can be measured to assess the link between two variables after removing the common eﬀect that a group of other variables has on them. Here, we measure the interaction between one target variable in a subnetwork and all variables in the other subnetwork, conditioning on the remaining variables in the ﬁrst subnetwork, as follows: F = RSSR−RSSF pF−pR RSSF N−pF , (12) (12) where RSSR and RSSF are the residual sum of squares of the reduced and full regression (leading to compute the ﬁrst and second V (· \| ·) terms, respectively), pR and pF are the number of coeﬃcients used in the reduced and full regression, and N is the time series length. The interaction measure is considered statistically signiﬁcant if F is larger than the critical value of the Fisher distribution with (pF −pR, N −pF) degrees of freedom at the signiﬁcance level α0.05. When it is necessary to solve only one linear regression problem, i.e., for the computation of R yj; Yj , R y; Yj, X , R xi; Xi , and R xi; Xi, Y , the RSSR reduces to the variance of the predicted variable, 6X. Lastly, for the computation of R (X; Y) in which X and Y are both multivariate, RSSR is the generalized variance of X, \|6X\|, and RSSR is the generalized partial variance of X given Y, \|6 X\|Y\|. Multivariate Interaction Analysis MENTAL, REST vs. GAME, MENTAL vs. GAME). Non-parametric tests were used because the hypothesis of normality of the distribution of each measure was rejected according to the Anderson–Darling test (Anderson and Darling, 1952). Finally, we deﬁne a measure of the “direct” interaction between two individual physiological processes zi, zj ∈Z conditioned to all other processes in the overall network as the quantity: R(zi; zj\|Z\{zi, zj}) = V zj Z\{zi, zj −V zj Z\{zj} , (11) which quantiﬁes the extent of common variability between zj and zi that is not shared with any other variable in the network Z. For instance, the direct interaction between the cardiac and respiratory variables is given by R (η; ϱ \|π,δ, θ, α,β) = ln(6η\|π,δ, θ,α,β) −ln(6η\|ϱ,π,δ, θ,α,β) (Figure 1C, middle). Frontiers in Neuroscience \| www.frontiersin.org MI Between a Target and All Other Processes in the Brain–Body Network y Figure 3 reports the spatial distribution on the scalp of the median values of the MI between a target i of the body subnetwork and all other processes, i.e., R(xi; Xi, Y) (a), alongside with the number of subjects which showed statistically signiﬁcant MI according to the F-test (b). This measure evaluates the degree of connectivity between the considered body process and all other processes in the overall network. Considering the cardiac variable η or the respiratory variable ρ as the target, the MI value was found to be high and statistically signiﬁcant in all subjects during REST and MENTAL (with a slight decrease in the median values during MENTAL), while it decreased markedly in magnitude during GAME, also resulting statistically signiﬁcant in a lower number of subjects. The decrease from REST to GAME was statistically signiﬁcant at all locations with target η, and at the locations of the electrodes AF3, F7, T7, FC5, FC6, P7, P8, O1, and O2 with target ρ. On the contrary, when the cardiovascular process π was taken as the target, the MI value was low and was signiﬁcant in a smaller number of subjects (around 50%), without displaying any signiﬁcant variations across conditions. The Kruskal–Wallis test showed homogeneity (p-value > 0.05) for spatial distributions of R(xi; Xi, Y) in all the cases. These results denote a high degree of connectivity between the cardiac and respiratory processes and the other network processes, decreasing with the GAME task, and an overall low connectivity for the cardiovascular process. RESULTS All the measures presented in the previous subsection were computed from the M = 7 time series collected from each of the 18 subjects in the three analyzed experimental conditions (REST, MENTAL, and GAME). Moreover, for all measures involving the brain processes (vector variable Y), the computation was repeated, for each of the 14 scalp electrodes, extracting the Q = 4 brain time series δ, θ, α, and β from the EEG signal acquired on that electrode while considering the same P = 3 body time series (see Figure 1A). For each set of time series, the analysis was computed using the ordinary vector least squares approach to identify the linear regression models needed for the computation Results are presented showing the median values, across the subjects, of the various interaction measures in the three considered conditions (REST, MENTAL, and GAME). The spatial distribution of each measure is obtained performing the analysis at every EEG electrode location, and is represented with color- coded values carrying out an interpolation over the schematic of the scalp. In addition, ﬁgures show the results of the statistical signiﬁcance analysis, reporting the number of subjects for which the measure was found to be signiﬁcantly larger than zero according to the Fisher F-test. We refer the reader to the Results are presented showing the median values, across the subjects, of the various interaction measures in the three considered conditions (REST, MENTAL, and GAME). The spatial distribution of each measure is obtained performing the analysis at every EEG electrode location, and is represented with color- coded values carrying out an interpolation over the schematic of the scalp. In addition, ﬁgures show the results of the statistical signiﬁcance analysis, reporting the number of subjects for which the measure was found to be signiﬁcantly larger than zero according to the Fisher F-test. We refer the reader to the February 2021 \| Volume 14 \| Article 602584 Frontiers in Neuroscience \| www.frontiersin.org 6 Multivariate Correlation and Brain–Body Networks Pernice et al. Figure 4 reports the spatial distribution on the scalp of the median values of the MI between a target j of the brain subnetwork and all other processes, i.e., R(yi; Yj, X) (a), alongside with the number of subjects which showed statistically signiﬁcant MI according to the F-test (b). The measure evaluates the connectivity between the considered brain rhythm and all other processes in the overall network. RESULTS The MI relevant to the δ, θ, and α brain variables showed a tendency to increase, when assessed for electrodes located in the frontal area of the scalp, during the mental arithmetic condition compared to the resting state, and to return to baseline values during the serious game condition. The index R(yi; Yj, X) increased signiﬁcantly at the AF3, AF4, and F7 electrodes for θ and at the AF3 and F7 electrodes for α, moving from REST to MENTAL, reﬂecting an increased interaction between such rhythms and the whole network during mental workload in the frontal region, and decreased signiﬁcantly at AF3, AF4, and F7 electrodes for δ, θ, and α moving from MENTAL to GAME; the decrease was statistically signiﬁcant also at the left parietal P7 and right occipital O2 electrodes when yj = α and comparing GAME to REST. A diﬀerent behavior was observed taking the process β as target, with no variations of the median MI values going from REST to MENTAL, a decrease at the P7, P8, and O2 electrodes going from REST to GAME, and a decrease at the O2 electrode going from GAME to MENTAL; this suggests a decreased connectivity between the β rhythm and all others localized to the parietal and right occipital regions. The Kruskal–Wallis test showed a heterogeneous spatial distribution of R(yi; Yj, X) (p-value < 0.05) when yi = θ during all three conditions, when yi = δ during REST and MENTAL, when yi = α during MENTAL, and when yi = β during GAME. The F-test showed statistically signiﬁcant values of R(yi; Yj, X) for almost all subjects when yi = θ, yi = α, and yi = β (in particular during REST and MENTAL), while it was signiﬁcant for a lower number of subjects (around 60%) when yi = δ (especially during GAME). Supplementary Material for the complete table of results in terms of median MI values, p-values of Kruskal–Wallis and post hoc pairwise comparison test, and of number of subjects with statistically signiﬁcant MI according to Fisher F-test for the Figures 2–8. MI Between Each Subnetwork as a Whole Figure 2 shows the median MI index R (X; Y) computed between the brain and body subnetworks (Figure 2A), and the number of subjects which showed statistically signiﬁcant MI (Figure 2B), mapped across the scalp in the three analyzed conditions. The index R (X; Y) can be thought as a measure of the overall connectivity between the body and brain subnetworks, each one considered as a whole. In almost all subjects and especially during the REST and MENTAL conditions, the two subnetworks share statistically signiﬁcant amounts of information at all the EEG electrodes positions (Figure 2B). In each condition, the Kruskal–Wallis test showed homogeneity (p-value > 0.05) for the spatial distribution of the MI index (the visually heterogeneous patterns in Figure 2A may be due to interpolation eﬀects due to the limited number of non-uniformly distributed electrodes). The overall connectivity tends to decrease going from REST to MENTAL and then to GAME (Figure 2A); compared to REST, the decrease is statistically signiﬁcant for the AF4 frontal electrode during MENTAL and for the F7 electrode during GAME. Frontiers in Neuroscience \| www.frontiersin.org MI Between a Target and All Other Processes in the Brain or Body Subnetwork REST or GAME vs. REST). FIGURE 3 \| Spatial distribution over the scalp of (A) the median multivariate interaction between a target xi of the body subnetwork and all remaining variables, R xi; Xi, Y , and (B) the number of subjects with statistically signiﬁcant values of the index, in the three analyzed conditions (REST, MENTAL, and GAME); the index is computed with target corresponding to the cardiac process η (upper row panels), to the respiratory process ρ (middle row panels), and to the cardiovascular process π (lower row panels). Markers are located at EEG electrode positions and in (A) are colored according to the results of statistical analysis (white: p < 0.05 MENTAL vs. REST or GAME vs. REST). Asterisk () on an electrode indicates p < 0.05 MENTAL vs. GAME. FIGURE 3 \| Spatial distribution over the scalp of (A) the median multivariate interaction between a target xi of the body subnetwork and all remaining variables, R xi; Xi, Y , and (B) the number of subjects with statistically signiﬁcant values of the index, in the three analyzed conditions (REST, MENTAL, and GAME); the index is computed with target corresponding to the cardiac process η (upper row panels), to the respiratory process ρ (middle row panels), and to the cardiovascular process π (lower row panels). Markers are located at EEG electrode positions and in (A) are colored according to the results of statistical analysis (white: p < 0.05 MENTAL vs. REST or GAME vs. REST). Asterisk () on an electrode indicates p < 0.05 MENTAL vs. GAME. yj = α involved the P7 and O2 electrodes when comparing GAME and REST, and for yj = θ the electrode FC5 when comparing GAME and MENTAL. These results indicate an increased connectivity of δ, α, and especially θ rhythms with all the others during mental workload in the frontal region, and a decreased connectivity of α with all the others during GAME in the left parietal and right occipital zones. Diﬀerent trends were shown when yj = β: the MI was substantially unchanged from REST to MENTAL, and decreased during GAME (with signiﬁcant changes at the electrodes P7, P8, and O2 when compared to REST, and at O2 when compared to MENTAL), thus showing a decreased connectivity of β rhythm with all the others during GAME in the parietal and right occipital regions. MI Between a Target and All Other Processes in the Brain or Body Subnetwork Figure 5 depicts the distribution of the MI between a target in the body subnetwork and the two other variables belonging to the same subnetwork, R(xi; Xi), in the three conditions, together with the number of subjects with statistically signiﬁcant MI. This index assesses the internal connectivity of the body subnetwork, measured between one process and the two others, while pairwise “direct” connectivity can be inferred from Figure 9. For each target node, its interaction within the body subnetwork was found high and signiﬁcant at REST and decreased progressively during the MENTAL and GAME conditions. The decrease of MI values from REST to GAME is statistically signiﬁcant for η and ρ taken as targets. The values of R(xi; Xi) computed with xi = η and xi = ρ were statistically signiﬁcant in almost all subjects during REST and MENTAL, and decreased slightly during GAME; when xi = π, the statistical signiﬁcance was lower in all conditions and reached the minimum of 50% of subjects during GAME. Overall, these results suggest a strong connectivity within February 2021 \| Volume 14 \| Article 602584 Frontiers in Neuroscience \| www.frontiersin.org 7 Multivariate Correlation and Brain–Body Networks Pernice et al. FIGURE 2 \| Spatial distribution of (A) the median multivariate interaction between brain and body, R(X; Y), and (B) the number of subjects with statistically signiﬁcant values of the index, in the three analyzed conditions (REST, MENTAL, and GAME). Markers are located at EEG electrode positions and in (A) are colored according to the results of statistical analysis (white: p < 0.05 MENTAL vs. REST or GAME vs. REST). FIGURE 2 \| Spatial distribution of (A) the median multivariate interaction between brain and body, R(X; Y), and (B) the number of subjects with statistically signiﬁcant values of the index, in the three analyzed conditions (REST, MENTAL, and GAME). Markers are located at EEG electrode positions and in (A) are colored according to the results of statistical analysis (white: p < 0.05 MENTAL vs. REST or GAME vs. REST). FIGURE 2 \| Spatial distribution of (A) the median multivariate interaction between brain and body, R(X; Y), and (B) the number of subjects with statistically signiﬁcant values of the index, in the three analyzed conditions (REST, MENTAL, and GAME). Markers are located at EEG electrode positions and in (A) are colored according to the results of statistical analysis (white: p < 0.05 MENTAL vs. MI Between a Target and All Other Processes in the Brain or Body Subnetwork The Kruskal–Wallis test showed spatial inhomogeneity (p-value < 0.05) with regard to δ and θ power in all the three conditions, only during MENTAL with regard to α power, and only during GAME with regard to β power. According to the F-test, the interaction values were the body subnetwork, mainly arising from cardiorespiratory interactions and declining during mental workload. Figure 6 depicts the distribution of the MI between a target in the brain subnetwork and the three other variables belonging to the same subnetwork, R(yj; Yj), as well as the number of subjects with statistically signiﬁcant MI. This index assesses the connection of the considered brain rhythm with all the others taken together, while the pairwise connectivity between rhythms can be inferred from Figure 9. For this measure, results are similar to those obtained for the global measure R(yj; Yj, X), showing a tendency of the measure to increase from REST to MENTAL in the frontal region of the scalp (statistically signiﬁcant at the AF3, AF4, and F7 electrodes when yj = θ, at the AF3 and F7 electrodes when yj = α, and at the AF3 electrode when yj = δ), and a tendency to decrease in the same region moving from MENTAL to GAME (signiﬁcant for AF3, AF4, and F7 when yj = δ and yj = α, and also for F8 when yj = θ); other signiﬁcant changes for February 2021 \| Volume 14 \| Article 602584 Frontiers in Neuroscience \| www.frontiersin.org 8 Multivariate Correlation and Brain–Body Networks Pernice et al. FIGURE 4 \| Spatial distribution over the scalp of (A) the median multivariate interaction between a target yj of the brain subnetwork and all remaining variables, R(yj; Yj, X), and (B) the number of subjects with statistically signiﬁcant values of the index, in the three analyzed conditions (REST, MENTAL, and GAME); the index is computed with target corresponding to the EEG power band processes δ, θ, α, β (from upper to lower row panels). Markers are located at EEG electrode positions and in (A) are colored according to the results of statistical analysis (white: p < 0.05 MENTAL vs. REST or GAME vs. REST). Asterisk () on an electrode indicates p < 0.05 MENTAL vs. GAME. Hash symbols indicate results of Kruskal–Wallis test (#: p < 0.05, non-homogeneity of the spatial distribution). MI Between a Target and All Other Processes in the Brain or Body Subnetwork FIGURE 4 \| Spatial distribution over the scalp of (A) the median multivariate interaction between a target yj of the brain subnetwork and all remaining variables, R(yj; Yj, X), and (B) the number of subjects with statistically signiﬁcant values of the index, in the three analyzed conditions (REST, MENTAL, and GAME); the index is computed with target corresponding to the EEG power band processes δ, θ, α, β (from upper to lower row panels). Markers are located at EEG electrode positions and in (A) are colored according to the results of statistical analysis (white: p < 0.05 MENTAL vs. REST or GAME vs. REST). Asterisk () on an electrode indicates p < 0.05 MENTAL vs. GAME. Hash symbols indicate results of Kruskal–Wallis test (#: p < 0.05, non-homogeneity of the spatial distribution). FIGURE 5 \| (A) Distributions of the multivariate interaction between a target xi of the body subnetwork and the two other variables of the same subnetwork, R xi; Xi , and (B) number of subjects with statistically signiﬁcant values of the index, in the three analyzed conditions (REST, MENTAL, and GAME); the index is computed with target corresponding to the cardiac, respiratory, and cardiovascular processes η, ρ, and π. In (A), pKW indicates results of Kruskal–Wallis test, while hash symbols indicate a p-value lower than 0.05 obtained using post-hoc test for the analysis between REST and the considered condition (#: p < 0.05 MENTAL vs. REST or GAME vs. REST). FIGURE 5 \| (A) Distributions of the multivariate interaction between a target xi of the body subnetwork and the two other variables of the same subnetwork, R xi; Xi , and (B) number of subjects with statistically signiﬁcant values of the index, in the three analyzed conditions (REST, MENTAL, and GAME); the index is computed with target corresponding to the cardiac, respiratory, and cardiovascular processes η, ρ, and π. In (A), pKW indicates results of Kruskal–Wallis test, while hash symbols indicate a p-value lower than 0.05 obtained using post-hoc test for the analysis between REST and the considered condition (#: p < 0.05 MENTAL vs. REST or GAME vs. REST). February 2021 \| Volume 14 \| Article 602584 Frontiers in Neuroscience \| www.frontiersin.org 9 Multivariate Correlation and Brain–Body Networks Pernice et al. MI Between a Target and All Other Processes in the Brain or Body Subnetwork FIGURE 6 \| Spatial distribution over the scalp of (A) the median multivariate interaction between a target yj of the brain subnetwork and the three other variables in the same subnetwork, R(yj; Yj), and (B) number of subjects with statistically signiﬁcant values of the index, in the three analyzed conditions (REST, MENTAL, and GAME); the index is computed with target corresponding to the EEG power band processes δ, θ, α, β (from upper to lower row panels). Markers are located at EEG electrode positions and in (A) are colored according to the results of statistical analysis (white: p < 0.05 MENTAL vs. REST or GAME vs. REST). Asterisk () on an electrode indicates p < 0.05 MENTAL vs. GAME. Hash symbols indicate results of Kruskal–Wallis test (#: p < 0.05, non-homogeneity of the spatial distribution). FIGURE 6 \| Spatial distribution over the scalp of (A) the median multivariate interaction between a target yj of the brain subnetwork and the three other variables in the same subnetwork, R(yj; Yj), and (B) number of subjects with statistically signiﬁcant values of the index, in the three analyzed conditions (REST, MENTAL, and GAME); the index is computed with target corresponding to the EEG power band processes δ, θ, α, β (from upper to lower row panels). Markers are located at EEG electrode positions and in (A) are colored according to the results of statistical analysis (white: p < 0.05 MENTAL vs. REST or GAME vs. REST). Asterisk () on an electrode indicates p < 0.05 MENTAL vs. GAME. Hash symbols indicate results of Kruskal–Wallis test (#: p < 0.05, non-homogeneity of the spatial distribution). statistically signiﬁcant in the large majority of subjects for all measures and conditions. P7 compared to MENTAL), while it was uniformly low when xi = π. The Kruskal–Wallis test showed homogeneity (p- value > 0.05) for the spatial distributions of R(xi; Y\|Xi) in the three considered conditions. Conditional MI Between a Target in a Subnetwork and the Whole Other Subnetwork Figure 8 reports the spatial distribution on the scalp of the median values of the conditional MI between a target j of the brain subnetwork and the whole body subnetwork, given the remaining variables in the brain subnetwork, i.e., R(yj; X\|Yj) (Figure 8A), alongside with the number of subjects which showed statistically signiﬁcant conditional MI according to the F-test (Figure 8B). This measure evaluates the strength of the connection of a brain rhythm with all the body processes, after conditioning on eﬀects of the other brain rhythms. Also in this case, the values of R(yj; X\|Yj) were much weaker than those of the unconditional measure R(yj; Yj) and exhibited markedly lower statistical signiﬁcance (compare Figure 8 with Figure 6). The conditional MI showed a tendency to increase during MENTAL when yj = δ and when yj = β (signiﬁcantly higher values compared to REST, respectively, at P7 and at AF3), and to decrease during GAME when yj = θ and when yj = α (signiﬁcantly lower values compared to REST, respectively, at F7 and at F7, O2); an increase from MENTAL to GAME was observed at AF4 when yj = θ. These results, together with those of Figure 7, highlight the presence of weak connectivity between the brain and body processes, with no precise trends Figure 7 reports the spatial distribution on the scalp of the median values of the conditional MI between a target i of the body subnetwork and the whole brain subnetwork, given the remaining variables in the body subnetwork, i.e., R(xi; Y\|Xi) (Figure 7A), alongside with the number of subjects which showed statistically signiﬁcant conditional MI according to the F-test (Figure 7B). This measure evaluates the strength of the connection of a body process with all the brain rhythms, after conditioning on eﬀects of the other body processes. Contrary to the MI measures previously analyzed, the conditional MI showed overall lower values, as R(xi; Y\|Xi) was on average an order of magnitude smaller than R(xi; Xi), and weaker statistical signiﬁcance, as the F-test rejected the null hypothesis only for few subjects (always less than 50%) in all the conditions and electrodes. Frontiers in Neuroscience \| www.frontiersin.org Conditional MI Between a Target in a Subnetwork and the Whole Other Subnetwork The conditional MI showed a tendency to decrease during GAME when xi = η (signiﬁcantly lower values at F8 compared to REST) and when xi = ρ (signiﬁcantly lower values at F7 compared to REST, and at F7, F3, and February 2021 \| Volume 14 \| Article 602584 Frontiers in Neuroscience \| www.frontiersin.org Frontiers in Neuroscience \| www.frontiersin.org 10 Multivariate Correlation and Brain–Body Networks Pernice et al. FIGURE 7 \| Spatial distribution over the scalp of (A) the median multivariate interaction between a target xi of the body subnetwork and all variables of the brain subnetwork, conditioned on the two remaining variables of the body subnetwork, R xi; Y\|Xi , and (B) the number of subjects with statistically signiﬁcant values of the index, in the three analyzed conditions (REST, MENTAL, and GAME); the index is computed with target corresponding to the cardiac process η (upper row panels), to the respiratory process ρ (middle row panels), and to the cardiovascular process π (lower row panels). Markers are located at EEG electrode positions and in (A) are colored according to the results of statistical analysis (white: p < 0.05 MENTAL vs. REST or GAME vs. REST). Asterisk () on an electrode indicates p < 0.05 MENTAL vs. GAME. Hash symbols indicate results of Kruskal–Wallis test (#: p < 0.05, non-homogeneity of the spatial distribution). FIGURE 7 \| Spatial distribution over the scalp of (A) the median multivariate interaction between a target xi of the body subnetwork and all variables of the brain subnetwork, conditioned on the two remaining variables of the body subnetwork, R xi; Y\|Xi , and (B) the number of subjects with statistically signiﬁcant values of the index, in the three analyzed conditions (REST, MENTAL, and GAME); the index is computed with target corresponding to the cardiac process η (upper row panels), to the respiratory process ρ (middle row panels), and to the cardiovascular process π (lower row panels). Markers are located at EEG electrode positions and in (A) are colored according to the results of statistical analysis (white: p < 0.05 MENTAL vs. REST or GAME vs. REST). Asterisk () on an electrode indicates p < 0.05 MENTAL vs. GAME. Hash symbols indicate results of Kruskal–Wallis test (#: p < 0.05, non-homogeneity of the spatial distribution). Conditional MI Between a Target in a Subnetwork and the Whole Other Subnetwork FIGURE 8 \| Spatial distribution over the scalp of (A) the median multivariate interaction between a target yj of the brain subnetwork and all variables of the body subnetwork, conditioned on the three remaining variables of the brain subnetwork, R(yj; X\|Yj), and (B) the number of subjects with statistically signiﬁcant values of the index, in the three analyzed conditions (REST, MENTAL, and GAME); the index is computed with target corresponding to the EEG power band processes δ, θ, α, β (from upper to lower row panels). Markers are located at EEG electrode positions and in (A) are colored according to the results of statistical analysis (white: p < 0.05 MENTAL vs. REST or GAME vs. REST). Asterisk () on an electrode indicates p < 0.05 MENTAL vs. GAME. Hash symbols indicate results of Kruskal–Wallis test (#: p < 0.05, non-homogeneity of the spatial distribution). FIGURE 8 \| Spatial distribution over the scalp of (A) the median multivariate interaction between a target yj of the brain subnetwork and all variables of the body subnetwork, conditioned on the three remaining variables of the brain subnetwork, R(yj; X\|Yj), and (B) the number of subjects with statistically signiﬁcant values of the index, in the three analyzed conditions (REST, MENTAL, and GAME); the index is computed with target corresponding to the EEG power band processes δ, θ, α, β (from upper to lower row panels). Markers are located at EEG electrode positions and in (A) are colored according to the results of statistical analysis (white: p < 0.05 MENTAL vs. REST or GAME vs. REST). Asterisk () on an electrode indicates p < 0.05 MENTAL vs. GAME. Hash symbols indicate results of Kruskal–Wallis test (#: p < 0.05, non-homogeneity of the spatial distribution). February 2021 \| Volume 14 \| Article 602584 11 Frontiers in Neuroscience \| www.frontiersin.org Multivariate Correlation and Brain–Body Networks Pernice et al. FIGURE 9 \| Topological representation of the interaction between pairs of nodes zi and zj of the physiological network (zi, zj ∈Z = {η, ρ, π, δ, θ, α, β}), provided by the statistically signiﬁcant values of the conditional MI measure R(zi; zj\|Z\{zi, zj}), during the three considered states (REST, MENTAL, and GAME). Thickness of the lines is proportional to the number of subjects for which the corresponding link is statistically signiﬁcant (p < 0.05, Fisher’s F-test). Conditional MI Between a Target in a Subnetwork and the Whole Other Subnetwork Red, blue, and black lines denote the links relevant to body–body, brain–brain, and brain–body interactions. FIGURE 9 \| Topological representation of the interaction between pairs of nodes zi and zj of the physiological network (zi, zj ∈Z = {η, ρ, π, δ, θ, α, β}), provided by the statistically signiﬁcant values of the conditional MI measure R(zi; zj\|Z\{zi, zj}), during the three considered states (REST, MENTAL, and GAME). Thickness of the lines is proportional to the number of subjects for which the corresponding link is statistically signiﬁcant (p < 0.05, Fisher’s F-test). Red, blue, and black lines denote the links relevant to body–body, brain–brain, and brain–body interactions. were observed in the previous analyses (frontal: AF3, AF4, F7; central: FC5; parietal: P7, P8; occipital: O2; temporal: T7, T8). in terms of spatial localization and just a few statistically signiﬁcant variations during MENTAL or GAME. The Kruskal– Wallis test showed homogeneity (p-value > 0.05) for the spatial distributions of R(yj; X\|Yj) in almost all of the cases, except than the case yj = α during GAME. The network analysis allows to investigate the topological structure underlying the MIs detected previously, as well as their changes across conditions. As regards the body subnetwork (red links), the topology is quite consistent across electrodes for any considered experimental condition. At REST, strong interconnections are observed between the η and ρ nodes, and signiﬁcant (though generally weaker) connections are also observed between η and π and between π and ρ. During MENTAL, the connection η–ρ remains signiﬁcant in almost all subjects, while the two other links (η–π and π–ρ) are generally less evident. The weakening of the links in the body subnetwork is even more evident during GAME, involving also a decrease in the number of the connections between η and ρ. Frontiers in Neuroscience \| www.frontiersin.org Direct Interactions Between Pairs of Processes Based on Conditional MI This fact is documented by the low absolute values and fraction of subjects with statistically signiﬁcant interaction observed for the conditional MI measures R(xi; Y\|Xi) and R(yj; X\|Yj) (see Figures 7, 8), as well as from the similar trends obtained for MI (Figures 3, 5) and conditional MI measures (Figures 4, 6). Weaker interactions between the brain and body subnetworks were observed in the same experimental settings also in recent studies performing dynamic analyses (Zanetti et al., 2019a; Antonacci et al., 2020). Brain–body interactions (black links) are less evident and more sparse, supporting in terms of the fully multivariate measure R(zi; zj\|Z\{zi, zj}) the results of Figures 7, 8 where a limited number of signiﬁcant values of R(xi; Y\|Xi) and R(yj; X\|Yj) were observed. Though weak, interactions between the brain and body subnetwork were almost always detected (the two subnetwork were isolated only at AF3 during REST, and at AF3, F7, P8, O2 during GAME). Such interactions were mostly involving the η node of the body subnetwork (in 29 out of the 40 brain–body connections shown in Figure 9), often linked to the β node of the brain subnetwork (in 13 cases), or the δ node of the brain subnetwork (14 connections), and only sporadically the remaining nodes. Overall, brain–body connections increased moving from REST to MENTAL (from 13 to 19 links shown in Figure 9) and decreased during GAME (eight links); the scalp electrodes where this behavior was more striking are located in the frontal (AF3, F7) and temporal (T7, T8) areas. The interactions occurring within the body subnetwork formed by cardiac, cardiovascular, and respiratory dynamics (Figure 5 and red links in Figure 9) were remarkable and quite consistent across conditions, evidencing a predominance of cardiorespiratory coupling and a weakening during mental stress and particularly during sustained attention. The strong link between the cardiac and respiratory variables, corresponding to the heart period and respiratory amplitude time series, is due to the respiratory sinus arrhythmia (RSA), a well- known physiological mechanism whereby the breathing activity modulates the variability of the heart rate (Yasuma and Hayano, 2004; Ben-Tal et al., 2012; Porta et al., 2012; Krohova et al., 2019). Our results are in agreement with those obtained in previous works using diﬀerent and more sophisticated techniques, e.g., in Zanetti et al. (2019a) computing the information the information exchanged dynamically between heart period and respiration, and in Krohova et al. Direct Interactions Between Pairs of Processes Based on Conditional MI Figure 9 reports the network representation of the direct interactions between pairs of variables of the physiological network across the three analyzed conditions, depicted on the basis of the conditional MI measure R(zi; zj\|Z\{zi, zj}). This measure evaluates the pairwise connectivity between two processes in the context of all other processes in the whole physiological network. In the ﬁgure, networks are constructed counting the subjects for which the measure was statistically signiﬁcant, and for visualization purposes are reported for the subset of the scalp electrodes for which most signiﬁcant variations Analyzing the brain subnetwork (blue links), we found that it is fully connected (i.e., it shows absence of isolated nodes) for any scalp electrode and experimental condition. The most evident connections are those involving the pairs of nodes δ–θ, θ–α, and February 2021 \| Volume 14 \| Article 602584 Frontiers in Neuroscience \| www.frontiersin.org 12 Multivariate Correlation and Brain–Body Networks Pernice et al. α–β, while the connections δ–α, δ–β, and θ–β are weaker and less consistent across spatial locations. This topology is present in all conditions at REST and persists in the other conditions, even though with some noticeable anatomical variations moving from one condition to another. During MENTAL, the connections δ–θ, θ–α, and α–β were reinforced in the frontal areas of the scalp (AF3, AF4, F7, FC5) and in the right temporal area (T8). A slightly larger variability in the topology was observed during GAME, with connection strength similar to REST although with some local diﬀerence (e.g., emergence of θ–β connections at F7, decrease of α–β connections at P7, increase of δ–θ connections at T7, and decrease at T8). the ﬁeld of network physiology showing that signiﬁcant degrees of interaction within and between organ systems sustain the physiological regulation in diﬀerent physiological states, e.g., including sleep stages (Ako et al., 2003; Bashan et al., 2012; Bartsch et al., 2015; Lin et al., 2020) or physiological stress (Faes et al., 2017b; Valente et al., 2018; Krohova et al., 2019). Nevertheless, exploiting the decomposition of the overall MI measure into measures eliciting the correlations relevant to a single target variable and selected groups of other variables, it has been possible to infer that the interactions within each subnetwork prevail over brain–body interactions. Direct Interactions Between Pairs of Processes Based on Conditional MI (2019) applying multiscale entropy methods. In the latter study, the weakening of the inﬂuence of respiration on heart rate has been ascribed to the inhibition of parasympathetic activity provoked by stress challenges and, when compared to other stressors like postural changes, to the lack of activation of baroreﬂex-mediated RSA mechanisms. We also found that the cardiovascular variable analyzed here, i.e., the PAT, strongly interacts with the cardiac period and the respiration amplitude. This link is mostly probably due to the known inﬂuence of heart rate on stroke volume and blood pressure that in turns varies the PAT, which is also inﬂuenced by respiration (Drinnan et al., 2001; Wang et al., 2014); the mechanism is such that respiration aﬀects the intra-thoracic pressure provoking changes in blood pressure and then also heart rate, with PAT variations following some beats later (Cavalcanti, 2000; Drinnan et al., 2001). Frontiers in Neuroscience \| www.frontiersin.org DISCUSSION Moreover, the decrease observed in the parietal and right occipital regions moving from rest to serious game when the MI term R(yj; Yj) was computed for the α and β EEG rhythms is in accordance with previous ﬁndings in the literature showing that parietal cortical regions, mainly in the right hemisphere, are involved in sustained attention tasks (Lawrence et al., 2003; Molteni et al., 2007; Klimesch, 2012; Saalmann et al., 2012; Behzadnia et al., 2017; Mitko et al., 2019); the modulation of EEG rhythms during sustained attention was previously observed regarding high-frequency waves (in the β and gamma ranges) in Molteni et al. (2007), with changes localized mostly in the right hemisphere and in the parietal region, and regarding the α rhythm in Behzadnia et al. (2017), showing that a greater decrease in the α power is associated with better performance during the task. Our study goes beyond the above described ﬁndings, also showing that the observed changes in the coupling strength between brain wave dynamics are supported by the topology of the brain–brain network and to its reorganization during mental stress and sustained attention. Remarkably, structured reorganizations of the connectivity and topology of physiological networks consequent to transitions across diﬀerent physiological states have been previously reported in the context of sleep analysis (Bashan et al., 2012; Faes et al., 2015b; Lin et al., 2020). Bashan et al. (2012) demonstrated that the strength of brain–brain links is high during light sleep and deep sleep, and is lower during rapid eye movement (REM) sleep; Lin et al. (2020) reported strong β–α and θ–α links in awake subjects and when signiﬁcant positive correlation is present between a pair of brain waves; in Faes et al. (2015b), strong dynamical interactions along the directions β->α and δ->θ were revealed during sleep employing time-lagged causality measures such as Granger causality and transfer entropy. accordance with what reported in Mather and Thayer (2018) where it is stated that oscillations in heart rate modulate brain oscillatory activities, especially in brain regions associated with emotion regulation, which can lead to enhanced functional connectivity. Other studies, mostly related to sleep analysis, also suggest the existence of relations between EEG rhythms and HRV arising from common eﬀects driven by the ANS (Ako et al., 2003; Faes et al., 2014; Kuo et al., 2016; Dzhebrailova et al., 2017). DISCUSSION In particular, the β waves seem to play a main role in mediating brain–heart interactions, likely due to their dependence on autonomic arousals and sympathetic activation (Faes et al., 2014; Kuo et al., 2016). In addition, considering that cardiorespiratory interactions are typically very strong, an indirect eﬀect (i.e., an eﬀect mediated by RSA) of respiration on the brain subnetwork seems also plausible. Such an eﬀect is also supported by evidences about the rhythmic modulation of the neuronal activity of the neocortex exerted by respiration-locked sensory inputs (Heck et al., 2017; Varga and Heck, 2017). On the contrary, the interaction between π and the other variables is quite limited (as demonstrated by the low MI values in Figure 3A and the few connections in Figure 9). This is also in agreement with recent results (Pernice et al., 2019d; Zanetti et al., 2019a) obtained using information-theoretic measures, suggesting a limited coupling between pulse wave velocity in the cardiovascular system and brain dynamics. Methodologically, the results of this work highlight the usefulness of the proposed MI measure to investigate the functional connection between diﬀerent subnetworks in the human body. The MI measure under certain assumptions is also directly proportional to mutual information (see Appendix), and this is useful to allow comparisons with other previous works in the ﬁeld, since information-theoretic-based measures have already been used in the past for this aim (Barnum et al., 2010; Faes et al., 2014, 2017a,b; Barrett, 2015). For example, in our previous work (Zanetti et al., 2019a) we have investigated the information generated, stored, and transferred among diﬀerent nodes in a physiological network taking into account only one electrode, while in Pernice et al. (2019b), we have carried out a multilevel stress assessment based on the concept of network physiology using time-domain measures (mean and standard deviation) and self-entropy. Also, in Antonacci et al. (2020), we have applied a more sophisticated technique consisting of a penalized regression performed through the Least Absolute Shrinkage and Selection Operator (LASSO) before calculating measures of information dynamics. All these approaches are dynamic, meaning that they account for time-lagged interactions. Compared to such approaches, the MI measures proposed here can be deﬁned “static,” since only instantaneous (zero-lag) interactions are taken into account. Static analysis in some sense subsumes dynamic analysis, since time lagged eﬀect typically determine zero-lag ones; moreover, the performed zero-lag correlation analysis is easier to implement and computationally eﬃcient. DISCUSSION The main results of this work can be summarized as follows: (a) the brain and body subnetworks of the human physiological network exhibit signiﬁcant degrees of internal and reciprocal interaction; (b) internal interactions (brain–brain and body– body) are predominant, conﬁrming the existence of signiﬁcantly correlated variations in the amplitude of the diﬀerent brain waves on one side (Lin et al., 2020), and of cardiovascular and cardiorespiratory interactions on the other side (Porta et al., 2012; Schulz et al., 2013); (c) cardiorespiratory interactions are the predominant form of interaction within the analyzed body subnetwork, and decrease signiﬁcantly during sustained attention (and less evidently during mental stress); (d) brain– brain interactions are sustained by a quite consistent topological structure, and are signiﬁcantly stronger in the frontal scalp areas during mental stress; (e) brain–body interactions are weaker than within-subnetwork interactions, but are often statistically signiﬁcant and are modulated by the physiological state, being stronger during the mental stress task and weaker during the sustained attention task. Our results suggest the presence of strong interactions within and between the brain and body subnetworks which vary according to the stress level elicited by the adopted protocol, as highlighted by the analysis of the MI measure R (X; Y) (Figure 2). This ﬁnding is in line with those of several investigations in Considering the interactions of the processes belonging to the brain subnetwork (Figure 6), our results highlight a marked increase in the frontal region occurring during the mental arithmetic task (but not during the attention task) for the links involving the δ, θ, and α EEG power time series. This ﬁnding February 2021 \| Volume 14 \| Article 602584 13 Multivariate Correlation and Brain–Body Networks Pernice et al. supports from the point of view of connectivity between diﬀerent brain rhythms the well-known fact that mental arithmetic tasks and operations with numbers produce an activation of speciﬁc prefrontal cortical areas (Inouye et al., 1993; Menon, 2010; Arsalidou and Taylor, 2011; Friedrich and Friederici, 2013). Frontiers in Neuroscience \| www.frontiersin.org DISCUSSION While instantaneous or-single lag interactions are the basis of the main studies in the ﬁeld of network physiology (Bashan et al., 2012; Lin et al., 2020), in this study, we have extended their investigation to the multivariate case, allowing the study of interactions between groups of sub-systems (through the g y py The analysis of brain–heart interactions, evidenced particularly by the network topology in Figure 9, documented an increased connectivity between the brain and body subnetworks during mental stress (especially in the frontal scalp areas), and a reduction during sustained attention when conditions of isolation of the two sub-networks were often encountered (e.g., at electrodes AF3, F7, P8, and O2). The increased brain–body connectivity during the mental arithmetic task is likely related to the widely studied compensatory responses co-occurring in the central and ANSs to the internal and environmental stimuli evoked by stress (see, e.g., Silvani et al., 2016 for a review on the topic). As regards the use of multivariate time series analysis techniques, ﬁndings similar to those reported here were obtained performing a dynamic analysis based on Granger causality in Zanetti et al. (2019a); moreover, stronger bidirectional interactions between brain and heart dynamics were reported during emotional elicitation (Greco et al., 2019). As regards the nature of brain–body interactions, we ﬁnd that those occurring more frequently are involving the variability of the heart period and of the β EEG waves. This ﬁnding is in February 2021 \| Volume 14 \| Article 602584 14 Multivariate Correlation and Brain–Body Networks Pernice et al. MI measures involving blocks of variables) and the distinction between direct and indirect/mediated connections (through conditional MI measures). Our results document how this approach leads to describe exhaustively not only the interactions occurring between diﬀerent subnetworks (brain–body), but also those occurring internally in a subnetwork (brain or body). DISCUSSION here performed also through a direct comparison with more sophisticated analysis techniques including the use of time- delayed techniques employing tools of information dynamics to retrieve directional information (Faes et al., 2014) and of non- linear model free entropy estimators (Faes et al., 2015a); (b) the frequency-speciﬁc decomposition of the proposed measures (e.g., following Faes et al., 2020) to investigate how MIs can reﬂect oscillatory rhythms with speciﬁc physiological meaning; and (c) the analysis on source-reconstructed signals to obtain better anatomically-localized estimates of the strength and topology of brain–body interactions (Lai et al., 2018; Van de Steen et al., 2019; Kotiuchyi et al., 2020). The main limitation of the current study consists in the fact that the analysis of EEG signals has been carried out on a scalp- level, as previously stated in Section “Time Series Extraction.” We are aware that recent studies have highlighted that particular care should be assumed making inferences about brain regions since EEG scalp level connectivity does not permit a perfectly reliable interpretation of interacting brain areas as they can be corrupted by volume conduction eﬀects or by confounding factors (Lai et al., 2018; Reid et al., 2019; Van de Steen et al., 2019). However, neural time series obtained starting from the oscillations recorded on the scalp—even if aﬀected by confounding factors—can still represent a starting point for estimating brain network interactions (Reid et al., 2019). From this point of view, the analysis carried out in this work represents a ﬁrst step to be conﬁrmed in the future using source-reconstructed signals (Van de Steen et al., 2019), or even exploiting frameworks for the computation of source connectivity measures directly from scalp recordings (Kotiuchyi et al., 2020). Other limitations of the current study consist in the relatively small number of subjects analyzed, in the possibility of a not so-clear distinction between the elicited level stress evoked by GAME and MENTAL situations which may aﬀect the obtained results and in the fact that blood pressure was not acquired on the subjects, which could give additional useful physiological indications. ETHICS STATEMENT The studies involving human participants were reviewed and approved by “Comitato Etico per la Sperimentazione con l’essere vivente dell’Università degli Studi di Trento”, via Calepina 14, 38122 Trento, Italy. The patients/participants provided their written informed consent to participate in this study. Frontiers in Neuroscience \| www.frontiersin.org FUNDING RP was supported by the Italian MIUR PON R&I 2014–2020 AIM project no. AIM1851228-2. LF and YA were supported by the Italian MIUR PRIN 2017 project 2017WZFTZP “Stochastic forecasting in complex systems”. The data acquisition and experimental setting was partially ﬁnanced by AUSILIA (co- funded project University of Trento, APSS Trento and Provincia Autonoma di Trento). Future developments consist in the implementation of a more complete protocol able to elicit other diﬀerent levels of mental stress to better investigate on the changes in the strength of the interactions between brain and peripheral subnetworks. Such protocol should also include intermediate resting phases between stressful situations to assess whether elicited stress still produces eﬀects during time in a consequent resting phase. Future methodological work is also envisaged regarding: (a) a thorough validation on simulations of the MI measures presented DATA AVAILABILITY STATEMENT The data are publicly available at the following link: http://www.lucafaes.net/its.html (“stress data for the scripts Example_BrainBodyStress”). CONCLUSION LF contributed to conceptualization and supervision. LF, RP, and DM contributed to methodology. RP and LF contributed to software and validation. MZ and GN contributed to data curation. RP and YA contributed to writing—original draft preparation. LF and AB contributed to writing—review and editing. RP, DM, and YA contributed to visualization. GN contributed to funding acquisition. All authors have read and agreed to the published version of the manuscript. The aim of this work was to extend the analysis of functional brain–body interactions based on simple correlation tools to the multivariate case, allowing to dissect such interactions into contributions originated within and between the two physiological districts. Taken together, the proposed measures of “MI” elicit transitions across diﬀerent physiological states as well as spatial features, and constitute a tool easy to implement and with low computational cost. Practical and clinical applications of this tool range from a better understanding of the links and working principles of central and autonomic neural regulation (Silvani et al., 2016), or of the physiological mechanisms underlying stressful conditions (Dimsdale, 2008), to the real-time and automatic classiﬁcation in real-life scenarios using non- invasive or wearable devices (Jovanov, 2019; Pernice et al., 2019c; Vinciguerra et al., 2019). REFERENCES Cao, J., and Worsley, K. (1999). 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Frontiers in Neuroscience \| www.frontiersin.org February 2021 \| Volume 14 \| Article 602584 APPENDIX In this Appendix, we report the derivation of the alternative deﬁnitions of squared correlation for two scalar variables (Eqs 2 and 3) and their generalization in the case of vector variables (Eq. 5), and we draw the connection between the interaction measures and information measures. information measures. In the case of scalar random variables x and y, the covariance of W = [xy] is the 2 × 2 matrix In the case of scalar random variables x and y, the covariance of W = [xy] is the 2 × 2 matrix 6W = 6x 6x;y 6y;x 6y , (A.1) 6W = 6x 6x;y 6y;x 6y , (A.1) [xy] = \|6W\| = 6x6y −62 x;y. Then, Eq. 2 follows easily inserting 62 x;y = 6x6y −\|6W\| in [ y] ;y ;y Eq. 1. Moreover, relating x and y through the linear regression model x = ay + b + u, under the typical assumptions that u has zero mean and is uncorrelated with y, the variance of x and the covariance between x and y can be written, respectively, as 6x = E (x −mx)2 = a26y + 6u, and 6x;y = E[(x −mx)(y −my)] = a6y, which combined together yield 62 x;y = 6x6y − 6u. This latter expression inserted into Eq. 1 yields Eq. 3. [ ] y y Eq. 1. Moreover, relating x and y through the linear regression model x = ay + b + u, under the typical assumptions that u has zero mean and is uncorrelated with y, the variance of x and the covariance between x and y can be written, respectively, as 6x = E (x −mx)2 = a26y + 6u, and 6x;y = E[(x −mx)(y −my)] = a6y, which combined together yield 62 x;y = 6x6y − 6u. This latter expression inserted into Eq. 1 yields Eq. 3. APPENDIX (A.3) (A.3) In the multivariate case when the jointly Gaussian vector variables X and Y are considered, the relations become H (X) = 0.5 ln (2πe)P\|6X\| and H (X\|Y) = 0.5 ln (2πe)P\|6X\|Y\| , which similarly yield In the multivariate case when the jointly Gaussian vector variables X and Y are considered, the relations become H (X) = 0.5 ln (2πe)P\|6X\| and H (X\|Y) = 0.5 ln (2πe)P\|6X\|Y\| , which similarly yield I (X; Y) = H (X) −H (X\|Y) = 0.5 ln \|6X\| \|6X\|Y\| = −0.5 ln 1 −ρ2 (X; Y) . (A.4) (A.4) Therefore, under the hypothesis of joint Gaussianity of X and Y the MI measure of Eq. 6 is equivalent, up to a factor of two, to the mutual information I (X; Y) between the two variables, i.e., R (X; Y) = 2I (X; Y), and the generalized variances appearing in Eq. 7 are related to the entropy H (X) and to the conditional entropy of H (X\|Y) via the equations V (X) = 2H (X) −Pln2πe and V (X\|Y) = 2H (X\|Y) −Pln2πe (Faes et al., 2015c, 2017b). These relations extend to all measures deﬁned in the following in the main text (Eqs 8–11); for instance, in the Gaussian case, the measure of direct interaction between two scalar variables conditioned on all other variables (Eq. 11) takes the form of a conditional mutual information, i.e., I (zi; zj\|Z\{zi, zj}) = H zj Z\{zi, zj − H zj Z\{zj} = 0.5V zj Z\{zi, zj −0.5V zj Z\{zj} = 0.5R(zi; zj\|Z\ {zi, zj}). Therefore, under the hypothesis of joint Gaussianity of X and Y the MI measure of Eq. 6 is equivalent, up to a factor of two, to the mutual information I (X; Y) between the two variables, i.e., R (X; Y) = 2I (X; Y), and the generalized variances appearing in Eq. 7 are related to the entropy H (X) and to the conditional entropy of H (X\|Y) via the equations V (X) = 2H (X) −Pln2πe and V (X\|Y) = 2H (X\|Y) −Pln2πe (Faes et al., 2015c, 2017b). These relations extend to all measures deﬁned in the following in the main text (Eqs 8–11); for instance, in the Gaussian case, the measure of direct interaction between two scalar variables conditioned on all other variables (Eq. APPENDIX Extending the above derivation to the multivariate case, the vector variables X and Y are related through the multivariate linear regression X = YA + B + U from which, assuming mU = 0 and E YTU = 0, the covariance of X and the cross-covariance between Y and X can be written, respectively, as 6X = E (X −mX)T(X −mX) = AT6YA + 6U and 6Y;X = E (Y −mY)T(X −mX) = 6YA, which combined together yield 6U ≡6X −6X;Y6−1 Y 6Y;X. Moreover, considering that the covariance matrix of the overall variable Z = [XY] is a block matrix with form 6Z = 6[X,Y] = 6X 6X;Y 6Y;X 6Y , (A.2) (A.2) its determinant can be obtained using the block determinant identity (Horn and Johnson, 2012) as 6[X,Y] = \|6Y\| 6X −6X;Y6−1 Y (6X;Y)T = \|6Y\| \|6U\|. This last expression leads easily to recover Eq. 5 from the deﬁnition of multivariate correlation of Eq. 4. its determinant can be obtained using the block determinant identity (Horn and Johnson, 2012) as 6[X,Y] = \|6Y\| 6X −6X;Y6−1 Y (6X;Y)T = \|6Y\| \|6U\|. This last expression leads easily to recover Eq. 5 from the deﬁnition of multivariate correlation of Eq. 4. Finally, we note that both the classic squared correlation and its multivariate extension proposed here have a link with information- theoretic measures when the observed variables have a joint Gaussian distribution. In fact, it is well known that, for scalar Gaussian variables, the variance of x is related to the entropy by the equation H (x) = 0.5 ln (2πe6x), and the partial variance of x given y is related to the conditional entropy of x given y by the equation H x\|y = 0.5 ln 2πe6x\|y (Faes et al., 2017a); as a consequence, the squared correlation between x and y is related to the mutual information by the equation I x; y = H (x) −H x\|y = 0.5 ln 6x 6x\|y = −0.5 ln 1 −ρ2 x; y . REFERENCES This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Varga, S., and Heck, D. H. (2017). Rhythms of the body, rhythms of the brain: respiration, neural oscillations, and embodied cognition. Conscious. Cogn. 56, 77–90. doi: 10.1016/j.concog.2017.09.008 February 2021 \| Volume 14 \| Article 602584 Frontiers in Neuroscience \| www.frontiersin.org 18 Multivariate Correlation and Brain–Body Networks Pernice et al. Frontiers in Neuroscience \| www.frontiersin.org APPENDIX 11) takes the form of a conditional mutual information, i.e., I (zi; zj\|Z\{zi, zj}) = H zj Z\{zi, zj − H zj Z\{zj} = 0.5V zj Z\{zi, zj −0.5V zj Z\{zj} = 0.5R(zi; zj\|Z\ {zi, zj}). February 2021 \| Volume 14 \| Article 602584 Frontiers in Neuroscience \| www.frontiersin.org 19
https://openalex.org/W3101462611	http://europepmc.org/articles/pmc3982566?pdf=render	English	null	Comment on "Force-field functor theory" [arXiv:1306.4332]	arXiv (Cornell University)	2,013	cc-by	1,399	A commentary on BPA? One can estimate this by consider- ing a single (N = 1) quantum harmonic oscillator, V(ˆq) = κˆq2/2, whose frequency is ω ≡√κ/m. Expanding V(q + ξ) in Equation (3) one ﬁnds Force-ﬁeld functor theory: classical force- ﬁelds which reproduce equilibrium quan- tum distributions by Babbush, R., Parkhill, J., and Aspuru- Guzik, A. (2013). Front. Chem. 1:26. doi: 10.3389/fchem.2013.00026 Force-ﬁeld functor theory: classical force- ﬁelds which reproduce equilibrium quan- tum distributions by Babbush, R., Parkhill, J., and Aspuru- Guzik, A. (2013). Front. Chem. 1:26. doi: 10.3389/fchem.2013.00026 WBPA(q) = ∞ n = 0 1 n! d2nV(q) dq2n β¯h2 12m n = V(q) + β(¯hω)2 12 , (4) This comment regards a recent paper by Babbush, Parkhill, and Aspuru- Guzik (henceforth BPA) (Babbush et al., 2013). The subject is the equilibrium thermodynamics of a system of many quantum particles with Hamiltonian ˆH = N i = 1 ˆp2 i /(2m) + V(ˆq), where ˆq = {ˆqi} and the commutator of the coordinate- and momentum operators is [ˆqi, ˆpj] = i¯hδij. In BPA it is correctly observed (see, e.g., Hillery et al., 1984; Cuccoli et al., 1995) that the coordinate distribution function at temperature T = β−1, namely (4) which does not improve upon the clas- sical result using Equation (2). From the known density for the quantum harmonic oscillator the exact functor for the class of harmonic potentials can be easily derived: V(q) = mω2 2 q2 −→ W(q) = 1 2β ln sinh β¯hω β¯hω + mω β¯h tanh β¯hω 2 q2. (5) η(q) = 1 Z ⟨q\|e−β ˆH\|q⟩ ≡1 Z m 2π¯h2β N/2 e−βW(q), (1) (5) WBPA(q) = V(q) −1 β ⟨U[r(τ)]⟩ = β¯h 0 dτ β¯h V[r(τ)] = dNξ (2πσ2) N 2 V(q + ξ) e−ξ2 2σ2 ,(3) For a linear functor, this expression should be proportional to mω2: evidently this is true only in the classical limit, β¯hω ≪1 or T ≫¯hω, where Equation (4) is recovered. However, the mapping V →W can surely be locally linear, namely V + εδV →W + εδW with δW independent of the small parameter ε. GENERAL COMMENTARY published: 24 December 2013 doi: 10.3389/fchem.2013.00034 Keywords: quantum thermodynamics, effective classical potential, path integration, Monte Carlo simulations, force-ﬁeld functor Keywords: quantum thermodynamics, effective classical potential, path integration, Monte Carlo simulations, forc It is convincingly proven in BPA that the mapping η(q) ↔W(q) (i.e., the exponential function) is bijective, as well as V(q) ↔η(q), and then it fol- lows that V(q) ↔W(q) is one-to-one. Furthermore, it is correctly pointed out that the Giachetti–Tognetti–Feynman– Kleinert (Giachetti and Tognetti, 1985; Feynman and Kleinert, 1986) (GTFK) effective potential Veff(q) differs from W(q). Indeed, Veff(q), which accounts exactly for any quadratic potential, entails that for approximating ⟨O(ˆq)⟩one has to include a further Gaussian average accounting for purely-quantum ﬂuctua- tions, as shown, e.g., in Vaia and Tognetti (1990); Cuccoli et al. (1995): there W(q) is also introduced and dubbed the local effective potential. However, in BPA it is not shown that Equation (29), the paper’s main result obtained as the Jensen’s approximation W(q) ≈WBPA(q) to the exact formula (26), can be calculated explicitly as Ruggero Vaia* Istituto dei Sistemi Complessi - Consiglio Nazionale delle Ricerche and Istituto Nazionale di Fisica Nucleare - Sezione di Firenze, Sesto Fiorentino, Italy *Correspondence: ruggero.vaia@isc.cnr.it Edi d b Edited by: d ted by Francesco Aquilante, Uppsala University, Sweden A commentary on Hence, WBPA(q) is reliable only when the temperature overcomes the typical quantum energy scale ¯hω; for instance, taking ω2 ∼V′′(qm)/m [qm being the minimum of V(q)], a pair of hydrogen molecules has typically ¯hω ∼ 102 K (Vaia and Tognetti, 1990 and refer- ences therein) and using WBPA(q) would only be reliable at very high T ≫102 K, i.e., just in the classical limit. Such an can be used to deﬁne the effective poten- tial W(q) (the kinetic part of the partition function Z is omitted in BPA) in terms of which the exact quantum equilibrium average of any operator O(ˆq) takes the classical form of a conﬁguration integral, i.e., the convolution between the potential V(q) and a Gaussian with variance σ2 = β¯h2/(6m) proportional to the squared de- Broglie wavelength. This is in agreement with the Wigner series (Wigner, 1932) up to lowest order, but lacks the nonlinear contributions to it. How accurate is the approximation made in Equation (29) of O(ˆq) = dq⟨q\|O(ˆq)e−β ˆH\|q⟩ = 1 Z m 2π¯h2β N/2 dq O(q) e−βW(q). (2) O(ˆq) = (2) December 2013 \| Volume 1 \| Article 34 \| 1 www.frontiersin.org Commentary on “Force-ﬁeld functor theory” Vaia and the quantum effective pair potential. J. Chem. Phys. 81, 2523–2527. doi: 10.1063/1.447985 and the quantum effective pair potential. J. Chem. Phys. 81, 2523–2527. doi: 10.1063/1.447985 approximation is indeed used in the high- T propagator of path-integral Monte Carlo algorithms in order to improve conver- gence in the Trotter number (Takahashi and Imada, 1984). Hence, the approxima- tion (29) of BPA can “reproduce quantum distributions” just when these are almost classical. Babbush, R., Parkhill, J. A., and Aspuru-Guzik, A. (2013). Force-ﬁeld functor theory: classi- cal force-ﬁelds which reproduce equilibrium quantum distributions. Front. Chem. 1:26. doi: 10.3389/fchem.2013.00026 Vaia, R., and Tognetti, V. (1990). Effective poten- tial for two-body interactions. Int. J. Modern Phys. B 4, 2005–2023. doi: 10.1142/S021797929 0001005 Wigner, E. P. (1932). On the quantum correction for thermodynamic equilibrium. Phys. Rev. 40, 749–759. doi: 10.1103/PhysRev.40.749 Cuccoli, A., Giachetti, R., Tognetti, V., Vaia, R., and Verrucchi, P. (1995). The effective poten- tial and effective Hamiltonian in quantum sta- tistical mechanics. J. Phys. 7, 7891–7938. A commentary on doi: 10.1088/0953-8984/7/41/003 On the other hand, the use of the exact effective pair-potential, rather than that obtained from Equation (29) of BPA, is a good starting point for treating a not too dense quantum ﬂuid by means of a classical-like simulation, as shown in the last section of BPA and as noted by sev- eral authors (see, e.g., Thirumalai et al., 1984 and many references cited in BPA). At variance with the procedure of BPA, based on the heavy calculation of a (locally) “linear functor” at ﬁxed T, it would be more practical to directly obtain the exact pair-potential W(q) for the chosen V(ˆq), a task that can easily be carried out at any T. Received: 29 October 2013; accepted: 23 November 2013; published online: 24 December 2013. Feynman, R. P., and Kleinert, H. (1986). Effective classical partition functions. Phys. Rev. A 34, 5080–5084. doi: 10.1103/PhysRevA.34.5080 Citation: Vaia R (2013) Commentary on “Force-ﬁeld functor theory: classical force-ﬁelds which reproduce equilibrium quantum distributions.” Front. Chem. 1:34. doi: 10.3389/fchem.2013.00034 Giachetti, R., and Tognetti, V. (1985). Variational approach to quantum statistical mechanics of nonlinear systems with application to sine- Gordon chains. Phys. Rev. Lett. 55, 912–915. doi: 10.1103/PhysRevLett.55.912 This article was submitted to Theoretical and Computational Chemistry, a section of the journal Frontiers in Chemistry. Hillery, M., O’Connell, R. F., Scully, M. O., and Wigner, E. P. (1984). Distribution functions in physics: fundamentals. Phys. Rep. 106, 121–167. doi: 10.1016/0370-1573(84)90160-1 Copyright © 2013 Vaia. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accor- dance with accepted academic practice. No use, distribu- tion or reproduction is permitted which does not comply with these terms. Copyright © 2013 Vaia. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accor- dance with accepted academic practice. No use, distribu- tion or reproduction is permitted which does not comply with these terms. Takahashi, M., and Imada, M. (1984). Monte Carlo calculation of quantum systems. II. Higher order correction. J. Phys. Soc. Jpn. 5381, 3765–3769. December 2013 \| Volume 1 \| Article 34 \| 2 Frontiers in Chemistry \| Theoretical and Computational Chemistry A commentary on doi: 10.1143/JPSJ.53.3765 Thirumalai, D., Hall, R. W., and Berne, B. J. (1984). A path integral Monte Carlo study of liquid neon December 2013 \| Volume 1 \| Article 34 \| 2 Frontiers in Chemistry \| Theoretical and Computational Chemistry Frontiers in Chemistry \| Theoretical and Computational Chemistry
https://openalex.org/W3033972602	https://link.springer.com/content/pdf/10.1007%2F978-3-030-43582-0_5.pdf	English	null	Output as Prophecy	Springer eBooks	2,020	cc-by	7,956	CHAPTER 5 © The Author(s) 2020 T. D. Grant and D. J. Wischik, On the path to AI, https://doi.org/10.1007/978-3-030-43582-0_5 © The Author(s) 2020 Output as Prophecy In the preceding chapter we considered some of the purposes to which machine learning might be put—for example, to predict the topic of a court case given words that the judge used in the court’s written judg- ment—and we described pattern ﬁnding as the method behind prediction. More important than the method however is the goal, in this example “to predict the topic,” and in particular the keyword predict. We introduced that word in the preceding chapter to begin to draw attention to how computer scientists use it when they engineer machine learning systems. In machine learning systems, predictive accuracy is the be-all and end- all—the way to formulate questions, the basis of learning algorithms, and the metric by which the systems are judged. In this chapter we consider prediction, both in Holmes’s view of law and in the machine learning approach to computing. In Holmes’s view of law, prediction is central. His answer to the ques- tion, What constitutes the law? has become one of the most famous epi- grams in all of law: The prophecies of what the courts will do in fact, and nothing more pre- tentious, are what I mean by the law.1 Holmes’s interest in the logic and philosophy of probability and statis- tics has come more to light thanks to recent scholarship2; he immersed 49 T. D. GRANT AND D. J. WISCHIK 50 himself early in his career in those subjects. Holmes’s use of the word “prophecy” was deliberate. It accorded with his overall view of law by getting away from the scientiﬁc and rational overtones of “prediction,” even as he used that word too. Arguably, given how elusive explanations have been of how machine learning systems arrive at the predictions they make, “prophecy” is a good term in that context too. We expand in this chapter on Holmes’s idea that prophecies constitute the law, and then we return to prediction in machine learning. 5.1 Prophecies Are What Law Is Holmes’s famous epigram has been widely repeated, but it is not widely understood. Taken in isolation from The Path of the Law, where Holmes set it down, and in isolation from Holmes’s development as a thinker, it might sound like no more than a piece of pragmatic advice to a practicing lawyer: don’t get carried away by the cleverness of your syllogisms; ask yourself, instead, what the judge is going to do in your client’s case. If that is all it meant, then it would be good advice, but it would not be a concept of law. Holmes had in mind a concept of law. The epigram needs to be read in context: The confusion with which I am dealing besets confessedly legal concep- tions. Take the fundamental question, What constitutes the law? You will ﬁnd some text writers telling you that it is something different from what is decided by the courts of Massachusetts or England, that it is a system of reason, that it is a deduction from principles of ethics or admitted axioms or what not, which may or may not coincide with the decisions. But if we take the view of our friend the bad man we shall ﬁnd that he does not care two straws for the axioms or deductions, but that he does want to know what the Massachusetts or English courts are likely to do in fact. I am much of this mind. The prophecies of what the courts will do in fact, and nothing more pretentious, are what I mean by the law.3 Holmes was contrasting “law as prophecy” to “law as axioms and deductions.” He saw an inductive approach to law—the pattern ﬁnding approach that starts with data or experience—not just to improve upon or augment legal formalism. He saw it as a corrective. The declaration in his dissent in the Lochner case a few years after The Path of the Law that “[g]eneral propositions do not decide concrete cases”4 was not just to 5 OUTPUT AS PROPHECY 51 say that the formal, deductive approach is insufﬁcient; it was to say that formalism gets in the way. The centrality of concern for Holmes was the reality of decision, the output that a court might produce. The realism or positivism in this understanding of law contrasted with the formalist school that had long prevailed. 5.1 Prophecies Are What Law Is To shift the concern of lawyers in this way was to lessen the role of doctrine, of formal rules, and to open a vista of social and histor- ical considerations heretofore not part of the law school curriculum and ignored, or at any rate not publicly acknowledged, by lawyers or judges. Jurists have been divided ever since as to whether the shift of conception was for better or worse. Whatever one’s assessment of it, the concept of law as Holmes expressed it continues to inﬂuence the law. p There is more still to Holmes’s epigram about prophecies. True, the contrast it entails between the inductive method and the deductive method alone has revolutionary implications. But Holmes was not merely concerned with what method “our friend the bad man” (or indeed the bad man’s lawyer) should employ to predict the outcome of a case. He wasn’t writing a law practice handbook. He was interested in individual encounters with the law to be sure,5 but this was because he sought to reach a general understanding of law as a system. Holmes’s invocation of prophecies, like his use of terms from logic and mathematics, was memo- rable use of language, but it was more than rhetoric: it was at the core of Holmes’s deﬁnition of law. He referred to the law as “systematized predic- tion.”6 This was to apply the term “prediction” broadly—indeed across the legal system as a whole. Holmes was not sparing in his use of the word “prophecy” when deﬁning the law. The word “prophesy” or its derivates appear nine times in The Path of the Law.7 He used it in the same sense when writing for the Supreme Court.8 Holmes’s concern with prediction is traceable in his other writings too.9 The heart of Holmes’s insight, and what has so affected jurisprudence since, is that the law is prediction.10 Prophecy does not refer solely to the method for predicting what courts will do. Prophecy is what constitutes the law. Prophecy of what, by whom, and on the basis of which input data? Holmes gave several illustrations. 5.1 Prophecies Are What Law Is For example, he famously described the law of contract as revolving around prediction: “The duty to keep a contract at common law means a prediction that you must pay damages if you do not keep it, and nothing else.”11 He stated his main thesis in similar terms: “a legal duty so called is nothing but a prediction that if a man does or omits certain things he will be made to suffer in this or that T. D. GRANT AND D. J. WISCHIK 52 way by judgment of the court.”12 This is a statement about “legal duty” irrespective of the content of the duty. It thus just as well describes any duty that exists in the legal system. We think that Holmes’s concept of law as prediction indeed is comprehensive. Many jurists don’t see it that way. Considering how Holmes understood law to relate to decisions taken by courts, one sees why his concept of law-as-prophecy often has received a more limited interpretation. Holmes wrote that “the object of [studying law], then, is prediction, the prediction of the incidence of the public force through the instrumen- tality of the courts.”13 Making the equation directly, he wrote, “Law is a statement of the circumstances, in which the public force will be brought to bear upon men through the courts….”; a “word commonly conﬁned to such prophecies… addressed to persons living within the power of the courts.”14 It is often assumed that Holmes’s description here does not account for the decisions of the highest courts in a jurisdiction, courts whose decisions are ﬁnal. After all, in a system of hierarchy, the organ at the apex expects its commands to be obeyed. To call decisions that emanate from such quarters “predictions” seems to ignore the reality of how a court system works. In a well-functioning legal system, a judgment by a ﬁnal court of appeal saying, for example, that the police are to release such and such a prisoner, should lead almost certainly to that outcome. The court commands it; the prisoner is released. In two respects, however, one perhaps trivial but the other assuredly signiﬁcant, the highest court’s statements, too, belong to the concept of law as prophecy. First, even in a well-functioning legal system, the court’s decision is still only a prediction. 5.1 Prophecies Are What Law Is As outlandish as the situation would be in which the police ignored the highest court, it is a physical possibility. A statisti- cian might say that the probability is very high (say, 99.9999%) that the highest court’s judgment that the law requires the prisoner to be released will in fact result in an exercise of public power in accordance with that judgment. We will say more below about the relation between probabil- ity and prediction.15 Leaving that relation aside for the moment, a judg- ment even of the highest court is a prediction in Holmes’s sense. It is a prediction in this way: the implementation of a judgment by agents of public power is an act of translation, and in that act the possibility exists for greater or lesser divergence from the best understanding of what the judge commanded. So the deﬁnition of law as prophecy is instanced in 5 OUTPUT AS PROPHECY 53 the chance that the “public force” will not properly implement the judi- cial decision. In a well-functioning legal system, the chance is remote. In legal systems that don’t function well, the predictive character of ﬁnal judgments is more immediate, because the risk in those systems is greater that the public force will not properly implement the courts’ commands. “Finality” in some judicial systems is more formal than real.16 The further respect in which the concept of law as prophecy is com- prehensive comes to view when we consider how judges decide cases and how advocates argue them. In deciding a case, a judge will have in mind how that decision is likely to be interpreted, relied upon, or rejected, by future courts and academics and public opinion, as well as by the instru- ments of public force. The barrister, for her part, in deciding what line of argument to pursue, will have in mind how the judge might be inﬂu- enced, and this in turn requires consideration of the judge’s predictions about posterity. 5.1 Prophecies Are What Law Is Holmes thus described a case after the highest court had decided it as still in the “early stages of law.”17 As Kellogg puts it, Holmes situated a case “not according to its place on the docket but rather in the continuum of inquiry into a broader problem.”18 The law is a self-referential system, whose rules and norms are conse- quences of predictions of what those rules and norms might be.19 Some people participate in the system in a basic and episodic way, for example the “bad man” who simply wants advice from a solicitor about likely out- comes in respect of his situation. Some people participate in a formative way. The apex example is the judge of the ﬁnal court of appeal whose pre- dictions about the future of the legal system are embodied in a judgment which she expects as a consequence of her authority in the legal system to be a perfect prediction of the exercise of public power in respect of the case. But her outlook, indeed her self-regard as a judge, entails more than that; a judge does more than participate in disconnected episodes of judging: she hopes that any judgment she gives in a case, because she strives for judgments that withstand the test of time, will be a more or less accurate prediction of how a future case bearing more or less likeness to the case will be decided. The judge describes her judgment as command, not as prophecy; but the process leading to it, and the process as the judge hopes it will unfold in the future, is predictive. Law-as-prophecy, understood this way, has no gap. Holmes’s claim, as we understand it, holds law to be predictive through and through. Prophecy is what law is made of. The predictive character of law, in this constitutive sense, is visible in the process of judicial decision, T. D. GRANT AND D. J. WISCHIK 54 regardless what level of the judiciary is deciding; and it is visible in all other forms of legal assertion as well. Prophecy embraces all parts of legal process. So everyone who touches the law is making predictions, from the self- interested “bad man” to the judge in the highest court of the land, and they make predictions about the full range of possible outcomes. 5.1 Prophecies Are What Law Is The experience that inﬂuences their predictions, as we saw in Chapter 3,20 Holmes understood to be wide, and the new situations that they make predictions about are unlimited. People on Holmes’s path of the law thus engage in tasks much broader than standard machine learning tasks. As we also discussed in Chapter 3,21 machine inputs, while they consist in very large datasets (“Big Data”), are limited to inputs that have been imparted a considerable degree of structure—a degree of structure almost certainly lacking in the wider (and wilder) environment from which expe- rience might be drawn. Machine outputs are correspondingly limited as well. Let us now further explore the machine learning side of the anal- ogy—and its limits. 5.2 Prediction Is What Machine Learning Output Is Holmes, writing in 1897, obviously did not have machine learning in mind. Nevertheless, his idea that prophecy constitutes the law has remark- able resonance with machine learning, a mechanism of computing that, like law as Holmes understood it, is best understood as constituted by prediction. The word prediction is a term of art in machine learning. It is used like this: In a typical scenario, we have an outcome measurement, usually quantita- tive (such as a stock price) or categorical (such as heart attack/no heart attack), that we wish to predict based on a set of features (such as diet and clinical measurements). We have a training set of data, in which we observe the outcome and feature measurements for a set of objects (such as people). Using this data we build a prediction model, or learner, which will enable us to predict the outcome for new unseen objects. A good learner is one that accurately predicts such an outcome.22 55 5 OUTPUT AS PROPHECY Though in common parlance the term “prediction” means forecasts— that is to say, statements about future events—in machine learning the term has a wider meaning. We have touched on the wider meaning in Chapter 4 and at the opening of the present chapter. Let us delve a little more into that wider meaning now. It is true that some machine learning outputs are “prediction” in the sense in which laypersons typically speak: “Storm Oliver will make landfall in North Carolina”23 or “the stock price will rise 10% within six months.” Other outputs are not predictions in the layperson’s sense. Indeed, the main purposes for which machine learning is used do not involve pre- dictions of that kind—purposes like classifying court cases by topic or controlling an autonomous vehicle. Whatever the purposes for which it is used, machine learning involves “prediction” of the more general kind computer scientists denote with that term. The essential feature of prediction in machine learning is that it should concern “the outcome for new unseen objects,” i.e. for objects not in the training set. 5.2 Prediction Is What Machine Learning Output Is Thus, for example, if the training set consists of labelled photographs, and if we treat the pixels of the photograph as features and the label as the outcome, then it is prediction when the machine learning system is given a new photograph as input data and it outputs the label “kitten.” In machine learning prediction, “pre-” simply refers to before the true outcome measurement has been revealed to the machine learning system. The sense of “pre-” in “prediction” holds even though other par- ties might well already know the outcome. For example, the computer scientist might well already know that the new photograph is of a tiger, not a kitten. That assignment of label-to-picture has already happened, but the machine learning system has not been told about it at the point in time when the system is asked to predict. Philosophers of science use the terms “postdiction” or “retrodiction” to refer to predicting things that have already happened.24 These words are not used in the machine learning community, but the concept behind them is much what that community has in mind when it talks about prediction. A signiﬁcant part of the craft of machine learning is to formulate a task as a prediction problem. We have already described how labelling a photograph can be described as prediction. A great many other examples may be given. Translation can be cast as prediction: “predict the French version of a sentence, given the English text,” where the training set is a human-translated corpus of sentences. Handwriting synthesis can as well. Given a dataset of handwritten text, recorded as the movements of a pen T. D. GRANT AND D. J. WISCHIK 56 nib, and given the same text transcribed into text in a word processor, the task of handwriting synthesis can be cast as prediction: “predict the movements of a pen nib, given text from a word processor.” As Judea Pearl observed in the interview with which we opened Chapter 4,25 it is truly remarkable how many tasks can be formulated this way. In the social sciences, it is “a rather new epistemological approach […] and research agendas based on predictive inference are just starting to emerge.”26 A theory of law based on predictive inference, however, emerged over a century ago: Holmes theorized law to be constituted by prophecy. So too might we say that machine learning is constituted by prediction. 5.2 Prediction Is What Machine Learning Output Is Moreover, prediction is not just the way that machine learning tasks are formulated. It is also the benchmark by which we train and evaluate machine learning systems in the performance of their tasks. The goal of training is to produce a “good learner,” i.e. a system that makes accurate predictions. Training is achieved by measuring the difference between the machine’s predictions (or postdictions, as the philosophers would say) and the actual outcomes in the training dataset; and iteratively tweaking the machine’s parameter values so as to minimize the difference. The machine that reliably labels tigers as “tigers” has learned well and, at least for that modest task, needs no more tweaking. The machine that labels a tiger as a “kitten” needs tweaking. The one that labels a tiger as “the forests of the night,” though laudable if its task had been to predict settings in which tigers are found in the poetry of William Blake, needs some further tweaking still to perform the task of labeling animals. This process of iterative tweaking, as we noted in Chapter 2, is what is known as gradient descent,27 the backbone of modern machine learning. Thus, a mechanism of induction, not algorithmic logic, is at the heart of machine learning, much as Holmes’s “inductive turn” is at the heart of his revolutionary idea of law. It is not machine learning’s fundamental characteristic that it can be used to forecast future events—when will the next hurricane occur, where will it make landfall? One doesn’t need machine learning to make fore- casts. One can make forecasts about hurricanes and the like with dice or by sacriﬁcing a sheep (or by consulting a ﬂock of dyspeptic parrots). One can also make such forecasts with classic algorithms, by simulating dynam- ical systems derived from atmospheric science. This sort of prediction is not the fundamental characteristic of machine learning. 57 5 OUTPUT AS PROPHECY The fundamental characteristic of machine learning is that the system is trained using a dataset consisting of examples of input features and out- come measurements; until, through the process of gradient descent, the machine’s parameter values are so reﬁned that the machine’s predictions, when we give it further inputs, differ only minimally from the actual out- comes in the training dataset. 5.2 Prediction Is What Machine Learning Output Is Judges, litigants, and their lawyers certainly try to align their predictive statements of law with what they discern to be the relevant pattern in law’s input data, that is to say in the collective experience that shapes the law. It is equally the case, in Holmes’s under- standing of the law, that we do not test court judgments by comparing against stipulated “correct” labels the way our spam email or tiger detec- tor was tested. Judgments are, however, tested against future judgments. This is to the point we made earlier about the judge’s aim that her judg- ments withstand the test of time. The test is whether future judgments show her judgment to have been an accurate prediction, or at least not so far off as to be set aside and forgotten. A machine learning system must be trained on a dataset of input fea- tures and outcome measurements. This is in contrast to the classic algo- rithmic approach, which starts instead from rules. For example the clas- sic approach to forecasting the weather works by solving equations that describe how the atmosphere and oceans behave; it is based on scientiﬁc laws (which are presumably the result of codifying data from earlier exper- iments and observation). Just as machine learning rejects rules and starts instead with training data, Holmes rejected the idea that law is deriving outcomes based on general principles, and he cast it instead as a predic- tion problem—prophesying what a court will do—to be performed on the basis of experience. 5.3 Limits of the Analogy As we noted in Chapter 3,28 the predictions made by a machine learning system must have the same form as the outcomes in the training dataset, and the input data for the object to be predicted must have the same form as objects already seen. In earlier applications of machine learning, “same form” was very narrowly construed: for example, the training set for the ImageNet challenge29 consists of images paired with labels; the machine learning task is to predict which one of these previously seen labels is the best ﬁt for a new image, and the new image is required to be the same dimensions as all the examples in the training set. Human ability to T. D. GRANT AND D. J. WISCHIK 58 make predictions about novel situations is far ahead of that of machines. A human lawyer can extrapolate from experience and make predictions about new cases that don’t conform to a narrow deﬁnition of “cases simi- lar to those already seen.” The distance is closing, however, as researchers develop techniques to broaden the meaning of “same form.” For exam- ple, an image captioning system30 is now able to generate descriptions of images, rather than just repeat labels it has already seen. Thus, it is well within their grasp for machines to label an image as “tiger on ﬁre in a forest,” but they are still a long way, probably, from describing, as the poet did, the tiger’s “fearful symmetry.” There is a more signiﬁcant difference between predictions in machine learning and in law. In machine learning, the paradigm is that there is something for the learning agent—i.e., the machine—to learn. The thing to learn is data, something that is given, not a changing environment affected by numerous factors—including by the learning agent. A machine for translating English to French can be trained using a human-translated corpus of texts, and its translations can be evaluated by how well they match the human translation. Whatever translations the machine comes up with they do not alter the English nor French languages. In law, by contrast, the judgment in a case becomes part of the body of experience to be used in subsequent cases. Herein, we think, Holmes’s concept of law as a system constituted from prediction may hold lessons for machine learning. 5.3 Limits of the Analogy In Chapters 6–8, we will consider some challenges that machine learning faces, and possible lessons from Holmes, as we discuss “explain- ability” of machine learning outputs31 and outputs that may have invidi- ous effects because they reﬂect patterns that emerge from the data (such as patterns of racial or gender discrimination).32 In Chapter 9,33 we will suggest that Holmes, because he understood law to be a self-referential process in which each new prediction shapes future predictions, might point the way for future advances in machine learning. Before we get to the challenges of machine learning and possible lessons for the future from Holmes, we will brieﬂy consider a question that prediction raises: does prediction, whether as the constitutive ele- ment of law or as the output of machine learning, necessarily involve the assessment of probabilities? 5 OUTPUT AS PROPHECY 59 5.4 Probabilistic Reasoning and Prediction WISCHIK 60 Are legal predictions expressed in the language of probability? Lawyers serving clients do not always give probability assessments when they give predictions, but sometimes they do.42 Some clients need such an assess- ment for purposes of internal controls, ﬁnancial reporting, and the like. Others ask for it for help in strategizing around legal risk. Modern empir- ical turns in law scholarship, it may be added, are much concerned with statistics.43 Attaching a probability to a prediction of a legal outcome is an inexact exercise, but it is not unfamiliar to lawyers. Holmes, when he referred to the prophecies of what courts will do, is often read to mean that the law should be made readily predictable.44 Though we don’t doubt he preferred stable judges to erratic ones, we don’t see that that was Holmes’s point. Courts whose decisions are hard to predict are no less sources of legal decision. Even when the lawyer has the privilege to argue in front of a “good” judge, whom for present pur- poses we deﬁne as a judge whose decisions are easy to predict, the closer the legal question, the harder it is to predict the answer. It is inherent that lawyers will be more conﬁdent in some of their predictions than in others. Judges, practically by deﬁnition of their role as legal authorities, do not proffer a view as to the chances that their judgments are correct. It is hard to see how the process of judgment would keep the conﬁdence of society, if every judgment were issued with a p-value!45 Yet reading judgments through a realist’s glasses, one may discern indicia of how likely it is that the judgment will be understood in the future to have stated the law. Judges do not shy from describing some cases as clear ones; others as close ones. They don’t call it hedging, but that’s very much what it’s like. When a judge refers to how ﬁnely balanced such and such a question was, it has the effect of qualifying the judgment. It thus may be that one can infer from a judgment’s text how much conﬁdence one should have in the judgment as a prediction of future results. The text, even where it does not express anything in terms about the closeness of a case, still may give clues. 5.4 Probabilistic Reasoning and Prediction “For the rational study of the law the blackletter man may be the man of the present, but the man of the future is the man of statistics,” said Holmes.34 It is not certain that Holmes thought that the predictive char- acter of law necessarily entails a probabilistic character for law. He was certainly interested in probability. In the time after his Civil War service, a period that Frederic Kellogg closely examined in Oliver Wendell Holmes, Jr. and Legal Logic, Holmes studied theories of probability and was much engaged in discussions about the phenomenon, including how it relates to logic and syllogism.35 Later, as a judge, he recognized the part played by probability in commercial life, for example in the functioning of the futures markets.36 In personal correspondence, Holmes said that early in his life he had learned “that I must not say necessary about the universe, that we don’t know whether anything is necessary or not. So that I describe myself as a bettabilitarian. I believe that we can bet on the behavior of the universe…”37 Holmes would have been comfortable with the idea that law, in its character as prediction, concerned prob- ability as well. Some jurists indeed have discerned in Holmes’s idea of law-as-prophecy just such a link.38 Predictions made by machine learning are not inherently probabilistic. For example, the “k nearest neighbors”39 machine learning algorithm is simply “To predict the outcome for a new case, ﬁnd the k most similar cases in the dataset, ﬁnd their average outcome, and report this as the prediction.” The system predicts a value, which may or may not turn out to be correct. Modern machine learning systems such as neural networks, however, are typically designed to generate predictions using the language of probability, for example “the probability that this given input image depicts a kitten is 93%.”40 Separately, we can classify machine learning systems by whether or not they employ probabilistic reasoning to generate their predictions: [One type of] Machine Learning seeks to learn [probabilistic] models of data: deﬁne a space of possible models, learn the parameters and structure of the models from data; make predictions and decisions. [The other type of] Machine Learning is a toolbox of methods for processing data: feed the data into one of many possible methods; choose methods that have good theoretical or empirical performance; make predictions and decisions.41 T. D. GRANT AND D. J. 5.4 Probabilistic Reasoning and Prediction The structure of the reasoning may be a clue: the more complex and particularistic a judge’s reasoning, the more the judgment might be questioned, or at least limited in its future application. Textual clues permit an inference as to how conﬁdent one should be that the judgment accurately reﬂects a pattern in the experience that was the input behind it.46 Does the law use probabilistic reasoning to arrive at a prediction? In other words, once a judgment has been made and it becomes part of the 5 OUTPUT AS PROPHECY 61 body of legal experience, do lawyers and judges reason about their level of conﬁdence that an earlier judgment is relevant for their predictions about a current case? Ex post, every judgment is in fact, to a greater or lesser extent, questioned or rejected or ignored—or afﬁrmed or relied upon. Nulliﬁcation, reversal, striking down—by whatever term the legal system refers to the process, a rejection of a judgment by a controlling authority is a formal expression that the judge got it wrong.47 Endorsement, too, is sometimes formal and explicit, the archetype being a decision on appeal that afﬁrms the judgment. Formal and explicit signals, whether of rejec- tion or of reliance, entail a signiﬁcant adjustment in how much conﬁdence we should have in a judgment as a prediction of a future case. j g p It is not just in appeals that we look for signals as to how conﬁdent we should be in a given judgment as a prediction of future cases. Rejection or endorsement might occur in a different case on different facts (i.e., not on appeal in the same case) and in that situation is therefore only an approximation: ignoring, rejecting, or “distinguishing” a past judgment; or invoking it with approval, a judge in a different case says or implies that the judge in the past judgment had the law wrong or he had it right, but indirect treatment in the new judgment, whether expressed or implied, says only so much about the past one. A jurist, considering such indirect treatment, would struggle to arrive at a numerical value to adjust how much conﬁdence to place in the past judgment.48 In evidence about judg- ments—evidence inferable from the words of the judgments themselves and evidence contained in their reception—one nevertheless discerns at least rough markers of the probability that they will be followed in the future. 5.4 Probabilistic Reasoning and Prediction There is no received view as to what Holmes thought the function of probability is in prediction. As is the case with machine learning, jurists make probabilistic as well as non-probabilistic predictions. You can state the law—i.e., give a prediction about the future exercise of public power—without giving an assessment of your conﬁdence that your pre- diction is right. Jurists also use both probabilistic and non-probabilistic reasoning. Holmes, when referring to prophecies, was not however telling courts how to reason (or for that matter, legislatures or juries; we will return to juries in Chapter 7). His concern was to state what it is that con- stitutes the law. True, we don’t call wobbly or inarticulate judges good judges. But Holmes was explicitly not concerned with the behavior of the “good” litigant; and, in his thinking about the legal system as a whole, his concern was not limited to the behavior of the “good” judge. T. D. GRANT AND D. J. WISCHIK 62 Notes 1. Holmes, The Path of the Law, 10 Harv. L. Rev. 457, 461 (1896–1897). 2. See Kellogg (2018) op. cit. 1. Holmes, The Path of the Law, 10 Harv. L. Rev. 457, 461 (1896–1897) See Kellogg (2018) op. cit. 2. See Kellogg (2018) op. cit. 3. 10 Harv. L. Rev. at 460–61 (1896–1897). 4. Lochner, 198 U.S. 45, 76 (1905) (Holmes, J., dissenting). 5. Though legal advice was not what Holmes was giving, his writings supply ample material for that purpose, and so have judges sometimes read him: see, e.g., Parker v. Citimortgage, Inc. et al., 987 F.Supp.2d 1224, 1232 n 19 (2013, Jenkins, SDJ). 6. 10 Harv. L. Rev. at 458 (emphasis added). 7. The text, which runs to 21 pages (less than 10,000 words), contains the word “prophecy,” “prophecies,” or the verb “to prophecy” on ﬁve pages: 10 Harv. L. Rev. at 457, 458, 461, 463, and 475. 8. American Banana Company v. United Fruit Company, 213 U.S. 347, 357, 29 S.Ct. 511, 513 (Holmes, J., 1909). 9. Moskowitz emphasized this line of Holmes’s thought in The Prediction Theory of Law, 39 Temp. L.Q. 413, 413–16 (1965–1966). 10. 10 Harv. L. Rev. at 462. 11. Id. at 462. 12. Id. 12. Id. 13. Id. at 457. 14. American Banana Company, 213 U.S. at 357, 29 S.Ct. at 513. 15. This chapter, pp. 59–61. 15. This chapter, pp. 59–61. 16. See for example White, Putting Aside the Rule of Law Myth: Corrup- tion and the Case for Juries in Emerging Democracies, 43 Corn. Int’l L.J. 307, 321 n. 118 (2010) (reporting doubt whether Mongolia’s other branches of government submit to judicial decisions, notwithstanding the Constitutional Court’s formal power of review). The predictive character of judgments of interstate tribunals, in this sense, is pronounced. Judg- ments of the International Court of Justice, to take the main example, under Article 60 of the Court’s Statute are “ﬁnal and without appeal,” but no executive apparatus is generally available for their enforcement, and the Court has no ancillary enforcement jurisdiction. Even in the best-functioning legal system, high courts may have an uneasy relation to the executive apparatus whose conduct they sit in judgment upon. Recall Justice Frankfurter’s concurrence in Korematsu v. United States where, agreeing not to overturn wartime measures against persons of Japanese, German, and Italian ancestry, he declared “[t]hat is their [the Governmen- t’s] business, not ours”: 323 U.S. 17. Vegelahn v. Guntner & others, 167 Mass. 92, 106 (1896) (Field, C.J. & Holmes, J., dissenting). Notes 214, 225, 65 S.Ct. 193, 198 (1944) (Frankfurter, J., concurring). 16. See for example White, Putting Aside the Rule of Law Myth: Corrup- tion and the Case for Juries in Emerging Democracies, 43 Corn. Int’l L.J. 307, 321 n. 118 (2010) (reporting doubt whether Mongolia’s other branches of government submit to judicial decisions, notwithstanding the Constitutional Court’s formal power of review). The predictive character of judgments of interstate tribunals, in this sense, is pronounced. Judg- ments of the International Court of Justice, to take the main example, under Article 60 of the Court’s Statute are “ﬁnal and without appeal,” but no executive apparatus is generally available for their enforcement, and the Court has no ancillary enforcement jurisdiction. Even in the best-functioning legal system, high courts may have an uneasy relation to the executive apparatus whose conduct they sit in judgment upon. Recall Justice Frankfurter’s concurrence in Korematsu v. United States where, agreeing not to overturn wartime measures against persons of Japanese, German, and Italian ancestry, he declared “[t]hat is their [the Governmen- t’s] business, not ours”: 323 U.S. 214, 225, 65 S.Ct. 193, 198 (1944) (Frankfurter, J., concurring). 17. Vegelahn v. Guntner & others, 167 Mass. 92, 106 (1896) (Field, C.J. & Holmes, J., dissenting). 5 OUTPUT AS PROPHECY 63 18. Kellogg (2018) at 82. 19. See also Kellogg at 92: “Prediction had a broader and longer-term refer- ence for [Holmes] than immediate judicial conduct, and was connected with his conception of legal ‘growth’.” See further Chapter 9. 19. See also Kellogg at 92: “Prediction had a broader and longer-term refer- ence for [Holmes] than immediate judicial conduct, and was connected with his conception of legal ‘growth’.” See further Chapter 9. 20. Chapter 3, pp. 34–35. 21. Chapter 3, p. 38. 22. Hastie et al. (2009) 1–2. 23. It is the use of machine learning to make “predictions” in this sense (“predictive analytics”) on which legal writers addressing the topic to date largely have focused. See, e.g., Berman, 98 B.U. L. Rev. 1277 (2018). 24. The terms “postdiction” and “retrodiction” are sometimes used in legal scholarship too, though writers who use them are more likely to do so in connection with other disciplines. See, e.g., Guttel & Harel, Uncer- tainty Revisited: Legal Prediction and Legal Postdiction, 107 Mich. L. Rev. 28. Chapter 3, p. 38. 29. Russakovsky, Deng et al., op. cit. (2015). 30. Hossain, Sohel, Shiratuddin & Laga, ACM CSUR 51 (2019). 31. Chapter 6, p. 70. 32 Chapter 7 pp 81–88; and Chapter 8 pp 89–100 29. Russakovsky, Deng et al., op. cit. (2015). 28. Chapter 3, p. 38. Notes 467–99 (2008), who considered ﬁndings from psychology that peo- ple are less conﬁdent about their postdictions (e.g., what was the outcome of the dice roll that I just performed?) than their predictions (e.g., what will be the outcome of the dice roll that I am about to perform?) id. 471–79. 25. See Chapter 4, p. 41. 26. Dumas & Frankenreiter, Text as Observational Data, in Livermore & Rockmore (eds.) (2019) at 63–64. 27. As to gradient descent, see Chapter 2, p. 23. Gradient descent is often coupled with another technique called cross validation, also based on prediction. The term derives from the idea of a “validation dataset.” When training a machine learn- ing system, it is not possible to measure prediction accuracy by testing predictions on the same dataset as was used to train the machine. (This can be shown mathematically.) Therefore, the train- ing dataset is split into two: one part for training parameter val- ues, the other part for measuring prediction accuracy. This latter part is called the “validation dataset.” Cross validation is totemic in machine learning: stats.stackexchange.com, a popular Internet Q&A site for machine learning, calls itself CrossValidated. It is also tech- nically subtle. See Hastie, Tibshirani & Friedman (2009) in §7.10, for a formal description 30. Hossain, Sohel, Shiratuddin & Laga, ACM CSUR 51 (2019). 31. Chapter 6, p. 70. 32. Chapter 7, pp. 81–88; and Chapter 8, pp. 89–100. T. D. GRANT AND D. J. WISCHIK 64 33. Chapter 9, pp. 103–111. 34. 10 Harv. L. Rev. at 469. 35. Kellogg (2018) 36–53. 36. Board of Trade of the City of Chicago v. Christie Grain & Stock Company et al., 198 U.S. 236, 247, 25 S.Ct. 637, 638 (Holmes, J., 1905). See also Ithaca Trust Co. v. United States, 279 U.S. 151, 155, 49 S.Ct. 291, 292 (Holmes, J., 1929) (mortality tables employed to calculate for purposes of tax liability the value of a life bequest as of the date it was made). 37. Letter from Holmes to Pollock (Aug. 30, 1929), reprinted De Wolfe Howe (ed.) (1942) vol. 2, p. 252 (emphasis original). Kellogg quotes this passage: Kellogg (2018) at 52. 38. See for example Coastal Orthopaedic Institute, P.C. v. Bongiorno & anthr, 807 N.E.2d 187, 191 (2004, Appeals Court of Massachusetts, Bristol, Berry J.). Cf. observing a relation between uncertainty and the predictive character of law, Swanson et al. v. Notes A “p-value” is a term familiar to courts, but not one they use to describe their own judgments. See Matrixx Initiatives, Inc. v. Siracusano, 563 U.S. 27, 39, 131 S.Ct. 1309, 1319 n. 6 (Sotomayor, J., 2011): 45. I.e., a numerical value representing the probability that a future court wil not treat the judgment as a correct statement of law. A “p-value” is a term familiar to courts, but not one they use to describe their own judgments See Matrixx Initiatives, Inc. v. Siracusano, 563 U.S. 27, 39, 131 S.Ct 1309, 1319 n. 6 (Sotomayor, J., 2011): ‘A study that is statistically signiﬁcant has results that are unlikely to be the result of random error….’ To test for signiﬁcance, a researcher develops a ‘null hypothesis’—e.g., the assertion that there is no relationship between Zicam use and anosmia… The researcher then calculates the probability of obtaining the observed data (or more extreme data) if the null hypothesis is true (called the p- value)… Small p-values are evidence that the null hypothesis is incorrect. (citations omitted) See also In re Abilify (Aripiprazole) Products Liability Litigation, 299 F.Supp.3d 1291, 1314–15; Abdul-Baaqiy v. Federal National Mortgage Association (Sept. 27, 2018) p. 7. See also In re Abilify (Aripiprazole) Products Liability Litigation, 299 F.Supp.3d 1291, 1314–15; Abdul-Baaqiy v. Federal National Mortgage Association (Sept. 27, 2018) p. 7. ( p , ) p 46. On a court consisting of more than one judge and on which it is open to members of the court to adopt separate or dissenting opinions, the existence and content of such opinions are another source of evidence as to how much conﬁdence one might place in the result. The possibility of assigning conﬁdence intervals to judgments on such evidence is sug- gested here: Posner & Vermeule, The Votes of Other Judges, 105 Geo. L.J. 159, 177–82 (2016). Regarding the inﬂuence of concurring opinions on future judgments, see Bennett, Friedman, Martin & Navarro Smelcer, Divide & Concur: Separate Opinions & Legal Change, 103 Corn. L. Rev. 817 (2018), and in particular the data presented id. at 854 and passim. Cf. Eber, Comment, When the Dissent Creates the Law: Cross- Cutting Majorities and the Prediction Model of Precedent, 58 Emory L.J. 207 (2008); Williams, QuestioningMarks: Plurality Decisions and Prece- dential Constraint, 69 Stan. L. Rev. 795 (2017); Plurality Decisions— The Marks Rule—Fourth Circuit Declines to Apply Justice White’s Concur- rence in Powell v. 48. A recent study, though for different purposes, makes an observation apt to our point: “It is one thing to say that the standards of juridical proof are to be explicated in probabilistic terms, it is another to provide such an explication.” Urbaniak (2018) 345 (emphasis added). 47. Or got something in the judgment wrong while having gotten other things right. We speak above, for sake of economy of expression, about a judgment struck down in toto. Notes Powers et al., 937 F.2d 965, 968 (1991, 4th Cir. Wilkinson, CJ): 38. See for example Coastal Orthopaedic Institute, P.C. v. Bongiorno & anthr, 807 N.E.2d 187, 191 (2004, Appeals Court of Massachusetts, Bristol, Berry J.). Cf. observing a relation between uncertainty and the predictive character of law, Swanson et al. v. Powers et al., 937 F.2d 965, 968 (1991, 4th Cir. Wilkinson, CJ): The dockets of courts are testaments… to the many questions that remain reasonably debatable. Holmes touched on this uncertain process when he deﬁned ‘the law’ as ‘[t]he prophecies of what the courts will do’. 39. Hastie et al., op. cit. (n. 27) § 2.3.2. This simple description of the k near- est neighbor algorithm does not reﬂect the real cleverness, which consists in inventing a useful similarity metric such that the simple algorithm pro- duces good predictions. Even cleverer is to use a neural network to learn a useful similarity metric from patterns in the data. 41. Lecture given by Zoubin Ghahramani at MIT, 2012. http://mlg.eng. cam.ac.uk/zoubin/talks/mit12csail.pdf. 42. Lawyers indeed give probability assessments to their clients often enough that behavioral decision theorists have studied the factors that inﬂuence lawyers’ views as to the chances of winning or losing in court. See, e.g., Craig R. Fox & Richard Birke, Forecasting Trial Outcomes: Lawyers Assign Higher Probability to Possibilities That Are Described in Greater Detail, 26(2) Law Hum. Behav. 159–73 (2002). 43. As to empiricism in legal scholarship generally, see Epstein, Friedman & Stone, Foreword: Testing the Constitution, 90 N.Y.U. L. Rev. 1001 and works cited id. at 1003 nn. 4, 5 and 1004 n. 6 (2015); in one of law’s subdisciplines, Shaffer & Ginsburg, The Empirical Turn in International Legal Scholarship, 106 AJIL 1 (2012). 44. See for example Kern v. Levolor Lorentzen, Inc., 899 F.2d 772, 781–82 (1989, 9th Cir., Kozinski, C.J., dissenting). 5 OUTPUT AS PROPHECY 65 45. I.e., a numerical value representing the probability that a future court will not treat the judgment as a correct statement of law. A “p-value” is a term familiar to courts, but not one they use to describe their own judgments. See Matrixx Initiatives, Inc. v. Siracusano, 563 U.S. 27, 39, 131 S.Ct. 1309, 1319 n. 6 (Sotomayor, J., 2011): 45. I.e., a numerical value representing the probability that a future court will not treat the judgment as a correct statement of law. Notes Texas as Binding Precedent—Manning v. Caldwell, 132 Harv. L. Rev. 1089 (2019). T. D. GRANT AND D. J. WISCHIK 66 Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. 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https://openalex.org/W2005770171	https://zenodo.org/records/1231834/files/article.pdf	English	null	Differentiation of topographical and chemical structures using an interfacial force microscope	Applied physics letters	1,992	public-domain	3,431	Differentiation of topographical and chemical structures using an interfacial force microscope Stephen A. Joyce, J. E. Houston, and T. A. Michalske Stephen A. Joyce, J. E. Houston, and T. A. Michalske Citation: Applied Physics Letters 60, 1175 (1992); doi: 10.1063/1.107396 View online: http://dx.doi.org/10.1063/1.107396 View Table of Contents: http://scitation.aip.org/content/aip/journal/apl/60/10?ver=pdfcov Published by the AIP Publishing Citation: Applied Physics Letters 60, 1175 (1992); doi: 10.1063/1.107396 View online: http://dx.doi.org/10.1063/1.107396 View Table of Contents: http://scitation.aip.org/content/aip/journal/apl/60/1 Published by the AIP Publishing Differentiation of topographical and chemical structures using an interfacial force microscope Stephen A. Joyce, J. E. Houston, and T. A. Michalske Sandia National Laboratories, Albuquerque, New Mexico 87185 (Received 30 September 1991; accepted for publication 6 January 1992) (Received 30 September 1991; accepted for publication 6 January 1992) The forces between a tungsten tip and a self-assembled monolayer of hexadecylthiol (C!t6H,,SH) on a thin gold film have been studied using a newly developed interfacial-force microscope. Imaging of the surface, combined with spatially resolved force versus separation measurements, allow for the distinction of topographical and chemical features of the surface. Several distinct regions are observed for this system. The first, characterized by a very weak interfacial interaction between tip and sample, is representative of the self- assembled monolayer. The other regions show relatively strong, long-range attractive forces, which are associated with gross defects in the film. The atomic force microscope (AFM) has recently been used to image the microscopic, three-dimensional structure of a large number of both insulating and conduct- ing samples. ‘T2 The imaging is generally accomplished by scanning a sharp tip across a surface while maintaining a constant force (or force gradient) between tip and sample. For a homogeneous surface, such measurements reflect the topography of the sample. In addition, one can measure the force between the tip and sample at a fixed location as a function of the tip-sample separation. These force-sepa- ration profiles are a direct measure of the interfacial inter- action and, as such, depend on the detailed chemical or material natures of both the tip and substrate surfaces. Combining both capabilities, imaging and force-separation profiles, allows the differentiation of the morphological and chemical features of heterogeneous samples. In this letter, we present such a study using the recently developed in- terfacial force microscope (IFM) to image the surface and measure selected area force-separation profiles for a tung- sten (W) tip interacting with a self-assembled monolayer of hexadecylthiol (C6H,,SH) adsorbed on a thin gold film. We find several regions on these surfaces: one is as- sociated with “flat” areas characteristic of the self-assem- bled monolayer; the others are associated with gross de- fects in the Au-substrate film and with contaminants on the Au-film surface. silicon substrate. Differentiation of topographical and chemical structures using an interfacial force microscope Stephen A. Joyce, J. E. Houston, and T. A. Michalske Sandia National Laboratories, Albuquerque, New Mexico 87185 (Received 30 September 1991; accepted for publication 6 January 1992) The substrates have a thin native-oxide layer and an intermediate thiolated silane layer is used to enhance the adhesion of the gold film.5 The interfacial force microscope has been described in detail elsewhere and will be discussed only briefly here.6 Of primary importance to the present work is the ability to measure the interfacial-force profiles, i.e., the force be- tween the tip and surface over the entire range of interfa- cial separations, from nanometers up to repulsive contact. Conventional cantilever-based force microscopes are not ideally suited for such measurements since they are me- chanically unstable when the attractive force gradient be- tween tip and sample exceeds the cantilever-force constant. This instability results in the discontinuous movement of the tip into contact with the sample. This “jump-to-con- tact” phenomenon has been observed frequently and de- scribed in detail by others.7V8 The instability prevents the measurement of interfacial forces over the range of sepa- rations necessary to fully characterize the interfacial inter- action. We have developed a zero-deflection/force-feedback controlled IFM that does not suffer from the “jumping” behavior.6 The mechanical instability is avoided by exter- nally balancing the interfacial force so that the sensor de- flection is minimized. The heart of the microscope is the differential capacitance-displacement sensor. A common capacitor plate, suspended on torsion arms about which it is free to rotate, is mounted above two fixed capacitor pads. When a sample is brought into the vicinity of a tip mounted on one end of the common plate, the interfacial force causes the deflection of the common plate. The re- sultant differential-capacitance change is detected by con- figuring the sensor as an ac impedance bridge. At this level, the unit can be operated as a conventional AFM. Force balancing can be achieved through the application of dc control voltages to the fixed-capacitor pads. The restoring force is of the form F,.== - Fi= 2CVA V/d, where Fi is the interfacial force, V is a constant bias voltage applied to both fixed-capacitor pads, d is the capacitor gap, and A V is the control voltage. Articles you may be interested in Measurement of nanomechanical properties of metals using the atomic force microscope J. Vac. Sci. Technol. B 12, 2211 (1994); 10.1116/1.587743 This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP: 129.22.67.107 On: Mon, 24 Nov 2014 07:35:14 This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP: 129.22.67.107 On: Mon, 24 Nov 2014 07:35:14 Differentiation of topographical and chemical structures using an interfacial force microscope Stephen A. Joyce, J. E. Houston, and T. A. Michalske Sandia National Laboratories, Albuquerque, New Mexico 87185 (Received 30 September 1991; accepted for publication 6 January 1992) 1175 Appl. Phys. Lett. 60 (lo), 9 March 1992 0003-6951/92/101175-03$03.00 @ 1 s article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http:/ 129 22 67 107 On: Mon 24 Nov 2014 07:35:14 Differentiation of topographical and chemical structures using an interfacial force microscope Stephen A. Joyce, J. E. Houston, and T. A. Michalske Sandia National Laboratories, Albuquerque, New Mexico 87185 (Received 30 September 1991; accepted for publication 6 January 1992) Negative separations refer to the distance between tip and sample on approach; positive separation indicates withdrawal. Attractive forces are negative, repulsive are positive. (A) Profile from the higher regions above the step. (B) Profile from the lower regions below the step. C,#G An electrolytically etched W tip’ was attached to one end of the sensor using silver epoxy. Since the tip is ex- posed to air, it is presumably covered with a thin-oxide film. The samples were mounted to the end of a quadrant piezoelectric-tube scanner. Two modes of operation are commonly employed. For imaging, the feedback control to the sensor is turned off and a second feedback controller for the piezotube is used to image the surface at a constant repulsive force of -2 X 10 - 7 N. For the selected-area force-profile measurements, the piezotube feedback con- troller is turned off and the sensor feedback on while the sample is scanned from -60 nm separation to repulsive contact and then back out. The inter-facial force is mea- sured both upon approach and withdrawal. The experi- ments were carried out both in air and in a turbomolecular- pumped vacuum system. No significant differences between air and vacuum operation were observed. phobic surface such as the methyl-terminated self-assem- bled monolayer. The lack of any attractive forces upon withdrawal indicates the monolayer prevents adhesion be- tween the tip and substrate even after repulsive-contact loading. The repulsive portions of the force profiles reflect both the elastic properties of the films and the compliance of the sensor. The hysteresis in the repulsive-force profiles is reproducible and corresponds to an anelastic response of the fi1m.i’ As shown of Fig. 2(B), strikingly different force pro- files are observed for the low regions adjacent to the steps. Strong, long-range attractive forces are seen. The attractive forces are longer range upon withdrawal, indicating a sig- nificant level of hysteresis. This behavior has been observed frequently and has been ascribed to capillary or meniscus forces between tip and sample.“-I3 Fluid layers on the surface or tip result in attractive forces by the formation of a meniscus when the tip and sample are brought into initial contact. Upon withdrawal, the attractive forces remain un- til the meniscus breaks. While the exact nature of the fluid layer responsible for these forces is not known, it is pre- sumably some form of adventitious contaminant such as water and/or hydrocarbons. Differentiation of topographical and chemical structures using an interfacial force microscope Stephen A. Joyce, J. E. Houston, and T. A. Michalske Sandia National Laboratories, Albuquerque, New Mexico 87185 (Received 30 September 1991; accepted for publication 6 January 1992) ( + A V is applied to one pad, -A V to Self-assembled monolayers of organic molecules have recently been the focus of a considerable research effort due to their many unique properties and potential applica- tions including, for example, use as nonlinear optical de- vices and electrochemical sensors.3 For the present study, we have used an n-alkyl thiol, hexadecylthiol, adsorbed on a thin gold film. The thiol end group strongly chemisorbs on Au surfaces, while the normal alkyl backbone interacts with neighboring molecules through long-range van der Waals forces to form densely packed, ordered films.4 The ideal result is a uniformly thick film ( -25 A) terminated with methyl groups. For this work, the thiol was deposited from a solution ( - 1 mM in ethanol) onto a thin ( - 100 nm) gold film which in turn had been deposited onto a 1175 Appl. Phys. Lett. 60 (lo), 9 March 1992 0003-6951/92/101175-03$03.00 @ 1992 Am article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitatio 129 22 67 107 On: Mon 24 Nov 2014 07:35:14 0003-6951/92/101175-03$03.00 @ 1992 American Institute of Physics 1175 of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded 22 67 107 On: Mon 24 Nov 2014 07:35:14 0003-6951/92/101175-03$03.00 of AIP content is subject to the terms a 2 67 107 On: Mon 24 Nov 2014 07:3 1175 nloade FIG. 1. Constant repulsive-force image of the self-assembled monolayer sample showing the edge of a large-scale defect. 8x10-‘-3 8 1, 11 1, 11 1, r 11, I 1 L , a 11 -4 b -I -6 -w,u”y”uj 60 Separation (nm) KG. 2. Force-separation profiles from two areas near a defect. Negative separations refer to the distance between tip and sample on approach; positive separation indicates withdrawal. Attractive forces are negative, repulsive are positive. (A) Profile from the higher regions above the step. (B) Profile from the lower regions below the step. 8x10-‘-3 8 1, 11 1, 11 1, r 11, I 1 L , a 11 -4 b -I -6 -w,u”y”uj 60 Separation (nm) FIG. 1. Constant repulsive-force image of the self-assembled monolayer sample showing the edge of a large-scale defect. the other.) Small, second-order corrections [of order C(A V)2/6] in the calculation of F, are necessary when C #G KG. 2. Force-separation profiles from two areas near a defect. Differentiation of topographical and chemical structures using an interfacial force microscope Stephen A. Joyce, J. E. Houston, and T. A. Michalske Sandia National Laboratories, Albuquerque, New Mexico 87185 (Received 30 September 1991; accepted for publication 6 January 1992) It is not unlikely, however, that~the hydrophobic self-assembled monolayer would sup- port such an interaction, suggesting that this region is not covered by the monolayer. between air and vacuum operation were observed. Figure 1 shows a constant repulsive-force image of a self-assembled monolayer on a thin gold tim. The 400 nm by 400 nm image reveals two relatively smooth areas (fea- tures up to a few nanometers high) separated by a 50-nm- wide step which is - 70 nm high. (As we will discuss later, this step is associated with defects in the substrate metal- lization.) The force images associated with such large-scale features are a convolution of the topographies of both the tip and sample. When the sample mount is physically moved so that other areas can be imaged, such large-scale features are observed about 20%-30% of the time. Smaller-scale protrusions (- 100 nm diam, 10-30 nm high) are occasionally observed, while the remainder of the surveys show relatively flat surfaces over the entire image. In order to better characterize these surfaces, force- profile measurements were performed over selected areas to determine whether any chemical effects are associated with the large-scale defects. Two such results are shown in Fig. 2 for areas above and below one of the large steps. Curve A in Fig. 2 is representative of the force measure- ments observed for the high regions above the steps. Note the absence of any strong, attractive forces between tip and sample upon approach. This behavior is consistent with the interaction of a metal tip and a chemically inert, hydro- between air and vacuum operation were observed. Figure 1 shows a constant repulsive-force image of a self-assembled monolayer on a thin gold tim. The 400 nm by 400 nm image reveals two relatively smooth areas (fea- tures up to a few nanometers high) separated by a 50-nm- wide step which is - 70 nm high. (As we will discuss later, this step is associated with defects in the substrate metal- lization.) The force images associated with such large-scale features are a convolution of the topographies of both the tip and sample. When the sample mount is physically moved so that other areas can be imaged, such large-scale features are observed about 20%-30% of the time. Joyce, Houston, and Michalske scitation.aip.org/termsconditions. Down 1176 Appl. Phys. Lett., Vol. 60, No. 10, 9 March 1992 article is copyrighted as indicated in the article Reuse of AIP c Differentiation of topographical and chemical structures using an interfacial force microscope Stephen A. Joyce, J. E. Houston, and T. A. Michalske Sandia National Laboratories, Albuquerque, New Mexico 87185 (Received 30 September 1991; accepted for publication 6 January 1992) Smaller-scale protrusions (- 100 nm diam, 10-30 nm high) are occasionally observed, while the remainder of the surveys show relatively flat surfaces over the entire image. surveys show relatively flat surfaces over the entire image. In order to better characterize these surfaces, force- profile measurements were performed over selected areas to determine whether any chemical effects are associated with the large-scale defects. Two such results are shown in Fig. 2 for areas above and below one of the large steps. Curve A in Fig. 2 is representative of the force measure- ments observed for the high regions above the steps. Note the absence of any strong, attractive forces between tip and sample upon approach. This behavior is consistent with the interaction of a metal tip and a chemically inert, hydro- y y Similar measurements were performed on other areas of the samples. For the large-step defects, force profiles from the upper regions are usually characterized by weak attraction, representative of the self-assembled monolayer, while for the lower regions, contaminant meniscus forces are seen. At the smaller protrusions, similar behavior is observed, except that here the meniscus forces are usually found at the protrusion and the weak forces on the lower Yat” regions. g Using the correlation of the type of defects observed 1176 ti l i 1176 nloade oxygen, while the bright areas are predominantly carbon- aceous with traces of oxygen and gold. f -.5 . * ‘ V i - : FIG. 3. SEM image of one of the samples. The darker rectangles in the center represent areas where the organic layer has been damaged by the larger electron-current densities used for high-magnification imaging. yg g In conclusion, we have characterized the topographical and chemical nature of a self-assembled monolayer ad- sorbed on a gold film using interfacial force microscopy. Imaging reveals a number of large-scale defects; selected- area force profiles show a chemically distinct nature of the sample in the vicinity of the defects. Over the majority of the surface, only weak interactions between tip and sample were observed, indicative of the low-energy surface pro- duced by the self-assembled monolayer. Contaminant-re- lated meniscus forces are associated with defects, i.e., areas not covered by the monolayer. This work has demon- strated the importance and utility of combining the imag- ing and force profiling capabilities of force microscopy for differentiating the microscopic structure and chemical na- ture of heterogeneous samples. ‘G. Binnig, C. F. Quate, and C. Gerber, Phys. Rev. Lett. 56,930 (1986). ‘D. Sarid, Scanning Force Microscopy (Oxford University, New York, 1991). ( ) 13G. S. Blackman, C. M. Mate, and M. R. Philpott, Phys. Rev. Lett. 65, 2270 (1990). 77 Appl. Phys. Lett., Vol. 60, No. 10, 9 March 1992 ticle is copyrighted as indicated in the article. Reuse of AIP ‘G. Binnig, C. F. Quate, and C. Gerber, Phys. Rev. Lett. 56,930 (1986). ‘D. Sarid, Scanning Force Microscopy (Oxford University, New York, 1991). “J. D. Swalen, D. L. Allara, J. D. Andrade, E. A. Chandross, S. Garoff, J. Israelachvili, T. J. McCarthy, R. Murray, R. F. Pease, J. F. Rablot, K. J. Wynne, and H. Yu, Langmuir 3, 932 (1987). 4R. G. Nuzzo and D. L. Allara, .I. Am. Chem. Sot. 105, 4481 (1983). “C. A. Goss, D. H. Charych, and M. Majda, Anal. Chem. 63,85 ( 1991). 6S. A. Joyce and J. E. Houston, Rev. Sci. Instrum. 62, 710 (1991). 7K. B. Lodge, Adv. Coll. 19, 27 (1983). ‘N. A. Burnham and R. J. Colton, J. Vat. Sci. Technol. A 7, 2906 (1989). ‘E. W. Muller and T. T. Tsong, Field Ion Microscopy (American Elsevier, New York, 1969), Chap. IV. ‘OS. A. Joyce, R. C. Thomas, J. E. Houston, T. A. Michalske, and R. M. Crooks (unpublished). “R. Erlandsson, G. Hadziioannou, C. M. Mate, G. M. McClelland, and S. Chiang, J. Chem. Phys. 89, 5190 (1988). “C M. Mate, M. R. Lorenz, and V. J. Novotny, J. Chem. Phys. 90, 7550 (i989). 13G. S. Blackman, C. M. Mate, and M. R. Philpott, Phys. Rev. Lett. 65, 2270 (1990). Joyce, Houston, and Michalske //scitation.aip.org/termsconditions. Differentiation of topographical and chemical structures using an interfacial force microscope Stephen A. Joyce, J. E. Houston, and T. A. Michalske Sandia National Laboratories, Albuquerque, New Mexico 87185 (Received 30 September 1991; accepted for publication 6 January 1992) FIG. 3. SEM image of one of the samples. The darker rectangles in the center represent areas where the organic layer has been damaged by the larger electron-current densities used for high-magnification imaging. g p The authors thank Professor R. Crooks (UNM) for providing the films and for many helpful discussions and R. Wayne Buttry (SNL) for performing the SEM and SAM analysis. during imaging and the associated force profiles, a simple picture of these surfaces can be drawn. Given that the height of the steps is about the nominal thickness of the Au film, these features most likely represent areas where gold was not deposited on the silicon substrate, perhaps due to contamination present during the Au deposition. The pro- trusions are presumably microscopic particles that adhered to the surfaces and either prevent thiol adsorption or sim- ply cover the self-assembled monolayer. p y y Ultrahigh-vacuum scanning electron and scanning Au- ger microscopies (SEM) and (SAM) were performed to confirm the nature of the defects on these samples. A rep- resentative SEM image is shown in Fig. 3. Again, several distinct regions were observed. These regions consist prin- cipally of a featureless surface upon which dark circular and small bright features are observed. Selected-area Au- ger electron analysis shows that the majority of the surface is gold covered with carbon. The larger dark areas consist, however, of silicon with significant amounts of carbon and g , , ( ) ‘N. A. Burnham and R. J. Colton, J. Vat. Sci. Technol. A 7, 2906 (1989). ( ) ‘E. W. Muller and T. T. Tsong, Field Ion Microscopy (American Elsevier, New York, 1969), Chap. IV. , , ), p ‘OS. A. Joyce, R. C. Thomas, J. E. Houston, T. A. Michalske, and R. M. Crooks (unpublished). ( p ) “R. Erlandsson, G. Hadziioannou, C. M. Mate, G. M. McClelland, and S. Chiang, J. Chem. Phys. 89, 5190 (1988). S C a g, J C e ys 89, 5 90 ( 988) “C M. Mate, M. R. Lorenz, and V. J. Novotny, J. Chem. Phys. 90, 7550 (i989). ( ) 13G. S. Blackman, C. M. Mate, and M. R. Philpott, Phys. Rev. Lett. 65, 2270 (1990). Joyce, Houston, and Michalske //scitation.aip.org/termsconditions. 1177 wnload 1177 is article
https://openalex.org/W2352678152	https://digitalcommons.uri.edu/cgi/viewcontent.cgi?article=1049&context=hdf_facpubs	English	null	The young and the restless: Socializing trumps sleep, fear of missing out, and technological distractions in first-year college students	International journal of adolescence and youth	2,016	cc-by	9,192	The young and the restless: Socializing trumps sleep, fear of The young and the restless: Socializing trumps sleep, fear of missing out, and technological distractions in first-year college missing out, and technological distractions in first-year college students students Sue K. Adams University of Rhode Island, suekadams@uri.edu Sue K. Adams University of Rhode Island, suekadams@uri.edu Desirée N. Williford University of Rhode Island See next page for additional authors Follow this and additional works at: https://digitalcommons.uri.edu/hdf_facpubs University of Rhode Island University of Rhode Island DigitalCommons@URI DigitalCommons@URI University of Rhode Island University of Rhode Island DigitalCommons@URI DigitalCommons@URI Human Development and Family Science Faculty Publications Human Development and Family Science Human Development and Family Science Citation/Publisher Attribution Citation/Publisher Attribution Adams, S.K., Williford, D., Vaccaro, A., Kisler, T.S., *Francis, A., & Newman, B. (2016). The Young and the restless: Socializing trumps sleep, fear of missing out, and technological distractions in first year college students. International Journal of Adolescence and Youth, 1-12. https://doi.org/10.1080/ 02673843.2016.1181557 Available at: https://doi.org/10.1080/02673843.2016.1181557 This Article is brought to you by the University of Rhode Island. 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Adams, Desirée N. Williford, Annemarie Vaccaro, Tiffani S. Kisler, Alyssa Francis, and Barbara M. Newman Creative Commons License Creative Commons License Creative Commons License Creative Commons License This work is licensed under a Creative Commons Attribution 4.0 License. International Journal of Adolescence and Youth ISSN: 0267-3843 (Print) 2164-4527 (Online) Journal homepage: https://www.tandfonline.com/loi/rady20 The young and the restless: Socializing trumps sleep, fear of missing out, and technological distractions in first-year college students Sue K. Adams, Desireé N. Williford, Annemarie Vaccaro, Tiffani S. Kisler, Alyssa Francis & Barbara Newman Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=rady20 Authors Authors ms, Desirée N. Williford, Annemarie Vaccaro, Tiffani S. Kisler, Alyssa Francis, and Barbara M Sue K. Adams, Desirée N. Williford, Annemarie Vaccaro, Tiffani S. Kisler, Alyssa Francis, and Barbara M. Newman This article is available at DigitalCommons@URI: https://digitalcommons.uri.edu/hdf_facpubs/49 Sue K. Adams, Desireé N. Williford, Annemarie Vaccaro, Tiffani S. Kisler, Alyssa Francis & Barbara Newman To cite this article: Sue K. Adams, Desireé N. Williford, Annemarie Vaccaro, Tiffani S. Kisler, Alyssa Francis & Barbara Newman (2017) The young and the restless: Socializing trumps sleep, fear of missing out, and technological distractions in first-year college students, International Journal of Adolescence and Youth, 22:3, 337-348, DOI: 10.1080/02673843.2016.1181557 To link to this article: https://doi.org/10.1080/02673843.2016.1181557 © 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 11 May 2016. Submit your article to this journal Article views: 6543 View Crossmark data Citing articles: 6 View citing articles © 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 11 May 2016. Submit your article to this journal Article views: 6543 View Crossmark data Citing articles: 6 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=rady20 International Journal of Adolescence and Youth, 2017 VOL. 22, NO. 3, 337–348 https://doi.org/10.1080/02673843.2016.1181557 OPEN ACCESS © 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. CONTACT Sue K. Adams suekadams@uri.edu ABSTRACT College students are a sleep-deprived population, with first-year students facing a number of specific challenges to sleep. As students transition into and through the first year of college, sleep may be sacrificed for a variety of reasons. Semi-structured interviews were conducted with fifteen first-year students, exploring factors that impacted sleep during the first semester of college. Study participants identified three unique but related themes that impacted their sleep: socializing trumps sleep; fear of missing out; and social/ technological distractions. Implications are provided for balancing social, academic and biological demands in emerging adulthood. College students, specifically first-year students, are extremely sleep deprived, with approximately 25% of students reporting less than six hours of sleep per night (Lund, Reider, Whiting, & Prichard, 2010). This statistic is alarming given that the average 18-year-old requires approximately 8.5 h of sleep for optimal functioning (National Sleep Foundation, 2013). Sleep deprivation is a troubling phenomenon because it negatively impacts the individual across all domains of functioning, including academic, social, phys- ical and emotional functioning. For instance, sleep deprivation can lead to negative outcomes such as impaired memory, decreased mood, decreased stress management, and impaired immune response (Curcio, Ferrara, & De Gennaro, 2006; Hill, 1994; Wolfson & Carskadon, 1998). While there is a growing body of literature about sleep during adolescence (e.g. Sadeh, Dahl, Shahar, & Rosenblat-Stein, 2009), there is limited research about sleep for first-year students during the initial semester at college – a time when sleep settings/environments (e.g. home versus residence hall) and patterns might change drastically. Moreover, almost no research has relied on qualitative data (e.g. student narratives) to high- light the ways students themselves describe the influences on their sleep, or lack thereof. This study begins to fill that gap. The young and the restless: Socializing trumps sleep, fear of missing out, and technological distractions in first-year college students Sue K. Adams, Desireé N. Williford, Annemarie Vaccaro, Tiffani S. Kisler, Alyssa Francis and Barbara Newman Department of Human Development and Family Studies, The University of Rhode Island, Kingston, RI, USA ARTICLE HISTORY Received 21 March 2016 Accepted 19 April 2016 KEYWORDS Sleep; college; transition; technology; social; fear of missing out Fear of missing out Several psychosocial factors contribute to sleep problems in adolescents, including the desire to stay up late to engage in social activities, self-determined bed times, and easy access to a vast range of stim- ulating activities (Dahl & Lewin, 2002). The latter may be particularly problematic in college residence halls, where individuals are constantly surrounded by others, and therefore are more likely to engage in social activities that may impact sleep schedules (Dahl & Lewin, 2002). One emerging construct that impacts the ability to set boundaries around sleep is termed the ‘FoMO’. FoMO is defined as a pervasive apprehension that a more exciting or interesting event is taking place elsewhere (Przybylski, Murayama, DeHaan, & Gladwell, 2013). Preliminary research exploring the prevalence of FoMO found nearly three-quarters of young adults reported they experienced the phenomenon (Alt, 2015). Przybylski et al. (2013) found FoMO is associated with greater Facebook use, and those high in FoMO were more likely to use Facebook immediately after waking in the morning and before falling asleep at night, which may impact the time it takes to fall asleep along with the quality of sleep. The generation of students currently enrolled in college is oftentimes characterized as possessing highly developed skills in information technology and multitasking (Alt, 2015). Such skills enable individ- uals to remain socially connected while engaged in independent work. Previous research has described this generation as focused on social interaction and connectedness with others through mobile phones, chat-rooms and email while simultaneously engaged in other tasks such as video games, listening to music and watching television (McMahon & Pospisil, 2005). Moreover, Pempek, Yermolayeva, and Calvert (2009) found 50% of their undergraduate sample used Facebook to communicate with friends not on campus (i.e. old friends and friends at other schools). Aside from social media use, Dahl and colleagues (2002) suggest social stresses; including fear, anxiety and emotional arousal interfere with the ability to fall asleep during adolescence. During adolescence factors associated with FoMO, such as rumination, stress and worry are likely to affect the time it takes to fall asleep as cognitive components related to the ability to fall asleep are changing (Dahl & Lewin, 2002). According to Chickering and Reisser (1993) emerging adults engage in seven developmental tasks during the collegiate years. While attempting to develop mature interpersonal relationships, emerging adults must be able to create equitable relationships based upon honesty, respect and equity. Fear of missing out Chickering and Reisser (1993, p. 48) also argue ‘development means more in-depth sharing and less clinging, more acceptance of flaws and appreciation of assets, more selectivity in choosing nurturing relationships, and more long-lasting relationships that endure crises, distance, and separation’. As emerging adults continue to develop mature interpersonal relationships, they would likely experience less fear that they are missing important social events during times of separation. Literature review The first year of college is a time filled with experimentation of how to handle newfound autonomy over the self (Arnett, 2000). Most high-schoolers have some degree of parental oversight when it comes to sleep patterns and making sure they are going to bed at a reasonable time. After beginning college, however, most students must adjust to their newfound autonomy regarding their schedules, as well CONTACT Sue K. Adams CONTACT Sue K. Adams S. K. Adams et al. 338 as balancing increasing social and academic demands in an environment with less externally imposed structure. There are a number of factors that may impact a student’s ability to get enough sleep during the first year of college, including a fear of missing out (FoMO), environmental stimuli, and technology. Environmental stimuli Environmental stimuli can also impact sleep throughout the first semester as students adjust to a new living environment. For example, in their sample of 1125 college students living in residence halls, Lund et al. (2010) found excess noise accounted for 33% of the responses when participants were asked ‘How often have you had trouble sleeping because of other reason(s)?’ Following closely behind, stress accounted for 35% of the responses. Sharing a bedroom with others (7%) and talking with friends (6%) were also commonly cited as reasons for disrupted sleep. This finding is supported by research suggesting constant contact with peers often interferes with going to bed at a healthy bed time (Dahl & Lewin, 2002). Additionally, when asked ‘if your sleep is at all compromised, to what one factor do you most strongly attribute this?’ 17% of respondents explained light or noise most strongly influenced sleep; 8% of respondents suggested light or noise most interfered with initiating International Journal of Adolescence and Youth 339 sleep. Similarly, Gellis and colleagues (2014) found students with higher levels of insomnia were more likely to engage in arousing behaviours near bedtime, report uncomfortable sleeping arrangements (including a noisy room) and engage in an improper sleep schedule – likely a result of residence hall living, social priorities, and newfound freedom. As emerging adults move through autonomy towards interdependence (Chickering & Reisser, 1993), they must learn to navigate autonomy and the lack of structure when they arrive at college. Through this transition, emerging adults ‘learn to function with relative self-sufficiency, to take responsibility for pursuing self-chosen goals, and to be less bound by others’ opinions’ (Chickering & Reisser, 1993, p. 47). One of the key aspects of this vector is the ability to set boundaries regarding other’s expecta- tions and a ‘freedom from continual and pressing needs for reassurance, affection or approval’ (p. 47). Setting boundaries that promote self-care may be difficult for first-year students, but they are critical to maintaining development, health and well-being. Purpose This paper was gleaned from an exploratory qualitative study about first-year students’ self-reported experiences with health, technology and belonging. This paper specifically focuses on the emergent themes related to one particular aspect of health (i.e. sleep) and technology. Our findings related to belonging and technology can be found elsewhere (Vaccaro et al., 2015). The purpose of this paper is to document the factors, described by students themselves, that influenced their sleep during the first semester of college. Technology Visually stimulating activity, such as technology use before bed, may also have negative consequences in regard to sleep quality. Despite this, in 2011, the Sleep in America Poll found adults ages 19–29 reported high levels of technology use (i.e. use of cell phones, computers and video games) before going to bed. In fact, 67% of this sample reported using their cell phone prior to sleeping at night, a behaviour which has been linked to difficulty falling asleep, repeated awakenings at night, or early wake times (Hershner & Chervin, 2014; National Sleep Foundation, 2013). Likewise, 60% of the sample reported using computers before bed, putting them at an increased likelihood of experiencing similar symptoms, such as feeling less rested upon waking and daytime drowsiness, particularly while driving a vehicle (Hershner & Chervin, 2014; National Sleep Foundation, 2013). Chronic night-time exposure to portable device lighting can create a misalignment of the circadian timing system (Cajochen et al., 2011) as well as disrupt the process of melatonin expression (Wood, Rea, Plitnick, & Figueiro, 2013). This is particularly noteworthy for college students, as they are already experiencing a mismatch between the pre-existing circadian rhythm shift of adolescence (an inherent drive to stay up later) and the requirement of an early wake time, such as for morning courses, before adequate sleep duration has been reached (Hershner & Chervin, 2014). This mismatch often causes college students to incur sleep debt during the week, which can lead to catching up on sleep during the weekend by sleeping in later. This, too, further delays the circadian system. Setting and participants The study was conducted at a mid-sized public institution in the United States with 13,000 enrolled undergraduates. The approximately 3000 first-year students who had recently transitioned into the university were eligible to participate in the study. Participants were recruited through the posting of flyers on campus and personal recruitment in first-year seminars and general education courses with high enrollments of first-year students. The sample consisted of 15 participants in their first year of undergraduate study. Fourteen women and one man volunteered to participate. All participants were traditional-aged students (i.e. 18 to 22 years of age) with the modal age being 18. Seven students had majors in the College of Human Science, one was a Pharmacy major, and seven were undeclared. Thirteen of the students lived in campus housing while one commuted from home. Theoretical and conceptual frameworks The biopsychosocial model is a theoretical and conceptual framework that elegantly bridges the dichot- omy between the social sciences and the medical sciences and considers the role of interpersonal and psychological dynamics for an individual’s health and well-being. George Engel (1977), the originator of the biopsychosocial model, proposes that simultaneous attention to biological, psychological and social aspects is necessary when considering health and pathology processes (Doherty & Campbell, 340 S. K. Adams et al. Sleep Technological Distractions Fear of Missing Out Social and Environmenta Stimuli Figure 1. Conceptual model for fear of missing out, socializing and technology use affecting sleep. 340 S. K. Adams et al. Fear of Missing Out Technological Distractions Social and Environmental Stimuli Social and Environmental Stimuli Technological Distractions gure 1. Conceptual model for fear of missing out, socializing and technology use affecting sleep. 1988). The biopsychosocial model operates by way of a family systems perspective to understand the multiple reciprocal factors from various facets of human experience. The biopsychosocial model, therefore, is a way of looking at the mind and body as two important systems that are interlinked. Our study employed the biopsychosocial model as a guiding framework for exploring factors that impeded sleep during the first semester of college, while also attending to Chickering and Reiser’s developmental considerations in the transition to college (Chickering & Reisser, 1993). See Figure 1. 1988). The biopsychosocial model operates by way of a family systems perspective to understand the multiple reciprocal factors from various facets of human experience. The biopsychosocial model, therefore, is a way of looking at the mind and body as two important systems that are interlinked. Our study employed the biopsychosocial model as a guiding framework for exploring factors that impeded sleep during the first semester of college, while also attending to Chickering and Reiser’s developmental considerations in the transition to college (Chickering & Reisser, 1993). See Figure 1. Data analysis Interviews were audio recorded and transcribed for analysis. Dual theoretical lenses (i.e. bio-psycho- logical, psycho-social) served as a foundation for our analysis. However, emergent themes through an in vivo coding process which are derived from participant words and meaning-making as opposed to a priori assumptions, were also analysed. Then, researchers searched within and between interviews for repetition, similarities, differences and linguistic connectors among the in vivo codes (Guest, MacQueen, & Namey, 2012, p. 50). y p Three forms of systematic coding were used in this study (Creswell, 2012; Miles & Huberman, 1994). During our first level of coding, each interviewer independently reviewed the transcripts and compiled lists of possible in vivo codes. Researchers then engaged in a second level of coding which included grouping first-level codes into ‘a smaller number of sets, themes or constructs’ (Miles & Huberman, 1994, p. 68). Second-level codes represented themes about sleep, social relationships, intimacy, belonging and technology. The research team then created a codebook containing a detailed coding frame which is ‘a structured compendium of codes that include[d] a description of [codes] and how the codes are related to each other’ (Guest et al., 2012, p. 50). In the final stage of analysis, we further examined emergent themes from second-level coding, seeking intersections and connections among them. Galletta (2013) explains how in this later stage of analysis, ‘relationship among codes is explored, and thematic clus- ters, or categories are formulated’ (p. 127). Then, researchers ‘test out ideas about relationships among codes as thematic clusters or categories, moving the research into an increasingly more abstract phase’ which was done during phase three of analysis (Galletta, 2013, p. 127). The research team tested the coding frame within single interviews and between interviews, paying special attention to the ways second-level codes related to one another. Through this process, interesting connections among sleep, social relationships, student fears of missing out, and social distractions related to communal living and our technological world emerged. In order to insure rigour of the study and address issues of credibility and trustworthiness, four strategies were used in this project (Jones, Torres, & Arminio, 2014). The researcher is considered the instrument in qualitative research (Galletta, 2013; Jones et al., 2014). Therefore, the first strategy was to analyse our positionality by recognizing the possible ways our interpretations were shaped by our experiences as individuals (Crotty, 1998). Second, we utilized negative case analysis. Procedure Qualitative interview research allows researchers to obtain ‘the intricate details about phenomena such as feelings, thought processes, and emotions that are difficult to extract’ (Strauss & Corbin, 1998, p. 11). The purpose of the study was to explore the feelings and processes related to sleep or the lack thereof, rather than medical or quantitative data regarding sleep. Thus, this study explored how uni- versity students thought about, felt, experienced and made meaning of their sleep patterns during the first semester of college. i To collect data, we utilized semi-structured interviews, roughly 60 min in length. Glesne (1999) argues that interviews are more than a vehicle for acquiring data, they are analytic acts that help a International Journal of Adolescence and Youth 341 researcher understand depth in meanings, explanations and relationships. In short, interview research is a systematic process for studying both the experiences of participants and how they make meaning of those experiences (Seidman, 1998). Interview questions were created broadly to allow first-year college students the ability to describe influences on their sleep or lack thereof. Sample interview questions included: ‘How have your sleep habits changed since beginning college?’ ‘What are the main reasons for the change?’; ‘If and how does wanting to feel connected to your friends motivate you to stay up later?’; ‘If and how does your intimate life have any effect on your sleep patterns and/or physical health?’ Probes, such as ‘tell me more about that’ and ‘can you provide an example’, were used to delve more deeply into student meaning-making about sleep. Results As described in the theoretical framework section, psychosocial development, including balancing autonomy and interdependence as well as forging of mature interpersonal relationships is an important part of college life. However, our findings suggest that the quest to be social came at a cost to sleep for most first-year students. In the following sections, we explicate three emergent themes related to the ways the social atmosphere of college and our technologically connected world deterred students from sleeping enough or sleeping well. We titled these three related, but somewhat unique themes: Socializing trumps sleep; FoMO; and social distractions. Each of these themes contains a combination of in-person, face-to-face interaction, as well as interactions mediated by technology. Data analysis In qualitative research, no code can perfectly describe the perspectives and experiences of all study participants. There are always a small number of cases (i.e. 1–2), called negative cases, that highlight the diversity in human behaviour and give oppositional context to an emergent theme. In this paper, we highlight student comments (e.g. negative cases) that ran counter to our codes and emergent themes. Third, we employed a form of peer review by inviting students to participate in peer debriefing sessions about the analyses and interpretations. They offered expert insight into our interpretations. Fourth, we uti- lized inter-rater reliability – only using codes and themes which yielded 100% agreement among the researchers (Tashakkori & Teddlie, 1998). 342 S. K. Adams et al. 342 S. K. Adams et al. Socializing trumps sleep It’s hard to find a time where she’s not doing homework and I’m not out doing something else where we can both sit down and talk … I usually Skype with her later on [in the evening] and it keeps me up – because … I need [to talk with] my friends from back home! Another respondent explained how she uses technology to keep in touch with her boyfriend from high school. As with her peers, she found the best time to connect with him was late at night. She shared, Another respondent explained how she uses technology to keep in touch with her boyfriend from high school. As with her peers, she found the best time to connect with him was late at night. She shared, I’m always texting, especially at night. Like, like my boyfriend will text me because like he works during the day and I go to school during the day … so like it’s a big time we have to talk before we go to sleep. I’m always texting, especially at night. Like, like my boyfriend will text me because like he works during the day and I go to school during the day … so like it’s a big time we have to talk before we go to sleep. One participant summed up the desire to swap sleep for socializing in person and via social media when she said, ‘I just wanna be able to talk to my friends and hang out. I’m [also] always on my laptop, like on Facebook or something and that, like, keeps me up sometimes’. It is important to note that most of our participants described how socializing with friends on and off campus led to delayed onset of sleep. We found it interesting that very few attributed lack of sleep to homework or academics. In fact, most participants described how they attempted to schedule in homework and ‘get it done’ so they could socialize. Some respondents reported how staying up late socializing led to exhaustion in the afternoon. So, when he got home from classes, they fell asleep only to wake early in the evening to socialize. Then, he would squeeze in a little homework after other people went to bed. He recognized this pattern, along with an average of four hours of sleep, was not healthy. But, he could not seem to break the cycle. Socializing trumps sleep The first theme was socializing trumps sleep. During the interviews, students spoke at length about the connections between socializing and sleep. All but one student admitted to delaying onset of sleep in order to socialize with peers on campus as well as friends and family from home and high school via social media. One respondent explained how socializing not only went late into the night, but left her restless and unable to fall asleep once she got into bed. I have friends who I wanna talk to and after coming home from classes and then having homework and then going to dinner. It’s like when I finally have time to sit down and talk to them. At that point it’s late, so it’s like my mind’s going. [After I socialize], it takes a little while for me to settle down to be able to sleep, so, I mean, I definitely go to bed later now than I used to at home. Another participant explained a similar phenomenon. She stayed up late connecting with friends from high school. Because of their hectic class schedules, the only time they could connect on social media was late at night. Another participant explained a similar phenomenon. She stayed up late connecting with friends from high school. Because of their hectic class schedules, the only time they could connect on social media was late at night. Well, my best friend is in Boston now. I haven’t seen her since, um, I came here on moving day. It’s like we have two different lives, we’re doing different things. It’s hard to find a time where she’s not doing homework and I’m not out doing something else where we can both sit down and talk … I usually Skype with her later on [in the evening] and it keeps me up – because … I need [to talk with] my friends from back home! Well, my best friend is in Boston now. I haven’t seen her since, um, I came here on moving day. It’s like we have two different lives, we’re doing different things. Socializing trumps sleep She explained how living at home did not offer the same type of social opportunities as those who lived in the residence hall. She valued her sleep and explained how no social temptations would ever trump sleep. She said, ‘I guess not living on campus affects, like, my involvement in, like, social activities … I wouldn’t trade sleep to stay up and talk to somebody’. Fear of missing out One shared, My friends were typing papers and we were all just staying up. We were all like, ‘I’m tired!’ but we didn’t wanna, like, leave the conversation and miss something. So, it was like, ‘Oh, I’ll still get seven hours of sleep.’ Or it’s like, ‘Oh, I’ll just stay up because I don’t get to see you guys’. My friends were typing papers and we were all just staying up. We were all like, ‘I’m tired!’ but we didn’t wanna, like, leave the conversation and miss something. So, it was like, ‘Oh, I’ll still get seven hours of sleep.’ Or it’s like, ‘Oh, I’ll just stay up because I don’t get to see you guys’. Similarly, other students did not want to end conversations via social media for FoMO on a ‘really good conversation’. One explained, If I’m, like, talking to someone on the computer and it’s a really good conversation … I don’t wanna be, like, ‘I just need to go to bed.’ Like, I just wanna continue talking. Then I won’t get enough sleep. While students who reported living in the residence halls experienced the FoMO, the student who lived off campus described less pressure to socialize and, in turn, less disruption to her sleep. She alluded to the FoMO phenomenon, but explained how being physically removed from the residence hall social life allowed her to resist the temptation to stay awake. She shared: I think it’s beneficial to commute … I don’t live in a dorm and have all those people, like, keeping me up at night and it’s help[ed], like, my studying and stuff like that … I don’t feel pressure to, like, not study if … they’re like, ‘Wanna just hang out or do other things or go out?’ Socializing trumps sleep Generally [I get] like 4 h of sleep a night. [I know] that’s not healthy [and I] need to go to sleep earlier [and] need to get work done earlier. But then I’d get in the pattern of staying up late, which meant that when I got home I’d be so tired I’d fall asleep and then I’d wake up [and] do my homework late. You know, it was just like a cycle that I couldn’t get out of. He knew sleep was important, but so was socializing and homework. When time was limited, he chose homework and socializing over a good night sleep. Similarly, other students reported socializing until very late at night and then trying to complete homework after peers went to bed. One explained, ‘I think it’s more like just hanging out earlier … and then I decide to work after everyone goes to bed’. International Journal of Adolescence and Youth 343 While many of our participants focused on night as a time to socialize, others explained how they made an effort to get homework done between classes during the day. This way, they were able to socialize at night and still get to bed at a reasonable hour. This effort, combined with a class schedule that did not begin until afternoon allowed one student to feel as if she slept enough. She reported, I really like the way that college is set up because I have a four-hour block in the afternoon. And I really kind of make an effort to get most of my homework done during that time … and classes start later so I have been getting a lot more sleep. It is important to note that the student who lived off campus was an exception to this theme. She explained how living at home did not offer the same type of social opportunities as those who lived in the residence hall. She valued her sleep and explained how no social temptations would ever trump sleep. She said, ‘I guess not living on campus affects, like, my involvement in, like, social activities … I wouldn’t trade sleep to stay up and talk to somebody’. It is important to note that the student who lived off campus was an exception to this theme. Fear of missing out The second and related theme that emerged from our data was a FoMO on something important. This fear is often related to social factors and can lead to a delay in the time students went to bed. While the prior section highlighted the self-reported ways that students delayed sleep for socializing, this theme shows that the lure of socializing was so strong that students often missed out on sleep because some- thing fun or important might happen. Nothing social could be happening, but they stayed awake just in case it did. One student shared, ‘if your roommate’s up or like there’s people talking in the hallways … you feel like you shouldn’t be asleep’. Another participant responded, ‘Everyone else is staying up, [so] you want to stay up later’. And similarly, ‘When you go to bed, you could be, like, awake with your friends and, like, be doing other things’. Furthermore, one participant explained how she was tempted to delay sleep so she could join peers who were watching a movie. It did not matter if she had already seen the film, or if the topic of the film was interesting, the impetus for delaying sleep was the FoMO on the socializing and binding that might happen during the course of the movie. She shared, In my dorm … most of the time we leave our doors open so if someone’s watching a movie they’ll be like, ‘Oh, we’re watching a movie.’ And it will be like 12:00 a.m. and it’s like a 2-h movie and you just go! In my dorm … most of the time we leave our doors open so if someone’s watching a movie they’ll be like, ‘Oh, we’re watching a movie.’ And it will be like 12:00 a.m. and it’s like a 2-h movie and you just go! In my dorm … most of the time we leave our doors open so if someone’s watching a movie they’ll be like, ‘Oh, we’re watching a movie.’ And it will be like 12:00 a.m. and it’s like a 2-h movie and you just go! Other participants explained how groups of students would resist going to bed even when they were tired because they did not want to‘miss something’. One shared, Other participants explained how groups of students would resist going to bed even when they were tired because they did not want to ‘miss something’. Social and technological distractions Like the other day I was trying to do homework and my cousin kept texting me and like every five minutes I would start reading something and he’d text me and I’d have to go back and re-read it and it makes the homework a lot longer which means you have to stay up longer. Other students noted they kept their cell phones within close proximity when they went to bed at night, potentially waking them when they receive a text message or an update from a social network site. In some cases, taking the potentially distracting cell phone to bed might have been related to the desire to be social and FoMO. For instance, one respondent shared, ‘I’ve been known to answer my [phone] or to answer texts while I’m sleeping’. For others, technological distractions were just part of everyday life. Many did not own an old-fashioned alarm clock. So, they relied on cell phones as their alarm. Participants reported keeping their phone (i.e. alarm) in close proximity (e.g. under their pillow) at night. Some found this to be more of a distraction and sleep interference than others. One respondent mentioned, ‘I set my alarm [on my phone] before I go to bed and I keep it under my pillow … sometimes I wake up if I get a text … sometimes I don’t’. Another student, however, did not feel her phone was a distraction at night. She seemed to be able to set boundaries and limit the distraction from social media, even when her phone was under her pillow. She shared, I kinda just put it under my pillow and like I use that as an alarm … I’m not, like, tempted to when I’m like going to bed [to], like, grab it and start texting people … I just like to put it down and go to sleep. We conclude this section with a final example that ties all three of our themes together. The incident shows how one young woman had a deep desire to connect socially with her long-distance boyfriend via skype. This young woman’s FoMO led her to stay on skype so late into the night that she (and her boyfriend) regularly fell asleep with the computer application open. Our study participant was then distracted by the noise of her roommate’s boyfriend snoring. Social and technological distractions Thus, the connections between social life trumping sleep, FoMO, and social living distractions can be seen in this final quote. The student explained, ‘My roommate falls asleep to her Skype … She Skypes with [her boyfriend] every second of the day … One time I was sleeping in the room and … I literally woke up because I heard her boyfriend snoring on Skype’. Social and technological distractions The third theme involved numerous distractions (e.g. noise in hallways, other people in room, etc.) as well as technological distractions. These distractions were related to the social nature of either the S. K. Adams et al. 344 residence halls or social media. One student compared the residence hall to home and explained how there were no distractions in high school to keep her awake. She said, ‘At home your parents went to bed and the house was quiet … [so] you’d be like, “Oh well, might as well go to bed too”, but [here there are lots of distractions]’. By contrast, another participant explained the multitude of distractions related to community living: It’s like there’s so much going on, there’s so much going on in the hallway, and, in my dorm, um, I know my friend’s dorm, their quiet hours start at ten, whereas mine start at eleven, so it’s–it’s a lot different because you hear the doors slamming and people aren’t trying to be quiet about it, like, they just let the doors slam, and it’s–it’s a big difference … because, to me, I wake up for, like, little things. Another student lamented the distractions, but admitted that she was also a culprit at times. She said, ‘People’s loudness [bothers me]. I feel like some people don’t have respect for other people in the hallway … [but] it’s not just other people’s fault, it’s me too.’ Another student lamented the distractions, but admitted that she was also a culprit at times. She said, ‘People’s loudness [bothers me]. I feel like some people don’t have respect for other people in the hallway … [but] it’s not just other people’s fault, it’s me too.’ hallway … [but] it’s not just other people’s fault, it’s me too.’ Physical distractions caused by living in a social environment were only part of the problem for our participants. Because we live in a technologically connected world, students also described how social media and other technological distractions kept them awake. One student explained how she was distracted from homework by her cousin who desired social connection. Instead of ignoring the texts or turning off her phone, she gave in to them, which delayed homework completion as well as sleep. Well also it’s like if I’m trying to do homework and my friend was texting me. Discussion This exploratory study examined student self-reported influences on sleep during the first semester of college. Findings revealed that students in their first year of college experienced impaired sleep due to a variety of factors. Three related themes emerged: socializing trumps sleep; FoMO; and technological distractions. International Journal of Adolescence and Youth 345 Choosing socializing over sleep was an important theme that emerged in the data. Some partici- pants identified difficulty balancing socializing and homework completion. Socializing would inevita- bly delay the start of homework, or vice versa, with both scenarios leading to sleep delays. However, because first-year students often have early morning classes, waking up for classes left students sleep deprived. This could also lead to napping in the afternoon, therefore perpetuating the cycle of delayed sleep onset at night. The results also suggest that an important factor in the ability to balance sleep, social and academic demands was the ability to structure one’s time. For instance, students who were able to use large blocks of time during the daytime to complete homework had more time at night to socialize. Conversely, students who used their free daytime hours to nap had to utilize the night-time to complete homework and socialize, therefore delaying sleep onset. Another related social factor that led to sleep delay was FoMO. Participants reported both in person and virtual pressures to stay awake and engage with peers. For instance, some participants did not want to go to sleep and miss social interactions with their peers. Others felt pressure to stay awake, as they did not want to be the first person to go to bed. These pressures also include virtual and technological pressures, such as not wanting to leave an online or skype conversation. This finding sheds insight into the vector moving through autonomy towards interdependence (Chickering & Reisser, 1993). To move through the autonomy and independence vector, students must learn time management as well as function self-sufficiently with little regard for other’s opinions. One of the key aspects of this vector is the ability to set boundaries regarding other’s expectations and a ‘freedom from continual and pressing needs for reassurance, affection or approval’ (p. 47). Our findings suggested that FoMO was so strong that it often led to lack of boundaries, which negatively impacted sleep. Discussion It seems as if students were so bound by other’s opinions, actions, and even potential actions that they delayed sleep to socialize or wait for socializing to happen. As college students engage in the process of identifying new peer groups, it may be anxiety provok- ing to be the first person to disengage from the group by deciding it is time to go to sleep. First-year students may not want to appear disinterested in their new friends and therefore overcompensate by over-engaging and setting few boundaries on their time. In this study, FoMO was so strong that they had difficulty ‘unplugging’ from each other both in person and through technology. Participants felt that they ‘shouldn’t sleep’ because they might miss something important. As emerging adults develop mature relationships characterized by increased separation, independence and distance from peers, they may be more able to unplug from peers, engage in self-care, and feel secure in the fact that their friends will still be present in their lives. The social nature of college life, and specifically residence hall living, also introduces newly matric- ulated students to multiple distractions that were not present when they lived at home. Our findings affirm prior studies that suggest environmental influences impact sleep (Lund et al., 2010). However, our data push beyond those studies to show how the environmental influences were not only physical (e.g. light, noise), but were inherently tied to the social nature of communal living (e.g. residence halls) and the ever-present social connections made possible by technology. Some distractions may have been present at home during high school, such as technology. It is common for adolescents of all ages to use cell phones before bed and/or in bed, thus interfering with their ability to fall asleep and remain asleep (National Sleep Foundation, 2013). Many college students will answer texts as they are trying to fall asleep or will wake up from sleep to answer texts. These behaviours may be related to a FoMO, or social norms that dictate the need to immediately acknowl- edge text messages. A few of our participants, however, were able to regulate their technology use and were not overly distracted by technological demands during sleep. Future research should explore this phenomenon further. Although three primary themes were identified, the associations among sleep and each individual theme were complex. Disclosure statement No potential conflict of interest was reported by the authors. Discussion As the final quote showed, there were connections between the act of socializ- ing, FoMO, and the social distractions of community living and our technologically connected world. While future research is needed to explicate the connections between these themes, our study adds to the literature by showing three unique, but interrelated reasons why first-semester college students S. K. Adams et al. 346 sacrificed sleep. Although we did not measure the type or quantity of sleep, all participants alluded to the fact that they did not get enough – some citing as little as four hours a night. Since inadequate sleep negatively impacts individuals across all domains of functioning (e.g. academic, physical, emo- tional), these findings offer scholars and practitioners rich qualitative information about why students report a deficit of sleep. In returning to our combined biopsychosocial and developmental theoretical perspectives, we argue that parents, scholars and practitioners must find ways to help college students develop mature interpersonal relationships and autonomy and interdependence in ways that do not require giving up sleep. The psycho-social and bio-psychical developments are both crucial to human development. In this study, we found, with one exception, that first-year students were not very suc- cessful at achieving optimal development in any of these areas. Acknowledgements Acknowledgements Special thanks to Jennifer Daly for her contributions to data collection and analysis. Limitations and future directions Several limitations to this study must be noted. To begin, because of the homogeneity (e.g. mostly women who lived in residence halls) and small size of the sample, results may not be transferrable to commuter students or other settings. Thus, future research should explore factors contributing to impaired sleep among a larger, more diverse population (e.g. males, athletes, students who lived off campus, students who are employed). Secondly, sleep duration and quality were not directly assessed as part of this study. Future research should combine student self-reported narratives about college life and sleep with precise measurements of sleep activity via technology or sleep diaries. It is possible that results may differ among first-year students based on their level of sleep problems. Future research should explore factors influencing sleep impairment among a population of students who report prob- lems with sleep deprivation in attempt to gain a greater understanding of factors impacting sleep impairment during emerging adulthood. These factors might include sociocultural factors that could impact sleep, such as pre-existing mental health issues and academic, occupational or environmental stressors that impact sleep and overall well-being during the first year of college. International Journal of Adolescence and Youth 347 Alyssa Francis, MA, graduated from Quinnipiac University with a BS in Psychology, as well as a MA degree in Human Development from the University of Rhode Island. She is currently enrolled as a doctoral graduate student in School Psychology at the University of Rhode Island. Barbara Newman, PhD, is a professor emeritus in the Department of Human Development and Family Studies at the University of Rhode Island. Her research areas focus on parent–child relationships in early adolescence, factors that pro- mote success in the transition to high school and the relationship of a sense of group belonging to positive adjustment. Dr. Newman has published numerous journal articles and is co-author of a lifespan textbook, ‘Development through life: A h i l h’ Barbara Newman, PhD, is a professor emeritus in the Department of Human Development and Family Studies at the University of Rhode Island. Her research areas focus on parent–child relationships in early adolescence, factors that pro- mote success in the transition to high school and the relationship of a sense of group belonging to positive adjustment. y University of Rhode Island. Her research areas focus on parent–child relationships in early adolescence, factors that pro- mote success in the transition to high school and the relationship of a sense of group belonging to positive adjustment. 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Adams, PhD, is an associate professor in Human Development and Family Studies at the University of Rhode Island. Sue's research interests include examining the role of technology use on sleep and mental health in college students. She has published articles in journals including Journal of Youth Development, Academic Pediatric and Cyberpsychology, Behavior and Social Networking. Desiree' N. Williford, BS, graduated from the University of Rhode Island with a degree in Human Development and Family Studies and Psychology. She is currently a doctoral graduate student in Clinical Psychology at West Virginia University. Annemarie Vaccaro, PhD, is an associate professor in the Department of Human Development and Family Studies at the University of Rhode Island. Annemarie's teaching and scholarly interests focus on intersectionality, diversity and social jus- tice issues in higher education. 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https://openalex.org/W3165322705	https://www.nature.com/articles/s41598-021-90254-y.pdf	English	null	A validated model for predicting live birth after embryo transfer	Scientific reports	2,021	cc-by	6,039	Michael S. Awadalla , Kristin A. Bendikson, Jacqueline R. Ho , Lynda K. McGinnis & Ali Ahmady Accurately predicting the probability of live birth and multiple gestations is important for determining a safe number of embryos to transfer after in vitro fertilization. We developed a model that can be fit to individual clinic data for predicting singleton, twin, and total live birth rates after human embryo transfer. The predicted and observed rates of singleton and twin deliveries were compared in a tenfold cross-validation study using data from a single clinic. The model presented accounts for patient age, embryo stage (cleavage or blastocyst), type of transfer cycle (fresh or frozen) and uterine/universal factors. The standardized errors for rates of singleton and twin deliveries were normally distributed and the mean errors were not significantly different from zero (all p > 0.05). The live birth rates per embryo varied from as high as 43% for fresh blastocysts in the 35-year-old age group to as low as 1% for frozen cleavage stage embryos in the 43-year-old age group. This quantitative model or a simplified version can be used for clinics to generate and analyze their own data to guide the number of embryos to transfer to limit the risk of multiple gestations. Although the goal of in vitro fertilization (IVF) and other fertility treatments is a single healthy intrauterine pregnancy and delivery, twins and higher order multiples are a frequent occurrence. According to the 2016 Center for Disease Control (CDC) Assisted Reproductive Technology (ART) National Summary Report, of the 19,137 live deliveries from ART cycles using fresh embryos with fresh nondonor eggs, 18.8% were twin deliver- ies and 0.6% were triplets or more1. Multiple gestations increase maternal and fetal morbidity and mortality not only through preterm labor and delivery but also preeclampsia, gestational diabetes, and other pregnancy complications2. A recent American Society for Reproductive Medicine (ASRM) practice committee document gives recommended limits to the number of embryos to transfer and encourages individual clinics to use their own data in order to minimize multiple gestations3. p g Many previous models have been developed to guide the number of embryos to transfer4–10. Typical mod- els build on the Speirs model which assumes that embryos implant independently when uterine factors are favorable4. Evidence supports the main assumption of the Speirs model that an individual embryo’s probability of implantation and live birth is not directly impacted by other embryos but there are universal factors that affect all embryos transferred concurrently4,9,11,12. www.nature.com/scientificreports www.nature.com/scientificreports Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Keck School of Medicine, University of Southern California, LAC+USC Medical Center, 2020 Zonal Avenue, IRD Room 533, Los Angeles, CA 90033, USA. email: michael.awadalla@med.usc.edu Scientific Reports \| (2021) 11:10800 A validated model for predicting live birth after embryo transfer OPEN Michael S. Awadalla , Kristin A. Bendikson, Jacqueline R. Ho , Lynda K. McGinnis & Ali Ahmady Michael S. Awadalla , Kristin A. Bendikson, Jacqueline R. Ho , Lynda K. McGinnis & Ali Ahmady These factors have been described in both general and specific terms such as “uterine receptivity”, “endometrial receptivity”, and “transfer efficiency”4,5. If universal factors are favorable all embryos are more likely to implant than if universal factors are not favorable. Taking universal fac- tors into account results in higher predicted rates of multiples and lower predicted rates of singleton pregnancy than would be expected if embryos simply combined according to individual embryo implantation rates (as if each embryo is modeled as a coin flip). This concept of universal factors is supported by previous research that found higher rates of multiple pregnancy than predicted by a model which did not account for universal factors9. g g y y Despite the availability of these models, routine use of prediction models to guide the number of embryos to transfer is not practiced and current guidelines are based on general rather than individual clinic data3. This begs the question: why have these models not been adopted for use in clinical practice? Several possible explanations include complexity that limits implementation, the information provided in some studies is not clinically use- ful, rapidly advancing IVF technology outdates models quickly, and reluctance to use a model that is not easy to understand. There is clearly a need for a simple, accurate, and quantitative model that can be updated with current data and easily applied to individual clinic data. y pp Single embryo transfer (SET) is a great option to limit the risk of multiple pregnancies but carries important limitations. Some patients may desire multiple embryo transfer for cost effectiveness, after failing one or more SETs, or for a cleavage stage embryo transfer. According to the CDC 2016 ART National Summary Report, the average number of embryos transferred for different age groups ranged from 1.4 to 1.6 embryos in autologous Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Keck School of Medicine, University of Southern California, LAC+USC Medical Center, 2020 Zonal Avenue, IRD Room 533, Los Angeles, CA 90033, USA. email: michael.awadalla@med.usc.edu Scientific Reports \| (2021) 11:10800 \| https://doi.org/10.1038/s41598-021-90254-y www.nature.com/scientificreports/ Figure 1. Embryo transfer cycles included and excluded. Figure 1. Embryo transfer cycles included and excluded. frozen embryo transfer (FET) cycles and from 1.5 to 2.4 embryos in autologous fresh cycles1. Michael S. Awadalla , Kristin A. Bendikson, Jacqueline R. Ho , Lynda K. McGinnis & Ali Ahmady Preimplantation genetic testing for aneuploidy (PGT-A) has aided in selecting embryos for SET but has limitations including cost, embryologist expertise required to perform a trophectoderm biopsy, turnaround time which makes fresh embryo transfer logistically challenging, and possible embryo damage from the biopsy. Given the fact that it took many years to determine that cleavage stage biopsies decrease implantation potential13, it is possible that the same may be discovered for blastocyst biopsy. Although a recent non-selection study did not detect a decreased sustained implantation rate after blastocyst biopsy14, results from a recent multicenter randomized clinical trial suggest that this is likely not the case at all clinics15. The recent multicenter trial showed no difference in ongoing pregnancy rate after single frozen embryo transfer based on selection by PGT-A versus morphology of unbiopsied blastocysts (50% vs 46%). This seems to indicate that blastocyst biopsy either decreased the live birth rate of an embryo, that viable embryos were not classified as euploid, or a combination of both. Blastocysts with mosaic and aneuploid biopsy results have indeed been shown to occasionally result in healthy liveborn children16–18. Additionally, the long-term effects of embryo biopsy on human growth and development are unknown. Thus, there is still a role for transferring untested cleavage and blastocyst stage embryos. The ability to use one’s own clinic data to guide the number of untested embryos to transfer is pertinent to modern practice.h g y p p The primary objective of this study is to develop and validate a practical model based on individual clinic data for predicting singleton, twin, and total live birth rates after IVF with transfer of one or more untested embryos. Additional objectives include demonstrating how a model can be fit to individual clinic data, reporting the results of fitting the model to our clinic data, providing a framework for future studies to include embryo morphology in a model, and to provide the necessary methods and computer code to fit the model to other datasets. Materials and methods For example, the predicted probability of twins in panel b is UNI × LBRe1 UNI × LBRe2 UNI . This same logic can be applied to transfer of more than two embryos. UNI, universal factors fraction; LBRe1, live birth rate for embryo 1; LBRe2, live birth rate for embryo 2. This figure is being reproduced from a previous publication19. Figure 2. Embryo transfer models demonstrated for transfer of two embryos. (a) Demonstrates logic for independent embryo implantation and (b) demonstrates logic for independent embryo implantation only when universal factors are favorable. The universal factors fraction is a fraction from 0 (never favorable) to 1 (always favorable). The probability of each outcome is equal to the product of the terms next to the corresponding arrows above and the corresponding terms on the perimeter of the square. For example, the predicted probability of twins in panel b is UNI × LBRe1 UNI × LBRe2 UNI . This same logic can be applied to transfer of more than two embryos. UNI, universal factors fraction; LBRe1, live birth rate for embryo 1; LBRe2, live birth rate for embryo 2. This figure is being reproduced from a previous publication19. transferred and when universal factors are always favorable (UNI = 1). The assumptions and logic in Fig. 2b are used in this analysis. transferred and when universal factors are always favorable (UNI = 1). The assumptions and logic in Fig. 2b are used in this analysis. Determination of live birth rates. Live birth rates (LBRs) per embryo were determined by age at oocyte retrieval and four categories of embryo transfer: fresh cleavage stage, frozen cleavage stage, fresh blastocyst stage, and frozen blastocyst stage. Patients aged 25–34 years were analyzed as a single group. For patients aged 35 years old and greater, live birth rates for an individual age were based off of a five-year group of patients cen- tered on the age of interest. The analysis was performed this way to smooth out random variation in the data. For example, all embryo transfer data from patients aged 33–37 years was used to generate live birth rates for patients 35 years old. Each embryo transfer was modeled as an equation as follows: number of embryos trans- ferred × LBR = number of live births that resulted. Materials and methods Study population. Patients undergoing IVF at a single center from May 2015 through April 2018 were included in the study. Day 3 embryos were included in the cleavage stage group while day 5 and day 6 embryos were included in the blastocyst stage group. Exclusion criteria were no transfer, gestational carrier, preimplanta- tion genetic testing, frozen oocyte embryo transfer, embryos frozen on different days, day 2/4/7 embryo trans- ferred, and one or more morulas transferred (Fig. 1). Our analysis included 684 embryo transfers of a total of 1675 embryos for an average of 2.4 embryos per transfer. All of the data was collected prospectively in real time. Demographics and cycle characteristics are included in Supplemental Table S1. Embryo transfer model. Our embryo transfer model is defined by the logic in Fig. 2 which is demon- strated for a transfer of two embryos. This logic can be easily extended to transfer of any number of embryos. Rather than each embryo implanting independently as shown in Fig. 2a, we assume that embryos implant inde- pendently only when universal factors are favorable as shown in Fig. 2b. To apply the embryo transfer model to predict outcomes, the live birth rate (LBR) for each embryo and universal factors fraction (UNI) for each transfer must be known. The LBRs are represented as fractions between 0 and 1. UNI is represented as a frac- tion between 0 (never favorable) and 1 (always favorable). By the logic in Fig. 2a the predicted rate of twins per transfer is LBRe1 × LBRe2 while the logic in Fig. 2b predicts a rate of UNI × LBRe1 UNI × LBRe2 UNI which simplifies to LBRe1×LBRe2 UNI . The model shown in Fig. 2b simplifies to that of Fig. 2a in two scenarios: when only one embryo is https://doi.org/10.1038/s41598-021-90254-y Scientific Reports \| (2021) 11:10800 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Embryo transfer models demonstrated for transfer of two embryos. (a) Demonstrates logic for independent embryo implantation and (b) demonstrates logic for independent embryo implantation only when universal factors are favorable. The universal factors fraction is a fraction from 0 (never favorable) to 1 (always favorable). The probability of each outcome is equal to the product of the terms next to the corresponding arrows above and the corresponding terms on the perimeter of the square. Discussionh The methods used in this analysis differ from those of previous models through the use of moving age groups, estimation of LBRs using linear algebra, a universal factors fraction, automated data processing and model validation through custom computer programs in MATLAB, and a simplified presentation of the end results in tables that can be used quickly in clinic (Fig. 4). The methods used in this analysis differ from those of previous models through the use of moving age groups, estimation of LBRs using linear algebra, a universal factors fraction, automated data processing and model validation through custom computer programs in MATLAB, and a simplified presentation of the end results in tables that can be used quickly in clinic (Fig. 4). In this analysis we introduce a “moving groups” analysis when determining embryo live birth rates. We group patients less than 35 years old into one age group because clinically we expect similar live birth rates and there were small numbers of patients in this group. For ages 35 through 43 years, 5-year moving groups centered on the age of interest best smoothed out random noise without over-smoothing. We assume that lower live birth rates associated with older patients in a group will balance out higher live birth rates from younger patients. We base our estimation of quality (live birth rate per embryo) on characteristics that can be determined prior to oocyte retrieval and can be easily compared between clinics: oocyte age at retrieval, embryo stage (cleavage or blastocyst) and cycle type (fresh or frozen). Including embryo morphology in a predictive model increases the complexity of validating the universal factors model and is more suited for a separate focused study. We have recently analyzed the association of cleavage stage and blastocyst morphology with live birth and described how morphology can be incorporated into the predictive model19,21. Using a live birth rate assuming aver- age embryo morphologic quality seems reasonable and is clinically useful for patient counseling both before retrieval and prior to transfer. We use a system of equations to determine best fit live birth rates by preference mostly because this approach is needed to evaluate data when embryos of different morphologic qualities are transferred concurrently. y We expand on the concept of “uterine receptivity” and present a new method for its estimation. www.nature.com/scientificreports/ was used to perform bootstrapping based on the logic in Fig. 2b to determine predicted outcome probability distributions for rates of singleton, twin, and total live births per embryo transfer for the embryo transfers in the excluded group. Observed, predicted, and residual live birth rates were graphed for the best fit model as well as for a model that assumes universal factors are always favorable (UNI = 1, Fig. 2a) for comparison (Supplemental Fig. S1). g Univariate analysis was performed to evaluate the means and distribution of the standardized residual errors for the fresh and frozen transfer predicted singleton and twin delivery rates. Two-tailed Z-tests were used to evaluate the mean standardized errors and quantile–quantile plots were used to evaluate the error distribu- tions. The mean standardized errors were not significantly different from zero for the fresh transfer predicted singleton delivery rate per transfer (p = 0.73) or twin delivery rate per transfer (p = 0.40). The mean standardized errors for frozen transfers were not different from zero for singleton delivery rate per transfer (p = 0.32) or twin delivery rate (p = 1.00). The observed standardized errors appeared to be approximately normally distributed on quantile–quantile plots (Supplemental Fig. S2). 95% confidence intervals (CIs) for the rates of live birth and ongoing pregnancy per embryo were determined using bootstrapping methods detailed in the supplemental methods section. Automation of data processing. We automate the process of data analysis so that data from an indi- vidual clinic (or multiple clinics) can be processed and live birth rates calculated quickly. Specifically, we use the “All Fields Export” feature in the clinic SART CORS database then extract pertinent cycles and cycle informa- tion with a SAS program (version 9.4, SAS Institute), and then perform the data analysis and validation studies through several custom MATLAB programs20. Since most of the analysis is automated, fitting a model to new data and validating the model takes only a few hours. Ethics approval. This study was approved by the University of Southern California IRB and was performed according to institutional guidelines. Informed consent. Per institutional guidelines, informed consent from participants was not required for this retrospective analysis of deidentified data. Resultsh The live birth rate per embryo varied from as high as 43% for fresh blastocysts in the 35-year-old age group to as low as 1% for frozen cleavage stage embryos in the 43-year-old age group as shown in Fig. 3a, b. 95% CIs and best fit ongoing pregnancy rates are shown in Supplemental Table S2. The best fit UNI was 0.68 for fresh embryo transfers and 0.75 for frozen embryo transfers (Supplemental Fig. S3). Best fit values of UNI for different group- ings of embryo transfers and based on ongoing pregnancy rates are shown in Supplemental Table S3. Based on these results and the logic in Fig. 2b, predicted embryo transfer outcomes are shown in Fig. 4 with the multiples column shaded green, yellow, red, or gray to indicate the risk of multiples at delivery (0–9%, 10–19%, 20–29%, or ≥ 30% respectively). Materials and methods The least squares solution for LBR was determined using linear algebra for all the transfers in a given age group and transfer category combination with the use of MATLAB (version 9.5, MathWorks). This analysis was also performed using ongoing pregnancy (defined as detection of a fetal heartbeat at 6–8 weeks gestation) as the outcome. Calculation of universal factors fraction. A computer simulation was performed to determine the best fit universal factors fraction (UNI) based on LBRs per embryo and actual transfer live birth outcomes using the logic in Fig. 2b 20. This was done by assuming that if universal factors are not favorable then no live births occur. However, if universal factors are favorable then each embryo behaves independently of the others and results in a live birth at a rate of LBR UNI . A best fit UNI was determined separately for fresh and frozen embryo transfers. The UNI was determined to best fit the number of observed singleton live births that resulted from multiple embryo transfers. The best fit solution was determined using a custom computer program in MATLAB which estimates the likelihood of outcomes of multiple embryo transfers for varying values of UNI 20. Values of UNI closer to 1 correspond to higher rates of singleton pregnancy and delivery. UNI values were also determined for transfer subgroups and using ongoing pregnancy as the outcome. Validation of the model and statistical analysis. To validate the model, a tenfold cross-validation study was performed separately for fresh and frozen embryo transfers using a custom computer program in MATLAB. Each embryo transfer was assigned to one of ten groups using a random number generator. To pre- dict the outcomes of group 1, groups 2 through 10 were used as training data to determine best fit rates of live birth for each age and embryo transfer category combination. Then the best fit UNI values were determined. This analysis was repeated 10 times, each time excluding a different group. A computer simulation in MATLAB https://doi.org/10.1038/s41598-021-90254-y Scientific Reports \| (2021) 11:10800 \| www.nature.com/scientificreports/ Discussionh In 1983 Speirs denoted uterine factors by a capital “U” and described the concept as the “upper limit of the overall pregnancy rate if a large number of healthy embryos were transferred”4. We use the same upper limit definition provided by Speirs but prefer the terminology “universal factors fraction” denoted by “UNI” to unambiguously refer to all factors that impact all embryos transferred concurrently. Universal factors include endometrial receptiv- ity, endometritis, anatomical uterine abnormalities, embryo transfer technique, uterine location of transferred embryos, embryo culture conditions, air quality, maternal medical comorbidities, parental genetic abnormalities, autoimmunity, and other factors. https://doi.org/10.1038/s41598-021-90254-y Scientific Reports \| (2021) 11:10800 \| www.nature.com/scientificreports/ Figure 3. Live Birth Rate per Embryo for (a) Blastocyst and (b) Cleavage Stage Embryos. 5-year moving groups are used to calculate live birth rates for ages 35 to 43. Figure 3. Live Birth Rate per Embryo for (a) Blastocyst and (b) Cleavage Stage Embryos. 5-year moving groups are used to calculate live birth rates for ages 35 to 43. The universal factors fraction can only be calculated through indirect measures such as outcomes of multiple embryo transfers. During transfer of multiple embryos with known live birth rates per embryo, a UNI closer to 1 will result in higher rates of singleton births and lower rates of twin births (Fig. 2b and Supplemental Fig. S3). In our analysis we decided to group fresh (UNI = 0.68) and frozen (UNI = 0.75) embryo transfers together when calculating the UNI and performing the validation studies. This grouping is based on current thought of uter- ine factors being more favorable in frozen transfers. For the frozen cleavage stage embryos there were only 88 total embryo transfers and our actual outcomes for this subgroup did not fit with a UNI value of 1 or less so the UNI value could not be determined for this subgroup (Supplemental Table S3). While this could be interpreted to mean that the implantation of one embryo impeded the implantation of additional embryos, a more likely explanation is that this is a result of random variation. We use best fit values of UNI since there was not enough data to determine UNI with any significant confidence. Approximately 5,000 transfers of multiple embryos are required to calculate UNI with a 95% CI of ± 0.05 20. Scientific Reports \| (2021) 11:10800 \| Limitations Due to the number of other factors considered and a limited dataset, we were unable to incorporate embryo morphology into this prediction model. The environment for embryo implantation may vary from patient to patient based on variables other than embryo stage and transfer cycle type (fresh or frozen) which are discussed here. While our model predicts rates of higher order multiples, our dataset contained only one case of triplet delivery and therefore we were not able evaluate use of this model for predicting higher order multiples. This model is meant to account only for dizygotic twinning. In clinical use, patients should be counseled that monozy- gotic twinning could add an additional 1–2% to the predicted rate of multiple gestation after transfer of a single embryo or multiple embryos. It is uncertain how applicable results from a single IVF clinic are to other clinics. Discussionh LBR, total live birth rate per embryo transfer; % mult., percentage of live deliveries that are multiples; % twins, percentage of live deliveries that are twin deliveries; % trip. or > , percentage of live deliveries that are triplets or greater. Figure 4. Predicted outcomes for (a) Fresh Blastocyst, (b) Fresh Cleavage Stage, (c) Frozen Blastocyst, and (d) Frozen Cleavage Stage Embryo Transfers. The multiples column results are shaded green, yellow, red, or gray to indicate the risk of multiples at delivery (0–9%, 10–19%, 20–29%, or ≥ 30% respectively). LBR, total live birth rate per embryo transfer; % mult., percentage of live deliveries that are multiples; % twins, percentage of live deliveries that are twin deliveries; % trip. or > , percentage of live deliveries that are triplets or greater. could be used for both fresh and frozen transfers. Predicted outcomes for different combinations of average live birth rate per embryo and number of embryos transferred are given for UNI = 0.70 in Supplemental Fig. S4 along with an example table which can be used for other clinics to easily analyze their own data. Conclusion We describe and validate a model for predicting live singleton and twin delivery rates after cleavage and blas- tocyst stage embryo transfers. We use a system of equations approach to concurrently analyze data from both single and multiple embryo transfers. Our quantitative estimation of universal factors is close to that estimated by Speirs in 1983. New datasets can be analyzed with these methods or a simplified version in hours. The model predictions are reported in easy-to-read tables that can be used to guide the number of embryos to transfer to maximize pregnancy rates while limiting multiple gestations. Discussionh Our calculated universal factors fraction of 0.68, or 68%, for fresh embryo transfers is similar to the value estimated by Speirs in 1983 of “about 70%”4. If we do not take universal factors into account and assume each embryo implants independently as if uni- versal factors are always favorable (UNI = 1, Fig. 2a) then our observed rate of twins is higher than predicted and the observed rate of singleton delivery is lower than predicted for fresh transfers (Supplemental Fig. S1D). This is corrected when we apply a best fit value of UNI to the same data (Supplemental Fig. S1B). This is less pronounced for frozen embryo transfers where the UNI value in our model is greater suggesting that universal factors are more favorable overall (Supplemental Fig. S1F and S1H). ( pp g ) Instead of presenting predicted outcomes by complicated equations involving exponentials and factorials, computer programs are used make the calculations and display the results in an easy-to-read format (Fig. 4)20. This incorporates the logic of concepts such as the binomial formula (which mathematically describes all pos- sible combinations of a given number of embryos) and the universal factors fraction (Fig. 2b). The predicted outcomes in Fig. 4 are similar to those observed by other investigators22–24. A simplified form of this model could be adopted by other clinics. Live birth rates for 5-year moving groups (centered on the age of interest) could be calculated simply as live births divided by number of embryos transferred. A universal factors fraction of 0.70 Scientific Reports \| (2021) 11:10800 \| https://doi.org/10.1038/s41598-021-90254-y www.nature.com/scientificreports/ Figure 4. Predicted outcomes for (a) Fresh Blastocyst, (b) Fresh Cleavage Stage, (c) Frozen Blastocyst, and (d) Frozen Cleavage Stage Embryo Transfers. The multiples column results are shaded green, yellow, red, or gray to indicate the risk of multiples at delivery (0–9%, 10–19%, 20–29%, or ≥ 30% respectively). LBR, total live birth rate per embryo transfer; % mult., percentage of live deliveries that are multiples; % twins, percentage of live deliveries that are twin deliveries; % trip. or > , percentage of live deliveries that are triplets or greater. Figure 4. Predicted outcomes for (a) Fresh Blastocyst, (b) Fresh Cleavage Stage, (c) Frozen Blastocyst, and (d) Frozen Cleavage Stage Embryo Transfers. The multiples column results are shaded green, yellow, red, or gray to indicate the risk of multiples at delivery (0–9%, 10–19%, 20–29%, or ≥ 30% respectively). Code availabilityh y The MATLAB code used to generate all of the data for figures and tables in this manuscript is being made avail- able through Mendeley Data at http://dx.doi.org/10.17632/mg8b5nv3g5. Received: 28 June 2020; Accepted: 7 May 2021 Received: 28 June 2020; Accepted: 7 May 2021 https://doi.org/10.1038/s41598-021-90254-y Scientific Reports \| (2021) 11:10800 \| www.nature.com/scientificreports/ References References 1. Centers for Disease Control and Prevention, American Society for Reproductive Medicine, Society for Assisted Reproductive Technology. 2016 Assisted Reproductive Technology National Summary Report. Atlanta (GA): US Dept of Health and Human Services. 5–18 (2018). 1. 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Does preimplantation genetic testing for aneuploidy (PGT-A) harm embryos? no—a multi-center, prospective, 14. Tiegs, A. W. et al. Does preimplantation genetic testing for aneuploidy (PGT-A) harm embryos? no—a multi-center, prospective, blinded, non-selection study evaluating the predictive value of an aneuploid diagnosis and impact of biopsy. Fertil. Steril. 112, e31 (2019). 5. Munné, S. et al. Preimplantation genetic testing for aneuploidy versus morphology as selection criteria for single frozen-thawed embryo transfer in good-prognosis patients: a multicenter randomized clinical trial. Fertil. Steril. 112, 1071–1079 (2019). 16. Fragouli, E. et al. Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid–aneuploid blas- tocysts. Hum. Genet. 136, 805–819 (2017). y 17. Scott, R. T. et al. Comprehensive chromosome screening is highly predictive of the reproductive potential of human embryos: A prospective, blinded, nonselection study. Fertil. Steril. 97, 870–875 (2012).t p p y 8. Greco, E., Minasi, M. & Fiorentino, F. Healthy babies after intrauterine transfer of mosaic aneuploid blastocysts [letter]. N. Engl J. Med. 373, 2089–2090 (2015).ft 19. Awadalla, M., Vestal, N., McGinnis, L. & Ahmady, A. Effect of age and morphology on live birth rate after cleavage stage embryo transfer. Reprod. Sci. 28, 43–51 (2021).t p 20. Awadalla, M. S. Author contributions M.A. performed the data analysis, modeling, and statistics. All authors contributed to the design, analysis, inter- pretation, and manuscript writing. All authors approved the final version of the manuscript. M.A. performed the data analysis, modeling, and statistics. All authors contributed to the design, analysis, inter- pretation, and manuscript writing. All authors approved the final version of the manuscript. M.A. performed the data analysis, modeling, and statistics. All authors contributed to the design, analysis, inter- pretation, and manuscript writing. 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https://openalex.org/W2564276977	http://revistas.unla.edu.ar/saludcolectiva/article/download/925/1098	Spanish; Castilian	null	El perfil general del excedente nutrimental en México en el periodo 1990-2013: un enfoque a partir del suministro energético de macronutrimentos y grupos de alimentos	Salud colectiva	2,016	cc-by	10,493	Artículo / Article 487 Artículo / Article 487 487 2Doctora en Ciencias Sociales, con especialidad en Antropología Médica. Profesora-Investigadora jubilada, Centro de Investigaciones Regionales Hideyo Noguchi, Universidad Autónoma de Yucatán, México. * El perfil general del excedente nutrimental en México en el periodo 1990-2013: un enfoque a partir del suministro energético de macronutrimentos y grupos de alimentos General profile of the nutrition surplus in Mexico from 1990-2013: An approach using the energy supplied by macronutrients and food groups José Cutberto Hernández Ramírez1, Judith Elena Ortega Canto2 1Doctor en Ciencias de la Salud, Universidad Autónoma de Yucatán, México. * RESUMEN En este texto se analiza la evolución del suministro excesivo de kilocalorías en México desde 1990 a 2013. Para cada año del periodo se estimó el requerimiento de energía y macronutrimentos de la población mexicana y se lo contrastó con el respectivo suministro per cápita para estimar la discrepancia entre requerimiento y suministro. Las discrepancias se analizaron como serie temporal. Así, el excedente energético osciló entre 700 y 800 kcal/día per cápita en todo el periodo y los azúcares-dulcificantes aporta- ron el mayor suministro energético por encima de su requerimiento. El exceso de lípidos se incrementó de modo intenso y constante principalmente por el aumento de lípidos de carne de aves de corral y cerdo. El exceso aportado por las bebidas alcohólicas tendió a polarizarse en el creciente consumo de cerveza. En suma, a partir de los azúcares- dulcificantes y la carne se tendió a configurar el suministro energético y su respectivo excedente. Esto tiene implicaciones directas en la prevalencia de enfermedades crónicas no transmisibles así como en el uso insostenible de la tierra, el agua y la energía. 2Doctora en Ciencias Sociales, con especialidad en Antropología Médica. Profesora-Investigadora jubilada, Centro de Investigaciones Regionales Hideyo Noguchi, Universidad Autónoma de Yucatán, México. * PALABRAS CLAVES Hipernutrición; Obesidad; Dieta Occidental; Nutrición, Alimenta- ción y Dieta; México. ABSTRACT This text analyzes the evolution of the excessive food energy supply in Mexico from 1990 to 2013. For each year, the energy and macronutrient requirements of the Mexican population were estimated and contrasted with the per capita energy supply. Discrepancies between requirement and supply were analyzed as a time series. The energy surplus ranged from 700 to 800 kcal per capita per day throughout the studied period and sugar/sweeteners contributed the highest above-requirement energy supply. Lipids excess increased steadily and intensely, mainly due to lipid increases from poultry and pork. Excess energy from alcoholic beverages tended to be concentrated into growing beer consumption. In summary, the energy supply and the corresponding surplus tended to be made up mainly of sugar/sweeteners and meat. This has direct implications for the prevalence of chronic non-communicable diseases as well as unsustainable use of land, water and energy. Salud Colectiva \| Universidad Nacional de Lanús \| ISSN 1669-2381 \| EISSN 1851-8265 \| doi: 10.18294/sc.2016.925 KEY WORDS Overnutrition; Obesity; Diet, Western; Diet, Food and Nutrition; Mexico. 1Doctor en Ciencias de la Salud, Universidad Autónoma de Yucatán, México. * INTRODUCCIÓN un 14% más que quienes comen una dieta más “tradicional” basada principalmente en maíz. Lo mismo ocurrió con las personas que ingirieron más lácteos, arroz, pasta, carnes, huevos, frutas y vegetales ya que presentaron un riesgo de obesidad de un 17% más(10). Por su parte, en las investigaciones basadas en cuestionarios de recordatorio de 24 hs se afirma que, en el periodo 1988-1999, el aporte de las grasas a la ingesta energética total diaria creció del 23,5% al 30,3% mientras que el aporte relativo de los cereales y las proteínas se redujo levemente(5). El engordamiento de las poblaciones humanas es un fenómeno registrado a partir de la segunda mitad del siglo XX. Según la Organización Mundial de la Salud (OMS) desde 1980 la prevalencia de obesidad se duplicó a nivel mundial y en el 2014 entre los mayores de 18 años en todo el mundo hay 13% y 39% de obesos y pre-obesos respectivamente(1). En otras palabras, el 52% de los individuos excede el límite del vo- lumen corporal normal. Gasto en alimentos auto declarado: Las investigaciones basadas en las encuestas na- cionales de ingresos y gastos en los hogares plantean que, entre 1984 y 1998, disminuyó el gasto total en alimentos; aunque el gasto relativo se incrementó en carbohidratos refi- nados (6,3%) y refrescos (37,2%)(5). Asimismo, se incrementó el gasto monetario en cereales, alimentos preparados para consumir en el hogar, bebidas sin alcohol y alimentos con- sumidos fuera de casa mientras que decreció el gasto en leguminosas y carnes(11). En ese contexto, una serie de investiga- ciones ha estudiado los cambios en la dieta de diversas poblaciones en el mundo durante las últimas cinco décadas. Así, se ha propuesto que en ese período en las poblaciones más obesas se incrementó la ingesta de alimentos refinados, azúcar, dulcificantes y grasas(2,3,4). En México, entre 1989 y 2012, la pre- valencia de exceso de peso creció(5,6,7) y los cambios en la alimentación presumiblemente vinculados a tal fenómeno se han estudiado desde diferentes enfoques aquí clasificados como: suministro disponible per cápita; consumo alimentario autodeclarado; gasto monetario en alimentos autodeclarado y ba- lanza comercial alimentaria. Balanza comercial alimentaria: Clark et al.(12) sostienen que con el Tratado de Libre Comercio de América del Norte la política comercial y agrícola estadounidense trans- formó el ambiente alimentario mexicano y que EE.UU. El perfil general del excedente nutrimental en México en el periodo 1990-2013: un enfoque a partir del suministro energético de macronutrimentos y grupos de alimentos KEY WORDS Overnutrition; Obesity; Diet, Western; Diet, Food and Nutrition; Mexico Salud Colectiva \| Universidad Nacional de Lanús \| ISSN 1669-2381 \| EISSN 1851-8265 \| doi: 10.18294/sc.2016.925 Hernández Ramírez JC, Ortega Canto JE. 488 SALUD COLECTIVA. 2016;12(4):487-504. doi: 10.18294/sc.2016.925 Salud Colectiva \| Licencia Creative Commons Reconocimiento — No Comercial 4.0 Internacional \| BY - NC INTRODUCCIÓN “exportó la obesidad” a México mediante dos vías complementarias: a) el in- cremento de las exportaciones de maíz, soya, azúcar y dulcificantes, carnes y productos listos para consumir, y b) la inversión cre- ciente de capital estadounidense en toda la cadena que conforma el sistema alimentario mexicano. Suministro disponible per cápita: Según este enfoque, en el periodo 1980-2000 se incrementó el suministro per cápita –y por tanto el consumo aparente– de grasas ani- males, verduras, frutas, bebidas alcohólicas, carnes, huevo y se redujo el de leguminosas, oleaginosas, aceites vegetales y mariscos. Asimismo de 1961 a 1981 la energía diaria disponible por habitante se incrementó sos- tenidamente y después se mantuvo constante en poco más de 3.000 kcal(8). Ahora bien, en los enfoques citados se describen los cambios en la dieta únicamente en términos de incrementos o decrementos, relativos o absolutos, en el consumo de ciertos alimentos, y no se describe ese consumo en términos de exceso. En otras palabras, no se especifica cuánto alimento es exceso y por qué lo es. Por ejemplo, aunque en México, de 1988 a 1999, el aporte energético diario de grasas pasó del 23,5% al 30,2%(5), la OMS recomienda un aporte de grasas del 15-30% de la ingesta energética total diaria(13); es Consumo alimentario autodeclarado: En las investigaciones basadas en cuestionarios de frecuencia de consumo semanal se afirma que de 1988 a 1999 se incrementó la ingesta de grasas y alimentos refinados(9). Además se propone que, en 2006, los mexicanos que consumieron más comida rápida, refrescos, bebidas alcohólicas, pan blanco, dulces y botanas tuvieron un riesgo de obesidad de 489 las hojas de balance elaboradas por la Organización de las Naciones Unidas para la Agricultura y la Alimentación (FAO), en las que se estima el suministro alimentario dis- ponible para el consumo humano en un país. Para ello, se suma la producción nacional más las importaciones, a las que se restan las exportaciones alimentarias, alimento para ganado, semillas, o destinado para usos no alimentarios, así como las pérdidas ocurridas por almacenamiento y transporte. La can- tidad de alimento disponible para consumo humano se divide entre la población existente en un año, y se obtienen estimaciones del su- ministro anual de alimentos por persona(14). decir, en ambos casos (23,5% y 30,2%) el consumo de grasas estuvo dentro del margen recomendado. INTRODUCCIÓN Además, en las discusiones y/o conclu- siones de los estudios pertenecientes a los primeros dos enfoques presentados no es clara la distinción entre macronutrimentos (proteínas, lípidos y carbohidratos), grupos de alimentos y alimentos concretos. Por tanto, la transmisión de resultados es más ambigua de lo que podría ser. En efecto, aquí se propone el concepto “excedente nutrimental” entendido como: la cantidad nutricionalmente innecesaria de kilocalorías ingeridas en un periodo esti- pulado y cuyo aporte constante se relaciona potencialmente con la acumulación de tejido adiposo. Así, proponemos que tal concepto es útil para articular explicaciones más pre- cisas respecto al exceso de peso en las po- blaciones y su relación con las tendencias y cambios coyunturales en el ambiente alimen- tario, es decir, qué tipo de comida está dispo- nible, dónde y a qué precio(12). p p Además, se realiza la conversión de los alimentos procesados a su equivalente en productos primarios con los cuales son elaborados, tal proceso se denomina estan- darización vertical. Por ejemplo, el pan es expresado en sus cantidades equivalentes de trigo, grasa vegetal y otros ingredientes uti- lizados para su preparación, de modo que los datos sobre disponibilidad de trigo in- cluyen su equivalente en trigo entero, harina de trigo y productos de harina de trigo. Asimismo, la clasificación de los productos específicos en grupos de alimentos, con sus respectivas estimaciones, se denomina estan- darización horizontal. Finalmente, los datos sobre el suministro alimentario per cápita se expresan cuantitativamente y mediante factores de composición alimentaria en tér- minos de energía, proteínas y lípidos ade- cuados tanto de productos primarios como de procesados. Así pues, el objetivo de esta investi- gación es describir los cambios generales del excedente nutrimental anual en México durante el periodo 1990-2013 en dos niveles de abstracción: macronutrimentos y grupos de alimentos. Esto proporcionará elementos que contribuirán a una mejor comprensión del suministro alimentario mexicano actual y su relación con diversas enfermedades cró- nicas vinculadas a la sobrealimentación. MÉTODOS Cabe resaltar que, en el caso de México, las hojas de balance de la FAO obtienen datos, principalmente, del Banco de Información Económica (BIE), depen- diente del Instituto Nacional de Estadística y Geografía (INEGI)(15). A su vez, el BIE presenta datos del sector alimentario prove- nientes de las encuestas de la Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación (SAGARPA), cuya esti- mación incluye, en el caso de maíz y trigo en granos, frijol, sorgo y arroz, la proporción no comercializable, o que no se lleva al mercado, que ciertos productores rurales usan para el Salud Colectiva \| Universidad Nacional de Lanús \| ISSN 1669-2381 \| EISSN 1851-8265 \| doi: 10.18294/sc.2016.925 Excesos absolutos Aunque el periodo de interés en este es- tudio es 1990-2013, en el cual se incrementó súbitamente la incidencia de exceso de peso en México, revisaremos brevemente ciertas tendencias previas del excedente nutrimental en tal país. Así, en la Figura 1 se observa cómo entre 1961 y 1980 el suministro ex- cesivo total diario de kilocalorías creció hasta casi duplicarse y luego, de la década de 1980 a 2010, se mantuvo casi estable. Datos Antes de estimar el excedente nutrimental en los años comprendidos entre 1990 y 2013 fue necesario: a) registrar el suministro ener- gético diario per cápita de macronutrimentos en cada año, y b) calcular el promedio del requerimiento energético diario per cápita de macronutrimentos de la población mexicana en cada año. Para registrar el suministro energético diario per cápita de alimentos se consultaron Salud Colectiva \| Universidad Nacional de Lanús \| ISSN 1669-2381 \| EISSN 1851-8265 \| doi: 10.18294/sc.2016.925 Hernández Ramírez JC, Ortega Canto JE. 490 cuatro pasos: 1) registro del suministro ener- gético diario per cápita de macronutrimentos en el año de interés; 2) estimación de los re- querimientos energéticos diarios de macro- nutrimentos para la población mexicana en dicho año; 3) sustracción del requerimiento energético diario de cada macronutrimento a su suministro energético diario per cápita en tal año y 4) sumar las diferencias obtenidas en el paso tres, más el aporte energético diario per cápita proveniente de las bebidas alcohólicas en el mismo año. autoconsumo(16,17). Pero para estimar el su- ministro de todos los demás alimentos, en el BIE solo se incluye la producción legal que se comercializa y paga impuestos en el circuito del mercado. Por otro lado, para obtener el promedio anual del requerimiento energético diario per cápita de macronutrimentos de la po- blación mexicana, se consultaron estándares antropométricos(18) para conocer el peso medio para la edad en grupos quinquenales por edad y sexo, y multiplicarlo por su factor correspondiente a la tasa metabólica basal por kg de peso corporal(19). En los mayores de 10 años tal producto se multiplicó por un co- eficiente de 1,55 relativo al gasto energético por actividad física. Análisis El excedente nutrimental anual durante el periodo 1990-2013 se estudió como serie temporal de la cual se analizaron, tanto en tér- minos absolutos como relativos, los cambios en los macronutrimentos que lo constituyen, así como sus respectivas variaciones anuales acumulativas y tendencias de suministro. Posteriormente se obtuvo un promedio ponderado del requerimiento energético diario de la población mexicana en cada año estudiado según las proporciones existentes de los grupos quinquenales por edad y sexo. Para ello se usaron las estimaciones y proyec- ciones poblacionales del Consejo Nacional de Población(20) de 1990 al 2013, y se añadió la revisión de los censos 1960, 1970 y 1980 del INEGI(21). Se estudiaron, además, las variaciones anuales acumulativas y cambios tendenciales de los grupos de alimentos que más contri- buyeron a la variabilidad en los suministros excedentes de azúcares y dulcificantes, carbohidratos y lípidos durante el periodo referido. Por último, los requerimientos energéticos de lípidos y proteínas se estimaron con base en el límite superior de los márgenes de las metas de ingestión de macronutrimentos propuestos por la OMS(13) en términos de porcentaje de la ingesta energética total, los cuales son: lí- pidos 15-30% y proteínas 10-15%. Respecto a la ingesta de carbohidratos, la OMS reco- mienda ajustar el porcentaje entre 55-75% (incluidos azúcares con menos del 10%) en función de la determinación previa de los re- querimientos de lípidos y proteínas. Por tanto, los porcentajes de la ingesta energética total considerados excedentes energéticos de ma- cronutrimentos fueron: lípidos >30%, car- bohidratos totales >55% (incluidos azúcares >10%) y proteínas >15%. Salud Colectiva \| Licencia Creative Commons Reconocimiento — No Comercial 4.0 Internacional \| BY - NC Cálculo del excedente nutrimental Además, durante las décadas de 1960 y 1970, el excedente nutrimental se conformó únicamente por energía de carbohidratos, azúcares-dulcificantes y bebidas alcohólicas. El excedente nutrimental total de cada año comprendido entre 1990 y 2013 se calculó en Salud Colectiva \| Licencia Creative Commons Reconocimiento — No Comercial 4.0 Internacional \| BY - N El perfil general del excedente nutrimental en México en el periodo 1990-2013 491 SALUD COLECTIVA. 2016;12(4):487-504. doi: 10.18294/sc.2016.925 0 100 200 300 400 500 600 700 800 900 1960 1970 1980 1990 2000 2010 kilocalorías excedentes por día Carbohidratos Azúcar y dulciicantes Lípidos Bebidas alcohólicas Proteínas Figura 1. Cambios en el excedente energético alimentario per cápita en cifras absolutas. México, 1960-2010. Fuente: Elaboración propia basada en datos del Consejo Nacional de Población (CONAPO)(17) y Food and Agriculture Organization Corporate Statistical Database (FAOSTAT)(14). 0 100 200 300 400 500 600 700 800 900 1960 1970 1980 1990 2000 2010 kilocalorías excedentes por día Carbohidratos Azúcar y dulciicantes Lípidos Bebidas alcohólicas Proteínas Figura 1. Cambios en el excedente energético alimentario per cápita en cifras absolutas. México, 1960-2010. Fuente: Elaboración propia basada en datos del Consejo Nacional de Población (CONAPO)(17) y Food and Agriculture Organization Corporate Statistical Database (FAOSTAT)(14). 0 100 200 300 400 500 600 700 800 900 1960 1970 1980 1990 2000 2010 kilocalorías excedentes por día Carbohidratos Azúcar y dulciicantes Lípidos Bebidas alcohólicas Proteínas Figura 1. Cambios en el excedente energético alimentario per cápita en cifras absolutas. México, 1960-2010. Fuente: Elaboración propia basada en datos del Consejo Nacional de Población (CONAPO)(17) y Food and Agriculture Organization Corporate Statistical Database (FAOSTAT)(14). Figura 1. Cambios en el excedente energético alimentario per cápita en cifras absolutas. México, 1960-2010. Fuente: Elaboración propia basada en datos del Consejo Nacional de Población (CONAPO)(17) y Food and Agriculture Organization Corporate Statistical Database (FAOSTAT)(14). Fuente: Elaboración propia basada en datos del Consejo Nacional de Población (CONAPO)(17) y Food and Agriculture Organization Corporate Statistical Database (FAOSTAT)(14). En síntesis, entre las observaciones más relevantes en el período 1990-2013 se en- cuentran: 1) la estabilidad del excedente total entre 700 y 800 kcal/día aproximadamente, 2) la permanencia de azúcar-dulcificantes y bebidas alcohólicas como elementos con- solidados del excedente nutrimental, y 3) se sugiere el inicio y la continuidad de una paulatina sustitución del aporte energético excesivo de carbohidratos por parte del cre- ciente exceso de lípidos. Cálculo del excedente nutrimental Y fue solo a partir de 1980 cuando las kiloca- lorías provenientes de lípidos se tornaron ex- cesivas para incrementarse constantemente después de 1990. Respecto a las kilocalorías de proteína, se integraron definitivamente como parte del excedente nutrimental desde el año 2000 en adelante (Figura 1). Entre 1990 y 2013 el excedente nutri- mental osciló entre 700 y 800 kcal/día, y en su composición proporcional desde el año 2000 se produjo la incorporación de un ligero exceso de energía proveniente de pro- teínas que presentó cambios irregulares –es decir, no mostró tendencia– y una variación media anual acumulativa del 1,8 % (Figura 2). Pero el cambio más sobresaliente fue el incre- mento lineal del exceso energético de lípidos (R2= 0,89) y la reducción lineal del exceso proveniente de carbohidratos (R2 = 0,68). Salud Colectiva \| Universidad Nacional de Lanús \| ISSN 1669-2381 \| EISSN 1851-8265 \| doi: 10.18294/sc.2016.925 Excesos relativos Cambios en el excedente energético alimentario per cápita en cifras absolutas. México, 1990-2013. uente: Elaboración propia basada en datos del Consejo Nacional de Población (CONAPO)(17) y Food and Agriculture rganization Corporate Statistical Database (FAOSTAT)(14). Nota: el valor 0 equivale al límite superior de la recomendación consuntiva diaria establecida por la OMS(13) para los respectivos ítems graficados. Todo valor superior a 0 aparece como excedente energético. Nota: el valor 0 equivale al límite superior de la recomendación consuntiva diaria establecida por la OMS(13) para los respectivos ítems graficados. Todo valor superior a 0 aparece como excedente energético. variación media anual acumulativa del -1,2% de 1990 a 2013; aunque aportó en promedio el 92% del suministro energético total de azú- cares y dulcificantes en tal periodo (Tabla 1). importancia respectivamente mientras que las proteínas aún muestran un exceso ligero (Figura 3). Además, los excesos energéticos rela- tivos de azúcares-dulcificantes y proteínas mostraron variaciones irregulares, mientras que los excesos energéticos relativos de car- bohidratos y lípidos sugieren una paulatina sustitución de los carbohidratos por los lí- pidos (Figura 3). Después del azúcar equivalente, los “dulcificantes”(14) (fructosa y maltosa quími- camente puras, glucosa, dextrosa, galactosa, isoglucosa, melazas, jarabe de fructosa y azúcar invertido) aportaron en promedio el 6,8% del suministro energético total de azú- cares y dulcificantes, no obstante su variación media anual acumulativa fue la mayor con un 9,9% (Tabla 1). Más aun, puede sugerirse que la reducción del aporte de azúcar equi- valente se compensó con el aumento ener- gético de los dulcificantes. Salud Colectiva \| Licencia Creative Commons Reconocimiento — No Comercial 4.0 Internacional \| BY - NC Excesos relativos Entre 1990 y 2013, el exceso energético per cápita de azúcares y dulcificantes se mantuvo alrededor del doble de su reque- rimiento específico según la ingesta reco- mendada por la OMS(13). Por tanto, puede plantearse que los azúcares y dulcificantes son el tipo de carbohidratos que se consumen más excesivamente en México. Después, los excesos de los demás carbohidratos y de los lípidos ocupan el segundo y tercer lugar de Por su parte, el aporte excesivo de azú- cares y dulcificantes se mantuvo con cambios irregulares y una variación media anual acu- mulativa del -0,8%. Asimismo, las bebidas al- cohólicas mostraron cambios irregulares y una variación media anual acumulativa del -0,3%. Salud Colectiva \| Universidad Nacional de Lanús \| ISSN 1669-2381 \| EISSN 1851-8265 \| doi: 10.18294/sc.2016.925 Hernández Ramírez JC, Ortega Canto JE. 492 SALUD COLECTIVA. 2016;12(4):487-504. doi: 10.18294/sc.2016.925 0 100 200 300 400 500 600 700 800 900 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 kilocalorías excedentes por día Carbohidratos Azúcar y dulciicantes Lípidos Alcohol Figura 2. Cambios en el excedente energético alimentario per cápita en c México, 1990-2013. Fuente: Elaboración propia basada en datos del Consejo Nacional de Población (CONAPO)(17) y Food Organization Corporate Statistical Database (FAOSTAT)(14). Nota: el valor 0 equivale al límite superior de la recomendación consuntiva diaria establecida por la respectivos ítems graficados. Todo valor superior a 0 aparece como excedente energético. SALUD COLECTIVA. 2016;12(4):487-504. doi: 10.18294/sc.2016.925 0 100 200 300 400 500 600 700 800 900 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 kilocalorías excedentes por día Carbohidratos Azúcar y dulciicantes Lípidos Alcohol Proteínas Figura 2. Cambios en el excedente energético alimentario per cápita en cifras absolutas. México, 1990-2013. Fuente: Elaboración propia basada en datos del Consejo Nacional de Población (CONAPO)(17) y Food and Agriculture Organization Corporate Statistical Database (FAOSTAT)(14). Nota: el valor 0 equivale al límite superior de la recomendación consuntiva diaria establecida por la OMS(13) para los respectivos ítems graficados. Todo valor superior a 0 aparece como excedente energético. 0 100 200 300 400 500 600 700 800 900 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 kilocalorías excedentes por día Figura 2. Cambios en el suministro energético total per cápita de azucares y dulcificantes La energía proveniente de “azúcar equivalente”(14) (azúcar centrifugado sin re- finar, azúcar refinado, azúcar para confi- tería y azúcares aromatizados) presentó una Por su parte, el azúcar no centrifugado y la miel aportaron en promedio el 0,8% y el 0,4% del suministro energético total de azú- cares y dulcificantes. Asimismo el azúcar no Salud Colectiva \| Licencia Creative Commons Reconocimiento — No Comercial 4.0 Internacional \| BY - N El perfil general del excedente nutrimental en México en el periodo 1990-2013 493 SALUD COLECTIVA. 2016;12(4):487-504. doi: 10.18294/sc.2016.925 R² = 0,713 R² = 0,886 0 20 40 60 80 100 120 140 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 % excedido sobre el requerimiento Azúcar y dulci-icantes Carbohidratos Lípidos Proteínas Lineal (Carbohidratos) Lineal (Lípidos) Figura 3. Cambios en los excesos relativos de macronutrimentos sobre su respectivo requerimiento. México, 1990-2013. Fuente: Elaboración propia basada en datos del Consejo Nacional de Población (CONAPO)(17) y Food and Agriculture Organization Corporate Statistical Database (FAOSTAT)(14). Nota: el valor 0% equivale al límite superior de la recomendación consuntiva diaria establecida por la OMS(13) para los respectivos ítems graficados. Todo valor superior a 0 aparece como excedente energético. 2(4):487-504. doi: 10.18294/sc.2016.925 Figura 3. Cambios en los excesos relativos de macronutrimentos sobre su respectivo requerimiento. México, 1990-2013. Fuente: Elaboración propia basada en datos del Consejo Nacional de Población (CONAPO)(17) y Food and Agriculture Organization Corporate Statistical Database (FAOSTAT)(14). Fuente: Elaboración propia basada en datos del Consejo Nacional de Población (CONAPO)(17) y Food and Agriculture Organization Corporate Statistical Database (FAOSTAT)(14). Nota: el valor 0% equivale al límite superior de la recomendación consuntiva diaria establecida por la OMS(13) para los respectivos ítems graficados. Todo valor superior a 0 aparece como excedente energético. su contribución al suministro energético total de carbohidratos; sin embargo, aun así, ambos conformaron la mayor parte de dicho sumi- nistro. De tal forma, los cereales mostraron un descenso con tendencia lineal (R2 = 0,8274) y una variación media acumulativa anual de -0,3%, mientras las leguminosas presen- taron una reducción con cambios irregulares y la respectiva variación media acumulativa anual del -0,3% (Tabla 1). centrifugado presentó una variación media anual acumulativa del -2,2%, mientras que la miel no mostró variaciones (Tabla 1). Cambios en el suministro energético total per cápita de azucares y dulcificantes En suma, el suministro energético per cápita diario de azúcares y dulcificantes en México durante el periodo 1990-2013 pre- sentó cambios irregulares sin tendencias fuertemente definidas, y una variación media anual acumulativa del -0,4%; no obstante, tales azúcares y dulcificantes se mantuvieron como el principal exceso relativo del exce- dente nutrimental. Cabe distinguir que la reducción del su- ministro energético proveniente de los ce- reales se debió a las disminuciones de los aportes de trigo, en primer lugar, y de maíz, en segundo; aunque ambos cereales apor- taron en promedio el 93,8% de las kiloca- lorías de cereales. En contraste, los demás cereales presentaron incrementos, incluso la avena mostró un aumento con buen ajuste lineal (R2 = 0,8011). Cambios en el suministro energético per cápita de carbohidratos En el periodo 1990-2013 se redujo el aporte energético de los carbohidratos al ex- cedente nutrimental en México debido a que los cereales y las leguminosas disminuyeron Salud Colectiva \| Universidad Nacional de Lanús \| ISSN 1669-2381 \| EISSN 1851-8265 \| doi: 10.18294/sc.2016.925 94 Hernández Ramírez JC, Ortega Canto JE. 494 Tabla 1. Proporciones promedio, variaciones y tendencias del suministro energético per cápita de macronutrimentos aportados por diversas fuentes. México, 1990-2013. Macronutrimentos y sus fuentes Proporción promedio en el periodo (%) Variación media anual acumulativa (%) Coeficiente de tendencia lineal (R2) Azúcares y dulcificantes Azúcar equivalente 92,0 -1,2 0,46 Dulcificantes 6,8 9,9 0,62 Azúcar no centrifugada 0,8 -2,2 0,64 Miel 0,4 0,0 0,03 Total 100,0 -0,4 0,01 Carbohidratos Cereales (total) 80,2 -0,3 0,83 Maíz 73,1 -0,3 0,60 Trigo 20,7 -0,6 0,78 Arroz 4,4 1,3 0,55 Avena 0,1 3,3 0,80 Otros 1,6 2,8 0,01 Frutas 6,4 0,4 0,14 Leguminosas 6,3 -0,3 0,39 Lácteos 3,1 0,1 0,26 Hortalizas 2,1 0,7 0,38 Tubérculos almidonados 1,9 0,2 0,54 Total 100,0 -0,2 0,55 Lípidos Animales 54,0 1,3 0,91 Vegetales 46,0 0,5 0,10 Total 100,0 0,9 0,92 Bebidas alcohólicas Cerveza 84,8 0,8 0,83 Bebidas alcohólicas destiladas 14,8 -4,8 0,81 Vino y bebidas fermentadas 0,4 - 0,4% Total 100,0 0,3 0,22 Proteínas Proteínas vegetales 57,0 -0,1 0,51 Proteínas animales 43,0 1,4 0,89 Total 100,0 0,5 0,74 Fuente: Elaboración propia basada en datos del Consejo Nacional de Población (CONAPO)(17) y Food and Agriculture Organization Corporate Statistical Database (FAOSTAT)(14). Tabla 1. Proporciones promedio, variaciones y tendencias del suministro energético per cápita de macronutrimentos aportados por diversas fuentes. México, 1990-2013 Fuente: Elaboración propia basada en datos del Consejo Nacional de Población (CONAPO)(17) y Food and Agriculture Organization Corporate Statistical Database (FAOSTAT)(14). Salud Colectiva \| Licencia Creative Commons Reconocimiento — No Comercial 4.0 Internacional \| BY - NC Cambios en el suministro energético per cápita de lípidos variación media anual acumulativa del 1,3%. Por su parte, los lípidos de origen vegetal pre- sentaron cambios irregulares con variación media anual acumulativa del 0,5% (Tabla 1). De 1990 a 2013 el incremento lineal del suministro energético per cápita de lípidos en México se debió al incremento lineal del aporte de lípidos de origen animal con Ahora bien, entre los lípidos de origen animal los contenidos en carnes y huevo fueron los que más contribuyeron al Salud Colectiva \| Licencia Creative Commons Reconocimiento — No Comercial 4.0 Internacional \| BY - N El perfil general del excedente nutrimental en México en el periodo 1990-2013 495 Tabla 2. Variaciones, tendencia y proporciones promedio del aporte energético per cápita de lípidos aportados por diversas fuentes animales en el periodo 1990-2013. Fuentes de lípidos animales Proporción promedio en el periodo (%) Variación media anual acumulativa (%) Coeficiente de tendencia lineal (R2) Lípidos de origen animal Carnes 49,0 2,4 0,97 Huevo 9,0 2,4 0,91 Lácteos 21,0 0,6 0,68 Grasas animales 18,0 -0,9 0,38 Pescado y frutos del mar 2,0 0,3 0,09 Vísceras 1,0 -0,2 0,41 Total 100,0 1,3 0,91 Lípidos de carnes Cerdo 51,6 1,5 0,93 Aves de corral 31,7 4,5 0,97 Vaca 14,2 0,5 0,21 Ovina y caprina 2,0 0,6 0,13 Otra 0,5 -2,1 0,78 Total 100,0 2,4 0,97 Fuente: Elaboración propia basada en datos del Consejo Nacional de Población (CONAPO)(17) y Food and Agriculture Organization Corporate Statistical Database (FAOSTAT)(14). Tabla 2. Variaciones, tendencia y proporciones promedio del aporte energético per cápita de lípidos aportados por diversas fuentes animales en el periodo 1990-2013. Fuente: Elaboración propia basada en datos del Consejo Nacional de Población (CONAPO)(17) y Food and Agriculture Organization Corporate Statistical Database (FAOSTAT)(14). incremento energético mencionado, ambos con una variación media anual acumulativa del 2,4% y una fuerte tendencia lineal de crecimiento expresada en un coeficiente R2 cercano a 1 (Tabla 2). Después, en tercer lugar aparecen los lípidos de lácteos, aunque lo hacen con una variación media anual acu- mulativa menor y una tendencia lineal con menos fuerza de asociación entre tiempo e incremento. aporte lipídico de pescados y frutos de mar, así como de las vísceras fue irrelevante, tanto en términos proporcionales como en tér- minos de contribución al incremento lineal observado en los lípidos de origen animal durante el periodo de estudio (Tabla 2). Salud Colectiva \| Universidad Nacional de Lanús \| ISSN 1669-2381 \| EISSN 1851-8265 \| doi: 10.18294/sc.2016.925 El tamaño del excedente nutrimental tipos de carne aportaron juntas el 83,3% del promedio total de lípidos provenientes de carnes en México durante el periodo estu- diado (Tabla 2). En México, desde 1961, el suministro de kcal/día supera el requerimiento de la po- blación; aunque debe distinguirse que de 1961 a 1980 tal exceso se incrementó de manera constante hasta casi duplicarse mientras que, entre 1980 y 2013, se mantuvo estable. Tales tendencias fueron descritas en un estudio en el que se plantea, sin profundizar, la “discre- pancia” entre suministro y requerimiento(8). Precisamente, nosotros profundizamos en ello al formular el concepto “excedente nutri- mental” y su respectivo análisis. En resumen, el crecimiento de la pro- porción de lípidos en el excedente nutrimental entre 1990 y 2013 se debió al incremento lineal del suministro de lípidos de origen animal y, específicamente, a los aportados por carne de aves de corral y de cerdo en primer lugar, seguidos por los provenientes de huevo y, finalmente, por los de lácteos. Cambios en el suministro energético per cápita de lípidos Pero entre las carnes cabe distinguir las que contribuyeron al incremento lineal de lípidos de origen animal. Así, fueron los lí- pidos provenientes de las aves de corral los que presentaron la mayor variación anual acumulativa con un 4,5% y una fuerte aso- ciación lineal tiempo-incremento (R2 = 0,97). Después de las aves de corral, el cerdo pre- sentó una variación anual acumulativa del 1,5% y una fuerte asociación lineal tiempo- incremento (R2= 0,93). Asimismo, ambos En términos proporcionales, los lípidos de las carnes aportaron, en promedio, el 49% del total de lípidos de origen animal. En segundo lugar, se encuentran los lácteos con el 21% y, en tercero y cuarto, las grasas animales y el huevo con promedios del 18% y del 9%, respectivamente. Por su parte, el Salud Colectiva \| Universidad Nacional de Lanús \| ISSN 1669-2381 \| EISSN 1851-8265 \| doi: 10.18294/sc.2016.925 Hernández Ramírez JC, Ortega Canto JE. 496 El exceso energético proveniente de las bebidas alcohólicas Pero la estimación del requerimiento energético promedio de la población mexicana realizada en esta investigación es un 27% mayor que la hecha por Ortiz et al.(8) y, por lo tanto, el tamaño de tal “discrepancia” o “excedente nutrimental”–en nuestros tér- minos– fue menor. En ese sentido, nuestra estimación presenta las siguientes caracterís- ticas y diferencias: El alcohol no es un alimento necesario dentro del requerimiento nutrimental de la población mexicana. En efecto, el 100% de la energía aportada por esta bebida se con- sideró innecesario y, por tanto, excedente. Así, en el periodo 1990-2013, la pro- porción de bebidas alcohólicas en el ex- cedente nutrimental se redujo, con una variación media anual acumulativa del -0,3% y cambios irregulares; pero tal reducción se debió solo a la disminución lineal (R2 = 0,81) del aporte de bebidas destiladas ya que, en contraste, el aporte energético de la cerveza, además de ser el mayor, se incrementó con su respectiva variación del 0,8% y una ten- dencia lineal con valor R2 = 0,83 (Tabla 1). Se basa en la Convención de Roma (19), la cual, a la luz de más evidencias científicas que su homóloga de Ginebra(22) –usada por Ortiz et al.–, presenta modificaciones en los requerimientos de niños y adolescentes. Para calcular el requerimiento energético de mayores de 18 usamos un factor de 1,55 que correponde al promedio para activi- dad física sedentaria. En contraste, según la Convención de Roma, el factor de 1,4 –usado por Ortiz et al.– es más adecuado para estimar requerimientos energéticos en intervenciones nutricias de corta duración dirigidas a personas dependientes con in- actividad total y en condiciones de crisis. Salud Colectiva \| Licencia Creative Commons Reconocimiento — No Comercial 4.0 Internacional \| BY - NC DISCUSIÓN La información aportada en esta inves- tigación presenta tanto convergencias como divergencias con los planteamientos pro- puestos en otros estudios. A continuación se discuten tales concordancias y contrastes en torno a cuatro elementos: 1) tamaño del exce- dente nutrimental, 2) cambios en los aportes de macronutrimentos, 3) el excedente nutri- mental y la industria de la alimentación y 4) el enfoque del suministro alimentario frente a las encuestas de alimentación. Las referencias antropométricas de peso para la edad que utilizamos están mucho más desagregadas por grupos de edad; por consiguiente, el cálculo del requerimiento es más específico para cada grupo de edad en función de su peso promedio estándar. Ciertamente, a excepción de Ortiz et al.(8), en algunas de las investigaciones más relevantes sobre la evolución de los pa- trones alimentarios en México, se obvia tal Salud Colectiva \| Licencia Creative Commons Reconocimiento — No Comercial 4.0 Internacional \| BY - NC 497 “discrepancia” y únicamente se describen los cambios porcentuales en la distribución de macronutrimentos en la dieta(9,5). Incluso, en algunas de ellas, basadas en recordatorios de 24 horas, se propone que la ingesta pro- medio diaria de las mexicanas mayores de 18 años en 1999 fue de 1.636 kcal diarias(5); mientras la FAO(14), en contraste, reportó que en tal año el suministro energético per cápita en México fue de 2.982 kcal diarias; es decir, casi el doble. incluso en los datos de 2012 en adelante la Encuesta Nacional Agropecuaria (ENA) im- plementa un método de verificación satelital de la superficie sembrada para cotejar lo de- clarado en las entrevistas. Esto último es im- portante para garantizar una mayor precisión de los datos a partir de tal año. Así las cosas, si se consideran las po- sibles fuentes de subregistro mencionadas, el tamaño del excedente nutrimental propuesto en este artículo –de por sí ya mayor a los excesos energéticos reportados en todas las investigaciones precedentes– podría ser to- davía más grande. Ahora bien, como se comentó más arriba, con excepción de los suministros de maíz y trigo en granos, frijol, sorgo y arroz en México, en los cuales se consideran tanto el consumo comercializado como el auto- consumo, para todos los demás suministros calculados respecto a tal país en las hojas de balance de la FAO solo se considera la pro- ducción legal que se comercializa y paga im- puestos. DISCUSIÓN Esto puede conllevar al subregistro del autoconsumo y la comercialización no registrada o “informal” de frutas, hortalizas y oleaginosas, así como de la caza y la pesca de variedades distintas a las domesticadas y que no se incluyen en las categorías del ca- tálogo de registro de información pecuaria en las encuestas mexicanas. Salud Colectiva \| Universidad Nacional de Lanús \| ISSN 1669-2381 \| EISSN 1851-8265 \| doi: 10.18294/sc.2016.925 Cambios en los aportes de macronutrimentos Tanto el método basado en el suministro alimentario como el apoyado en encuestas de consumo de alimentos autodeclarado coinciden en un punto: la ingesta de grasas en la población mexicana se incrementó de manera constante durante la última década del siglo XX. Más aún, en esta investigación, basada en el suministro alimentario per cápita, se evidencia que, hasta el año 2013, persistió un aumento lineal del aporte excesivo de lí- pidos tendiente a sustituir el exceso provisto por los carbohidratos. En efecto, es posible sugerir que el valor total del excedente nutrimental puede ser aún mayor a nuestra estimación, especialmente, por un subregistro de la energía proveniente de proteínas y grasas aportadas por ciertos productos pecuarios y oleaginosas, carbohi- dratos de frutas y hortalizas así como por los tipos de bebidas alcohólicas que no tienen control fiscal o sanitario. Asimismo, respecto al azúcar de caña, los datos más recientes obtenidos mediante información satelital e instrumentos de recolección mejorados muestran que, al menos en 2014, del 100% de la producción nacional de caña de azúcar solo el 3,5% se destina al autoconsumo. Además, nuestros resultados muestran que el suministro per cápita de azúcares y dulcifi- cantes en México es, por mucho, el que más excede las recomendaciones dietéticas de la OMS(13); sin embargo, en los estudios basados en encuestas de consumo alimentario autode- clarado el análisis de la ingesta de azúcares es escaso y unidimensional. Escaso porque solo alude al azúcar indirectamente al comentar que se incrementó el consumo de carbohidratos refinados(5), sin ir más allá. Unidimensional, porque se enfoca en el consumo de bebidas azucaradas(23) las cuales, incluidas como dul- cificantes en las estimaciones de la FAO(14), re- presentan menos del 6,8% del suministro total de azúcares y dulcificantes mientras el 92% proviene de otras fuentes. Otra fuente de posible subregistro puede ser la discrepancia entre lo que los productores declaran producir (cifra normalmente usada para el cálculo del suministro de alimentos) y la producción real; sin embargo, hasta ahora en México no se cuenta con estudios que de- muestren la existencia de tal discrepancia e Así, dichas investigaciones resaltan el incremento de lípidos en la dieta y tienden Salud Colectiva \| Universidad Nacional de Lanús \| ISSN 1669-2381 \| EISSN 1851-8265 \| doi: 10.18294/sc.2016.925 Hernández Ramírez JC, Ortega Canto JE. 498 SALUD COLECTIVA. 2016;12(4):487-504. Cambios en los aportes de macronutrimentos doi: 10.18294/sc.2016.925 a subregistrar y minimizar el consumo de azúcar en México y, tal vez, ese énfasis en los lípidos se debe a su coincidencia tem- poral con el aumento de obesidad en la po- blación. Ciertamente, el suministro excesivo de azúcar y dulcificantes antecede por casi 10 años al primer registro de dicho aumento de obesidad y lo mismo ocurre con los ex- cesos de carbohidratos y bebidas alcohó- licas. Entonces, parece ser que, en 1990, cuando los lípidos fueron incluidos entre los excesos y comenzaron a sustituir a los carbo- hidratos, la obesidad comenzó a incremen- tarse; sin embargo, es importante advertir el peso cuantitativo y constante del azúcar y las bebidas alcohólicas en el excedente alimen- tario y su posible efecto aditivo a partir de la inclusión del exceso de lípidos. variaciones medias anuales. Y dichos datos ejemplifican ciertas tendencias internacio- nales de la industria cárnica, tales como la proyección de que para el 2022 casi la mitad de la carne adicional consumida en el mundo será de pollo; o bien que actual- mente solo crece el mercado para productos avícolas y cerdo porque ambas especies usan comederos y pueden criarse en espacios con- finados, lo cual permite satisfacer la creciente demanda de carne barata(24). En efecto, el incremento desde los años ochenta en las exportaciones de maíz ama- rillo y soja de EE.UU. hacia México(12) corres- ponde a la demanda creciente de alimento para aves y cerdos por parte de tales industrias cárnicas. Por ende, esas exportaciones son correlato del incremento excesivo de lípidos de origen animal en la dieta mexicana. Finalmente, según la teoría de la tran- sición nutricional(2), el perfil actual del estado nutricio de la población mexicana se co- rrespondería, entre otros elementos, con una reducción del consumo de cereales no “industrializados”. Sin embargo, Ortiz et al.(8) afirman que el consumo alimentario en México no se apega a tal planteamiento ya que allí la disponibilidad de cereales no solo no se redujo, sino que se incrementó, con au- mentos de trigo y arroz. Pero tal afirmación es muy apresurada porque obvia una tendencia: tanto desde la década de 1980 como en el periodo estudiado, el suministro de cereales y, especialmente, el de trigo, presenta un des- censo lineal (Tabla 1). Cambios en los aportes de macronutrimentos Así, nuestro estudio muestra cómo el uso de algunos elementos del análisis de series temporales permite rea- lizar observaciones menos superficiales de los datos del suministro de alimentos. g Por otro lado, el exceso promedio per cápita de 243 kcal/día de azúcar y dulcifi- cantes entre 1990 y 2013 equivale a un peso neto de 58 g diarios(25). Considérese que esos 58 g extra pueden consumirse en diversas formas ya sea como azúcar “de mesa”, sa- carosa integrada en alimentos dulces y salados procesados industrialmente o dulcificantes usados en la industria alimentaria para mo- dificar las propiedades organolépticas (color, olor, sabor y textura) de los alimentos. Dado que son útiles para postergar la caducidad de múltiples productos alimenticios “industriali- zados” y/o potenciar su atractivo al paladar, los azúcares y dulcificantes forman parte de la dieta cotidiana aunque a veces “no se vean” ni se perciban conscientemente. Así pues, en el perfil general del exce- dente nutrimental en México entre 1993 y 2013 resaltan, principalmente, el creciente exceso de lípidos provenientes de la carne producida industrialmente y el exceso re- lativo mayoritario y consolidado de azúca- res-dulcificantes. Tales hechos coinciden con cierta hipótesis según la cual, así como los cereales fueron el centro de los sistemas alimentarios de las grandes civilizaciones del pasado (el arroz de la civilización china, el trigo de la persa y griega, el maíz de la mesoamericana), el núcleo alimentario de la sociedades industriales contemporáneas se Salud Colectiva \| Licencia Creative Commons Reconocimiento — No Comercial 4.0 Internacional \| BY - NC El excedente nutrimental y la industria de la alimentación El aumento del suministro per cápita de lípidos en México –y del excedente de lí- pidos– desde 1990 se debe a la expansión de la industria avícola seguido de la in- dustria porcina. De ahí que los crecimientos lineales de los lípidos de carne de aves de corral, huevo y cerdo presenten las mayores 499 donde el suministro alimentario puede com- plementarse con encuestas de alimentación. conforma elípticamente a partir de dos focos que configuran el sistema alimentario: azúcar refinada y carne(26). Los métodos de encuesta comúnmente usados (recordatorio de 24 horas y fre- cuencias de consumo) ofrecen información más desagregada respecto al tipo de ali- mentos concretos que las personas dicen consumir y permiten hacer clasificaciones por edad, sexo y área geográfica. Pero tal in- formación es útil hasta cierto punto, ya que habría que considerarla con precaución si carece de verificación empírica más allá del registro escrito de los hábitos alimentarios. Después de los azúcares-dulcificantes y los lípidos, la creciente cantidad de energía proveniente de la cerveza proyecta su propia permanencia relevante en el excedente nutri- mental, mientras que el aporte energético de las bebidas destiladas tiende a la baja (Tabla 1). Y tales cambios en la composición del su- ministro de bebidas alcohólicas en México son análogos a los reportados en un estudio que abarcó el período 1970-1989(27); sin em- bargo, los datos relativos a las bebidas sin control fiscal o sanitario no se registran en las hojas de balance alimentario de la FAO, por lo tanto, existe un subregistro en el consumo total de alcohol. g Y he ahí la cuestión, los recordatorios de 24 horas o las frecuencias de consumo son discursos codificados y no datos “empíricos”, aunque se los presenta como tales, esto es: re- gistran lo que las personas dicen que comen y no necesariamente lo que realmente comen. Entonces, tales encuestas son discursos que requieren contrastarse con las respectivas conductas que refieren y, de no hacer tal contraste, se corre el riesgo de confundir los discursos con las conductas y hacer una ge- neralización sesgada por sobreregistro o, más frecuentemente, por subregistro. En tales tér- minos, una de las principales desventajas de las encuestas es el sesgo en la cantidad de ali- mentos consumida; no obstante, en algún es- tudio se propone algo parcialmente opuesto, es decir, que las encuestas dietéticas propor- cionan información detallada especialmente respecto al tamaño de las porciones(31). El excedente nutrimental y la industria de la alimentación Respecto al aumento del consumo de cerveza en México cabe agregar que, a fi- nales del siglo XIX, la cerveza era un bien suntuario importado de Alemania, EE.UU. e Inglaterra y consumido por las comu- nidades de extranjeros en el país; sin em- bargo, en la primera mitad del siglo XX la cerveza mexicana desplazó a su homóloga importada y pasó de ser un bien suntuario a una bebida popular(28). Así, México es hoy el mayor exportador mundial de cerveza(29) y, desde 1960, el capital productor de cerveza en ese país tiende a concentrarse oligo- pólicamente en empresas que controlan desde la producción de insumos hasta la distribución(30). Para ejemplificar tal contradicción con- sidérese que en EE.UU. cierta encuesta nacional registró solo el 50% del alcohol consumido según los registros de venta de bebidas alcohólicas(32); o bien que, mientras aquí mostramos cómo el suministro de azú- cares y dulcificantes en México es el más ex- cesivo en términos relativos, en un estudio nacional reciente basado en encuestas se afirma que el 72% de la población mexicana ingiere la cantidad adecuada de azúcar y que el 80,4% declaró haber comido azúcares y dulces al menos uno de los tres días que abarcó un cuestionario tipo recordatorio de 24 horas(33,34). Dieta sostenible y excedente nutrimental en México tener presente que son útiles para registrar los cambios y tendencias no en la dieta sino en el gasto monetario en comida. De otra forma se corre el riesgo de asimilar –o con- fundir– el gasto con el consumo y llegar a conclusiones erróneas, tales como decir que durante los últimos treinta años en México se ha reducido la ingesta de carnes porque en los hogares se gasta menos dinero en su compra(35); no obstante, el suministro casi se duplicó en tal periodo(14). Pero resulta que también los datos sobre el suministro alimentario son calculados con base en di- versas encuestas, es decir, se basan en de- claraciones de diversos actores sociales y no necesariamente en el dato empírico directo referido por dichos actores. Según un informe de la FAO(36) las dietas sostenibles son: …aquellas dietas con bajo impacto ambiental que contribuyen a la segu- ridad alimentaria y nutrimental y a una vida sana de las generaciones presentes y futuras. Las dietas sostenibles son pro- tectoras y respetuosas de la biodiversidad y de los ecosistemas, son culturalmente aceptables, accesibles, económicamente justas y asequibles; nutricionalmente adecuadas, inocuas y saludables; a la vez que optimizan los recursos naturales y humanos. Así las cosas, no se trata de invalidar la posibilidad de acercarse a la realidad me- diante las encuestas sino de señalar sus lí- mites, resaltar la necesidad de contrastar diversas encuestas y, cuando es posible, ve- rificar el dato empírico referido por los en- trevistados. Ahora bien, la elección de datos de suministro alimentario o de encuestas dietéticas depende, entonces, de qué es lo que se desea saber y del nivel de análisis. Por ejemplo, proponemos que, por un lado, los tipos de encuesta dietética citados son útiles para responder qué se come y/o con qué frecuencia, tanto en poblaciones como en subgrupos poblacionales y, por otro, los datos de suministro alimentario sirven para responder cuánto se come en términos de macronutrimentos y grupos de alimentos en una población nacional y los cambios de esas cantidades a través del tiempo. Por tanto, no son métodos intercambiables, aunque sí son complementarios y, como toda indagación científica en alimentación, cada uno por se- parado o incluso ambos combinados, solo nos brindan un saber aproximado y parcial. Suministro alimentario versus encuestas dietéticas La pertinencia de los datos de suministro alimentario de las hojas de balance de la FAO para deducir el consumo de macronu- trimentos y grupos de alimentos de una po- blación depende del nivel de análisis. Si se trata de una población nacional, entonces esos datos son un indicador imprescindible que ofrece información fiable. Pero su homo- geneidad impide transpolar ipso facto tales de- ducciones hacia subgrupos –por edad, sexo o área geográfica– de dicha población y es allí Por otro lado, respecto a las encuestas de gasto monetario en alimentos conviene Salud Colectiva \| Universidad Nacional de Lanús \| ISSN 1669-2381 \| EISSN 1851-8265 \| doi: 10.18294/sc.2016.925 Hernández Ramírez JC, Ortega Canto JE. 500 Salud Colectiva \| Licencia Creative Commons Reconocimiento — No Comercial 4.0 Internacional \| BY - NC Dieta sostenible y excedente nutrimental en México En efecto, es recomendable contrastar los aportes de las encuestas dietéticas con otras fuentes de información cualitativas (obser- vación participante) y, en el caso de pobla- ciones nacionales, cuantitativas (suministro de alimentos). Ahora bien, en México, el mayor consumo de alimentos procesados indus- trialmente conllevó una ingesta excesiva de azúcares-dulcificantes y grasas incluidas en carnes de pollo y cerdo. Así, tanto la diver- sificación de los azúcares-dulcificantes po- tencialmente adictivos incluidos en múltiples alimentos como el incremento excesivo de lí- pidos aportados por tales carnes detonaron la sobrealimentación y, por tanto, la obesidad, patologías metabólicas y cardiovasculares. Además, el suministro creciente de productos de la industria avícola y porcina incrementa el consumo de piensos transgénicos cultivados con herbicidas potencialmente nocivos (por ejemplo, el glifosato) para el ganado y los consumidores de carne(24). Pero hay más, producir un gramo de proteína animal requiere en promedio ocho gramos de proteína vegetal. Por con- siguiente, cada gramo extra de proteína de aves y cerdo producidos intensivamente incrementa ocho veces la demanda de ce- reales y soja y, por ende, el mayor uso de tierra destinada a cultivos que satisfacen indirectamente la creciente demanda de carne por parte de los consumidores. Esto conlleva consecuencias adversas para la biodiversidad ya sea por la deforestación para destinar tierras al cultivo, por el uso de semillas transgénicas o ambas. 501 Por su parte, ligado a lo anterior, la pro- ducción industrial de carne implica el uso excesivo y la contaminación del agua. Por ejemplo, mientras producir 1 kilo de papas o trigo requiere alrededor de 150 y 1.400 litros de agua respectivamente, producir un kilo de pollo requiere 4.000 litros de agua(37). Asimismo, la sobrefertilización del suelo ocasionada por el exceso de abonos líquidos y estiércol deriva en la filtración de fósforo y nitratos hacia aguas subterráneas, ríos y lagos, pozos y manantiales, esto es: daños al suelo y la biodiversidad así como un mayor riesgo de cáncer por parte de quienes beben esa agua contaminada(24). décadas, en ese país la proporción de produc- tores-autoconsumidores es cada vez menor debido a fenómenos tales como la migración del campo a la ciudad o a EE.UU. Junto a esto, la introducción creciente del dinero y diversas mercancías –incluidos los alimentos procesados industrialmente– en las comu- nidades rurales durante las últimas cuatro décadas las ha integrado paulatinamente en el circuito de consumo mercantil capita- lista. Dieta sostenible y excedente nutrimental en México En breve, las personas consumen cada vez menos alimentos producidos por ellas mismas y la producción agropecuaria y de alimentos, en general, tiende a concentrarse en grandes productores(39,40), esto es: cada vez hay menos espacios que den cabida a los subregistros referidos arriba. Así pues, por las tendencias histórico-económicas en México (integración creciente de la economía rural al circuito mercantil capitalista), el indicador suministro alimentario provee información suficientemente confiable (por supuesto me- jorable) respecto a los cambios generales de la alimentación en dicho país. A tales efectos sanitarios y ambientales derivados del consumo creciente de carnes, cabe añadir que su producción intensiva genera casi un tercio de los gases de efecto invernadero global(24) y que las emisiones in- directas de CO2 ocasionadas por una dieta basada en vegetales son menores que las de una dieta mixta con vegetales y productos de origen animal(38). Con todo, así es como, según tal con- cepto de “dieta saludable”, el suministro alimentario en México caracterizado por ex- cesos de azúcares-dulcificantes y grasas de origen animal así como por el crecimiento lineal intenso del consumo de carne, leche y huevo, y el decrecimiento del consumo de maíz y frijol, corresponde a una dieta que carece de inocuidad, es inadecuada para los requerimientos de la población y, por ende, no contribuye a la seguridad nutricional ni a una vida sana de las generaciones presentes y futuras. No es una dieta protectora y res- petuosa de la biodiversidad ni de los eco- sistemas y tampoco optimiza los recursos naturales y humanos. Los cambios en el perfil general del ex- cedente nutrimental en México en el periodo 1990-2013 son correlato de la paulatina consolidación de un sistema alimentario in- dustrial configurado a partir de dos focos: azúcares-dulcificantes y carne. Esto conlleva potenciales efectos adversos tanto para la salud de los consumidores de alimentos como para el medio ambiente. En efecto, la dieta mexicana en el periodo de estudio fue insustentable y tiende a mantenerse así. Mientras el excedente nutrimental des- cripto en este trabajo exista, posiblemente el perfil epidemiológico de la población mexicana seguirá presentando una alta y creciente prevalencia de enfermedades cró- nicas no transmisibles relacionadas con la sobrealimentación, tales como diabetes, enfermedades cardiovasculares y obesidad, entre otras. Dieta sostenible y excedente nutrimental en México Ni qué decir respecto al dete- rioro que el incremento lineal del consumo de carne industrial implicará en la conta- minación del agua por nitratos –asociados con el cáncer– y en los riesgos sanitarios, ecológicos y económicos resultantes de la producción-consumo masivo de piensos Salud Colectiva \| Universidad Nacional de Lanús \| ISSN 1669-2381 \| EISSN 1851-8265 \| doi: 10.18294/sc.2016.925 CONCLUSIONES Aunque las hojas de balance de la FAO posiblemente subregistren el autoconsumo y la producción circulante en mercados “in- formales” que están al margen del fisco en México, vale decir que, desde hace siete Salud Colectiva \| Universidad Nacional de Lanús \| ISSN 1669-2381 \| EISSN 1851-8265 \| doi: 10.18294/sc.2016.925 Hernández Ramírez JC, Ortega Canto JE. 502 SALUD COLECTIVA. 2016;12(4):487-504. doi: 10.18294/sc.2016.925 elaborados con soya y maíz transgénico, que son riesgos: 1) sanitarios, por la expo- sición a sustancias tales como el herbicida glifosato; 2) ecológicos, por la reducción de la biodiversidad; y 3) económicos, por la concentración, centralización y acapa- ramiento de la producción alimentaria por parte de los propietarios de ciertos paquetes tecnológicos de producción. 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Eshel G, Martin PA. Diet, energy and global warming. Earth Interactions. 2006;10:1-17. 39. Appendini K. De la milpa a los tortibonos: la restructuración de la política alimentaria en Salud colectiva \| Licencia Creative Commons reconocimiento — No Comercial 4.0 Internacional \| BY - NC FORMA DE CITAR Hernández ramírez JC, Ortega Canto JE. El perfi l general del excedente nutrimental en México en el periodo 1990- 2013: un enfoque a partir del suministro energético de macronutrimentos y grupos de alimentos. Salud Colectiva. 2016;12(4):487-504. doi: 10.18294/sc.2016.925. Hernández ramírez JC, Ortega Canto JE. El perfi l general del excedente nutrimental en México en el periodo 1990- 2013: un enfoque a partir del suministro energético de macronutrimentos y grupos de alimentos. Salud Colectiva. 2016;12(4):487-504. doi: 10.18294/sc.2016.925. recibido: 4 de abril de 2016 \| Versión fi nal: 24 de agosto de 2016 \| Aprobado: 7 de octubre de 2016 recibido: 4 de abril de 2016 \| Versión fi nal: 24 de agosto de 2016 \| Aprobado: 7 de octubre de 2016 http://dx.doi.org/10.18294/sc.2016.925
https://openalex.org/W4281611446	https://hal.inrae.fr/hal-03630410/file/Ecography%20-%202022%20-%20Desaegher%20-%20How%20far%20is%20enough%20Prediction%20of%20the%20scale%20of%20effect%20for%20wild%20bees.pdf	English	null	How far is enough? Prediction of the scale of effect for wild bees	Ecography	2,022	cc-by	9,282	How far is enough? Prediction of the scale of effect for wild bees James Desaegher, Annie Ouin, David Sheeren To cite this version: James Desaegher, Annie Ouin, David Sheeren. How far is enough? Prediction of the scale of effect for wild bees. Ecography, 2022, 5, 10.1111/ecog.05758. hal-03630410 Distributed under a Creative Commons Attribution 4.0 International License James Desaegher, Annie Ouin and David Sheeren J. Desaegher (https://orcid.org/0000-0001-9205-0184) ✉ (jamesdesaegher@gmail.com), A. Ouin and D. Sheeren, Univ. de Toulouse, INRAE, UMR DYNAFOR, Castanet-Tolosan, France and LTSER Zone Atelier ‘PYRÉNÉES GARONNE’, Auzeville-Tolosane, France. Ecography Subject Editor: Michel Baguette Editor-in-Chief: Dominique Gravel Accepted 11 February 2022 doi: 10.1111/ecog.05758 2022: e05758 A crucial issue for landscape ecologists is identifying the spatial extents at which a land scape affects species occurrence. Multi-scale analyses are usually conducted to iden tify the ‘scale of effect’, that is, the spatial extent associated with the best relationship between landscape variables and species occurrence, which is assumed to be related to species traits. However, few guidelines exist to determine the range of distances to be investigated. g Based on the foraging distances of wild bee species, our main goal was to estimate the maximum distance of effect, that is, the distance beyond which the scale of effect for wild bee species is unlikely to be detected. Using the InVEST pollination model, we 1) modelled bee categories with distinct foraging distances and identified the scale of effect on their simulated abundance 2) defined an index, noted λ, that estimates the distance beyond which landscape compo sition has only negligible effects on simulated abundances. We validated our results by identifying the scale of effect on the abundances of 16 bee species collected in south- western France. We detected a significant positive relationship between the average foraging dis tance (α) of the modelled bees and their scale of effect. The λ index was linearly related to the average foraging distances of bees (λ = 5.4α + 253) and was above the identified scale of effect for the modelled bees. The λ was also found to be above the scale of effect for 93% of the observed bee species. p Our results suggest that the λ index is a good estimator of the upper limit of the scale of effect for wild bees. The λ index could be used to identify the minimum dis tance between sampling sites before setting up an experiment and the maximum buffer size required in multi-scale analysis to detect the scale of effect. Keywords: bee, InVEST, landscape, pollination, scale of effect Keywords: bee, InVEST, landscape, pollination, scale of effect ECOGRAPHY ECOGRAPHY HAL Id: hal-03630410 https://hal.inrae.fr/hal-03630410v1 Submitted on 5 Apr 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Introduction ‘To understand the drama [in the ‘ecological theatre’ (Hutchinson 1965)], we must view it on the appropriate scale.’ (Wiens 1989) www.ecography.org www.ecography.org © 2022 The Authors. Ecography published by John Wiley & Sons Ltd on behalf of Nordic Society Oikos © 2022 The Authors. Ecography published by John Wiley & Sons Ltd on behalf of Nordic Society Oikos This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 1 Landscape ecologists aim to study how landscape hetero geneity influences (the processes that affect) the abundance and distribution of organisms (Fahrig 2005). They study the relationships between biological responses collected at dif ferent locations (e.g. species abundance), and the environ mental variables in the surrounding area (e.g. proportion of habitats). However, choosing the spatial extent at which to measure the landscape variables is crucial but complex (Levin 1992). Indeed, it depends not only on the organism under investigation, and its mobility, but also on habitat fragmenta tion, matrix quality or topography (Miguet et al. 2016). An inappropriate choice of spatial extent may result in failure to detect the ecological pattern or even in the detection of artefacts (Wiens 1989). One common way to identify the ‘appropriate’ spatial extent of investigation related to the behavioural ecology of species is to measure landscape variables at multiple cir cular extents around sampling sites and then to choose the extent associated with the best statistical relationship between the landscape variables and the biological response (Fig. 1; Jackson and Fahrig 2012). This procedure is called ‘multi- scale analysis’ and the optimal spatial extent is often called the ‘scale of effect’ (Jackson and Fahrig 2012, 2014, Miguet et al. 2016). Here, the term ‘scale’ refers to the geographic scale and is used as a synonym for geographical extent.f Identifying the scale of effect is an essential step before formulating any explanations concerning a species–habitat relationship at landscape scale (Thornton and Fletcher 2014). However, a meta-analysis of the practices of ecologists per forming multi-scale analyses revealed that the range of scales explored is too narrow, since 44% of studies reported a scale of effect at the smallest or the largest scale evaluated (Jackson and Fahrig 2014). To our knowledge, only the general guide line resulting from the simulation work of Jackson and Fahrig (2012) is available, which expects the scale of effect to be 4–9 times the median dispersal distance of the organism studied. www.ecography.org There is a critical need to improve our ability to predict the scale of effect for a given species, and to validate the predic tions with empirical data. Estimating a priori of the range in which the scale of effect would fall into, could help landscape ecologists choose sampling sites located sufficiently far away from one another to reduce the spatial pseudo-replication often identified a posteriori (Miguet et al. 2016). Of course, it would also improve the accuracy of the identification of the scale of effect associated with a species before analys ing a species–habitat relationship. Better predictions of the scale of effect would help landscape managers and conser vation biologists improve the efficiency of their actions, for example, scaling and designing nature reserves according to the focus organisms (Wiens 1989, Jackson and Fahrig 2012, Miguet et al. 2016, Moll et al. 2020).f Figure 1. Design of a fictive multi-scale analysis (a) relating the abundance of a bee species to the proportion of a habitat (e.g. grass land) measured for different landscape extents (i.e. buffer radii) cen tred on the sampling sites (yellow dots). Based on the linear relationships between species abundance and the proportion of habitat calculated within the different landscape extents (b), the scale of effect (S), is identified as the buffer radius with the best statistical relationship, for instance based on the R2 (c). (Stuber et al. 2018, Moll et al. 2020). Despite the ecological and economic importance of pollination (Gallai et al. 2009, Ollerton et al. 2011), quantitative relationships between bee species traits related to foraging distance (Gathmann and Tscharntke 2002, Greenleaf et al. 2007) and their specific scale of effect, have never been identified in wild bees.f fi One way to predict the scale of effect for a bee species could consist in restricting the upper limit of the range of can didate scales by identifying a distance beyond which the scale of effect for the species is biologically unlikely to be detected based on their estimated foraging distances. A conservative hypothesis would be that the scale of effect must necessarily be less than the distance beyond which any landscape compo sition (e.g. floral or nesting plots) only has negligible effects g An early considered approach to predict the scale of effect of an organism is using allometric relationships related to spe cies mobility (Wiens 1989). Study area The study area is located in south-western France in the Vallées et Coteaux de Gascogne site in the Figure 2. Illustration of the distinction between the average foraging distance (α), the scale of effect (S) and the λ distance and their hypothetical relationships. Bees are assumed to locate their nests in sites surrounded by floral resources within their foraging range (blue area). The scale of effect (S), identified with a multi-scale analysis, is represented by the dashed line. The λ distance represents the distance beyond which landscape composition only has negli gible effects on the abundance observed in the sampling site (yellow flower in the centre). www.ecography.org However, predicting the scale of effect based on species traits, mainly tested on birds (Thornton and Fletcher 2014), often failed to produce significant results 2 long term socio-ecological research area (LTSER ZA PYGAR) ‘Pyrénées Garonne’ (Ouin et al. 2022) and covers approximatively 2500 km2 (50 × 50 km, Fig. 3). This agricultural area is characterised by hilly landscapes with a traditional cropping system combin ing woodlots and mixed crop-livestock farming. The major ity of grasslands are located on hillsides while annual crops (cereals, oilseed rape and sunflower, maize) are grown in the valleys (Choisis et al. 2010). on bee species abundance, hereafter called the maximum dis tance of landscape effect (abbreviated λ, Fig. 2). The λ dis tance can be estimated using spatially explicit models that predict pollinator abundances in landscapes. In this study, we used the InVEST pollination model (Sharp et al. 2018) to predict the relative abundance of bees in a landscape with the aim of 1) defining and estimating the λ distances associated with different modelled bee cat egories characterised by different average foraging distances through simulations, 2) assessing if the scale of effect for the modelled bees are smaller than their λ distance and exploring the relationships between the scale of effect, average foraging distance and λ distance (Fig. 2) and 3) validating our simula tion results by estimating the λ distance and the scale of effect associated with actual ground-nesting bee species collected in south-western France. The land use/land cover (LULC) map For the purpose of our simulations and analyses, we pro duced a LULC map of the study area by applying a series of basic GIS operations to three different spatial datasets available in vector format: 1) the RPG (Registre Parcellaire Graphique) from the Common Agricultural Policy (CAP) declarations (<www.data.gouv.fr>), 2) the BDTOPO (2010) nationwide database, produced by the French national mapping agency (IGN) (<https://geoservices.ign. fr/documentation/diffusion/telechargement-donnees-libres. html>), 3) the OCS-GE (OCcupation du Sol à Grande Échelle) database also obtained from IGN. The three datasets were topologically corrected and combined in a single vector layer to produce a map with the highest possible accuracy. The resulting map was composed of 92 LULC categories with 81 agricultural land use categories (e.g. wheat, oilseed rape, sunflower) and 11 other LULC categories (e.g. build ings, hedgerows, water areas) (Supporting information). The vector layer was rasterized in a 4-m spatial resolution raster to take advantage of a good spatial resolution while keep ing the volume of data manageable during the simulations. The pollination model and parameterization The model is defined as follows: 3 3 Supply Nest AFR Poll Nest Flower p s p s p s s p s p D j p e , , , , , = ´ ´ = ´ ´ ¢ ¢ - å å a j s j p D s e ´ ( ) æ è ç ç ç ç ö ø ÷ ÷ ÷ ÷ ´ ¢ - å Activity Poll , a (1) Visitation Flower Activity AFR Sup p j s p j s j p s p , , , , , = ´ æ è çç ö ø ÷÷ ´ ¢ å ply ¢ ¢ - - ´ å p s D p D e e , a a (2) Supply Nest AFR Poll Nest p s p s p s s p s , , , , = ´ ´ = landscape on the visitation rate of modelled bees can be con sidered as negligible, according to the bees’ average foraging distance; 2) to predict visitation rates for different sampling sites that can be used to explore the relationship between the scale of effect for the modelled bees and their average forag ing distance. upply Nest AFR Poll Nest Flower p s p s p s s p s p D j p e , , , , , = ´ ´ = ´ ´ ¢ ¢ - å å a j s j p D s e ´ ( ) æ è ç ç ç ç ö ø ÷ ÷ ÷ ÷ ´ ¢ - å Activity Poll , a (1) g We simulated the visitation rate of the 16 modelled bees in 50 target sunflower fields. We chose sunflower to have 1) a sufficient quantity of target plots for spatial sampling distribution as sunflower is the most frequently cultivated entomophilous crop in the study area, and 2) target plots with the same floral resource abundance, making visitation rates comparable among plots. We randomly selected fields between 10 000 m2 and 20 000 m2 in size, located at least 5 km from the border of the study area, and separated from each other by at least 4 km (Fig. 3). To assess the distance- weighted effects of landscape composition on the visitation rate of modelled bees, i.e. The pollination model and parameterization how the landscape effects decline with increasing distance from the sampling sites, we cropped the LULC input map around the centres of the 50 sunflower fields according to different buffer radii. For each sunflower field, 108 circular extents were defined with a buffer radius ranging from 10 to 90 m (10-m increment) and from 100 to 5000 m (50-m increment). (2) where 𝐷 is the Euclidean distance between pixel p and p′.h where 𝐷 is the Euclidean distance between pixel p and p′.h p p p The model requires three input data: 1) a LULC map in a raster format (section ‘The land use/land cover (LULC) map’), 2) a table of LULC attributes (indices: Nest, Flowerj) and 3) a table of pollinator attributes (indices: α, Poll, Activityj).h We ran the InVEST pollination model using Python ver. 2.7.16 programming language iteratively with the 5400 cir cular input maps (50 fields × 108 buffer radii) and for the 16 modelled bees (average foraging distance ranging from 50 to 800 m). Since sunflowers flower in July in the study area, we simulated the visitation rate in the 50 sunflower fields during July. From the 86 400 output maps of visitation rates (50 fields × 108 buffer radii × 16 modelled bees), we extracted the visitation rate for the pixel located closest to the centroid of the sunflower field (i.e. map centre). p yj The table of LULC attributes combines indices ranging from 0 to 1 for the 92 LULC categories concerning 1) the relative availability of nests and 2) the relative availability of floral resources during the seasons or months studied. We decided to study bee species nesting in belowground nests since the majority of wild bee species are ground nesters (Cane 1991, Michener 2007). For each LULC category, the availability of belowground nests was attributed using expert knowledge. Concerning the availability of floral resources, we defined 12 indices associated with the 12 months of the year. For 70% of the LULC categories, the values of the flo ral resource indices were based on information reported in existing databases containing records of pollen and nectar production of plant taxa (mainly EFSA 2013, Baude et al. 2016). For the remaining LULC categories (30%) the floral resource indices were attributed using expert knowledge (see the Supporting information for details).i The pollination model and parameterization In this study, we used the InVEST pollination model (Sharp et al. 2018) mainly based on the equations of the Lonsdorf model (Lonsdorf et al. 2009). The model is spatially explicit and predicts the abundance of wild bees through their visitation rate on floral resources.h l The visitation rate of a species s in a map pixel p is calcu lated in two steps. In a first step, the model identify where the species actually nests in the landscape, through the mapping of an index called ‘pollinator supply’, Supplyp,s. The pollinator supply in pixel p, depends on 1) the availability of suitable nesting substrate for the species in the pixel, Nestp,s, 2) the accessible floral resources in the neighbourhood of pixel p, AFRp,s, 3) the pollinator species abundance, Polls. The acces sible floral resources, AFRp,s, depends on 1) the average forag ing distance of bees, α, 2) the abundance of floral resources in all the surrounding pixels p′, during season j, Flowerp′,j and 3) the activity of species 𝑠 in season 𝑗, Activitys,j. In a second step, based on the pollinator supply map, the model calcu lates the ‘visitation rates’ to the flowering resources surround ing the original nests.hi Figure 2. Illustration of the distinction between the average foraging distance (α), the scale of effect (S) and the λ distance and their hypothetical relationships. Bees are assumed to locate their nests in sites surrounded by floral resources within their foraging range (blue area). The scale of effect (S), identified with a multi-scale analysis, is represented by the dashed line. The λ distance represents the distance beyond which landscape composition only has negli gible effects on the abundance observed in the sampling site (yellow flower in the centre). Figure 2. Illustration of the distinction between the average foraging distance (α), the scale of effect (S) and the λ distance and their hypothetical relationships. Bees are assumed to locate their nests in sites surrounded by floral resources within their foraging range (blue area). The scale of effect (S), identified with a multi-scale analysis, is represented by the dashed line. The λ distance represents the distance beyond which landscape composition only has negli gible effects on the abundance observed in the sampling site (yellow flower in the centre). Distance-weighted effects of landscape and λ distances To assess how the InVEST pollination model weights the landscape effects on the visitation rates of modelled bees with increasing distance from the map centre, we calculated the percentage change in visitation rates according to the radius of the circular input maps.i Concerning the table of pollinator attributes, we defined 16 modelled bee categories that only differed in their aver age foraging distance (α-index). The α-index ranged from 50 to 800 m with a 50-m increment. This range was rep resentative of most bee species (Gathmann and Tscharntke 2002, Greenleaf et al. 2007). Since the 16 modelled bees were simulated separately, during one month, and were all below ground nesters, we arbitrarily set to 1, the relative abundance (Polls), the foraging activity during that month (Activityj) and the preference for belowground nests (Nestp,s). p p For each combination of a modelled bee, a field and a buf fer radius, we calculated the percentage change in visitation rates as follows: %DVisitation Visitation Visitation Visitation max m r r r r ( ) = ( ) - ( ) ax ( ) where Visitation(r) is the visitation rate for the pixel in the centre of the field with a buffer radius r, and Visitation(rmax) is the visitation rate for the pixel in the centre of the field with the maximum buffer radius (5000 m). The simulation Our simulation using InVEST pollination model had two goals: 1) to quantify the distance at which the effects of the f For each modelled bee (i.e. each α value), we calculated the %ΔVisitation(r) functions for the 50 target fields. Using 4 Figure 3. The left-hand map shows the French administrative regions and the right-hand map is the land use/land cover (LULC) map of the study area. The yellow dots represent the 50 target sunflower fields used in the simulation and the blue dots represent the 30 pan trap ping sites. Figure 3. The left-hand map shows the French administrative regions and the right-hand map is the land use/land cover (LULC) map of the study area. The yellow dots represent the 50 target sunflower fields used in the simulation and the blue dots represent the 30 pan trap ping sites. Figure 3. The left-hand map shows the French administrative regions and the right-hand map is the land use/land cover (LULC) map of the study area. The yellow dots represent the 50 target sunflower fields used in the simulation and the blue dots represent the 30 pan trap ping sites. to have a ‘scale-free’ visitation rate estimation with no map border effects.i the 50 %ΔVisitation(r) functions we identified the maximum percentage change for each radius, noted %ΔVisitationMAX(r). The %ΔVisitationMAX function is expected to decrease with increasing buffer radius and should reflect how the effects of the landscape decrease with distance. Finally, for the 16 modelled bees, we identified the radius abbreviated λ1% thereby verifying the equation: %ΔVisitationMAX(λ1%) = 1%. Therefore, λ1% represents the distance beyond which land scape composition (i.e. flower and nesting plots) is respon sible for less than 1% change in the visitation rates of the modelled bee in the centre of the sunflower field. f We fitted 1728 linear models (16 modelled bees × 108 buffer radii) to link the visitation rate of the modelled bees in the sunflower fields to the two landscape variables, after checking the assumptions for linear regressions (nor mality, homoscedasticity and independence of residuals) (Zuur et al. 2010). By construction in the pollination model, the mean floral and nesting resource availability are key variables for visitation predictions. Therefore, we only considered the two statistical models including both vari ables. The simulation Using a selection procedure based on the corrected Akaike information criterion (AICc) (Barton 2022), we selected the model most frequently identified as the best model across 108 buffer radii. li We used a linear model to establish the statistical relation ship between the average foraging distances of the 16 mod elled bees (α) and their λ1% distances. f For each modelled bee, we compared the explanatory performance (goodness of fit) of the 108 models using the amount of explained variance (R2). The buffer radius associ ated with the best R2 was considered to be the scale of effect for the modelled bee. The scale of effect for the 16 modelled bees was related to their average foraging distance using linear models. An additional sensitivity analysis was performed for each scale of effect using a delete-five-observations Jackknife. This approach consisted in randomly removing five sampling sites from the data (10% of the data) before calculating the scale of effect. The procedure was repeated 1000 times. Scale of effect for the modelled bees To identify the scale of effect for the 16 modelled bees, we performed multi-scale analyses (Fig. 1) with two landscape variables calculated within 108 buffer radii (from 10 to 5000 m) as the explanatory variables, and the simulated visita tion rate of modelled bees in the 50 sunflower fields as the response variable. The two landscape variables were 1) the mean availabil ity of floral resources in July, and 2) the mean availability of belowground nests. These two variables were weighted means obtained by summing the indices of floral resource or nest ing resource availability in each LULC category occurring around the 50 fields within the 108 buffer radii (from 10 to 90 m by 10-m and from 100 to 5000 m by 50-m), weighted by the proportion of each LULC category in the different buffer zones. Sensitivity analysis The aim of this analysis was to assess how a variation of InVEST model parameters related to the floral and nesting We performed generalized linear models (GLM) since spe cies abundances are count data. For each species, we selected Table 1. Summary data associated with each bee species. The first column shows total abundance, all sampling sites combined, the second column correspond to the number of sites in which the species was found, the third column the intertegular distance (ITD) from Carrié et al. (2017, 2018), the fourth column correspond to the predicted typical homing distance (THD) according to Greenleaf et al. (2007), the fifth columns correspond to our index of maximum distance of effect λ1%. The sixth and seventh columns correspond to the statistical models used to study the scale of effect. ‘P’ and ‘NB’ stand for Poisson and Negative Binomial distributions, ‘Ab’, ‘F’ and ‘N’ respectively stand for ‘Abundance’, ‘mean availability of floral resources’ and ‘mean availability of belowground nests’. Species Ab Sites ITD (mm) THD (m) λ1% (m) Distrib. Scale of effect for the observed bee species We performed our analyses on a subset of 16 bee species, selected according to the observed abundance (occurrence in at least three different sites), and the availability of informa tion about their nesting preference and intertegular distance (ITD; distance between the wing’s insertions) in Carrié et al. (2017, 2018). The foraging distance of the species was esti mated using the allometric relationship between the inter tegular distance and the typical homing distances (THD) proposed by Greenleaf et al. (2007, reported R2 = 0.72). The THD is the distance where 50% of the bees return home when released at this distance. This is only a proxy measure ment for foraging range. Hereafter, we distinguish the ‘aver age foraging distance’ associated with the modelled bees (α parameter), from the ‘typical homing distance (THD)’ asso ciated with the observed bee species. We limited our analyses to bee species with a typical homing distances of less than 1000 m (80% of the observed bee species) to be consis tent with the average foraging distances considered for the modelled bees. We tested for global spatial autocorrelation in model residuals (Kühn and Dormann 2012) using the Moran test (Sokal and Oden 1978). When global spatial autocorrelation was detected at least at one radius, we identified the sampling sites causing autocorrelation using the local indicators of spa tial associations (LISA) (Anselin 1995), and then removed the sampling sites from the analysis. For each species, the explanatory quality of the model was evaluated across the 108 buffer radii using the AICc (Jackson and Fahrig 2012, 2014). The buffer radius associated with the lowest AIC was considered to be the scale of effect for the species. Finally, we used a delete-two-observations jackknife of the scale of effect to estimate sensitivity to subsampling. To identify the scale of effect for the 16 observed bee species, we performed multi-scale analyses with the same landscape variables as those used for the modelled bees and calculated within the same 108 buffer radii (from 10 to 5000 m). The two explanatory variables were the mean availability of floral resources in April (month of pan trapping), and the mean availability of belowground nests. As response variable, we used the observed abundance of each species in pan traps at each sampling site. Pan trapping of bee speciesf In mid-April 2016, 30 different sampling sites were selected in the margins of cereal fields in the study areas. In each site, three coloured pan traps (blue, white, yellow), filled with soapy water were set up and left for four days. The collected bees were then counted and identified to the species level f Concerning the response variable, we used the visitation rate calculated with the largest circular map extent (buf fer radius = 5000 m). We chose the 5000 m buffer radius 5 5 by experienced entomologists (for protocol details see the Supporting information). between the Poisson and the negative Binomial distributions according to the algorithm convergence warnings and the overdispersion of residuals (i.e residual deviance > residual degrees of freedom, following Hervé (2016)). For most species not all four models (abbreviated M1–4 in Table 1) were suit able for scale of effect analysis. For each species, we identified all suitable models for scale of effect analysis with two criteria 1) ∆AICc < 2 compared to the model with the best AICc (i.e. ∆AICcinter-model, Supporting information) and 2) ∆AICc > 4 between the ‘worst’ and the ‘best’ scale (i.e. ∆AICcinter-scale, Supporting information), in order to have enough AICc vari ability among scales to distinct the scale of effect. We also veri fied the existence of significant landscape effects at least at one of the 108 studied scales. Finally, among the suitable mod els, we selected the most complete model (see the Supporting information for detailed methods). Sensitivity analysis Selected model Pseudo R2 (McFadden) Scale of effect (m) 1-Andrena haemorrhoa 7 6 2.64 529.5 311 P M2: Ab ~ F+ N 0.36 1100 2-Andrena nigroaenea 6 6 2.84 670.9 3876 P M2: Ab ~ F + N 0.31 150 3-Andrena nigroolivacea 12 10 2.46 421.1 2527 P M1: Ab ~ F × N 0.37 1800 4-Andrena nitida 7 5 2.97 775.7 4442 P M1: Ab ~ F × N 0.66 2550 5-Andrena ranunculi 4 3 1.75 139.6 1007 P M2: Ab ~ F + N 0.32 750 6-Andrena ventricosa 5 5 1.63 110.9 852 P M1: Ab ~ F × N 0.45 10 7-Halictus maculatus 3 3 1.55 94.2 762 P M1: Ab ~ F × N 0.88 50 8-Halictus scabiosae 10 9 2.44 410.1 2468 P M1: Ab ~ F × N 0.36 20 9-Halictus simplex 16 12 1.9 182.3 1237 NB M3: Ab ~ F 0.11 NA 10-Lasioglossum albipes 5 4 1.64 113.1 863 P M2: Ab ~ F + N 0.39 650 11-Lasioglossum malachurum 82 23 1.79 150.2 1064 NB M1: Ab ~ F × N 0.42 1550 12-Lasioglossum marginatum 48 11 1.59 102.3 805 NB M3: Ab ~ F 0.43 10 13-Lasioglossum pallens 6 5 1.59 102.3 805 P M2: Ab ~ F + N 0.21 NA 14-Lasioglossum pauxillum 20 11 1.24 45.7 500 NB M4: Ab ~ N 0.16 20 15-Lasioglossum puncticolle 24 15 1.49 82.9 701 NB M2: Ab ~ F + N 0.23 350 16-Lasioglossum villosulum 16 10 1.33 57.3 562 NB M2: Ab ~ F + N 0.24 100 6 resource availability affected the scale of effect for five dif ferent modelled bees (α = 50, 100, 200, 400, 800 m), and for the 16 observed bees. Following the same methodol ogy as the sensitivity analysis in Lonsdorf et al. (2009), for each parameter, we generated a random variation of ± 0.1 drawn from a uniform distribution. This variation appeared as a reasonable interval relative to the range in the floral and nesting resource availability. To keep the duration of simulation and the volume of data manageable, we gener ated 100 new parameter combinations and assessed for each combination how it affected the scale of effect for the mod elled and the observed bees (see the Supporting information for details). modelled bee. Importantly, no scale of effect values was found above the λ1% equation. Scale of effect for the modelled bees Importantly, the scale of effect for 13 out of the 14 bee species were below the λ1% equation. Only the scale of effect for L. malachurum (11), was 1.5 times above λ1% (Fig. 5b). The sensitivity analysis on floral and nesting resource param eters revealed a very similar pattern of scale of effect varia tion compared to jackknife subsampling (Supporting information). For all the modelled bees, we selected the linear model with no interaction between the mean availability of floral resources and the mean availability of belowground nests, as it was most frequently identified as the best model across 108 buffer radii. Using multi-scale analyses (Fig. 1), based on R2 comparisons across buffer radii, we then detected the scale of effect for each modelled bee. The scale of effect increased significantly with increasing average foraging distance of the modelled bee (p-value < 0.001, Fig. 5a). The linear relation ship between the average foraging distance (α) and the scale of effect (S), is given by the following equation: S* = 1.8α − 35 (rpearson = 0.99, R2 = 0.97). The results of the jackknife subsampling revealed that removing five observations mod erately changed the scale of effect (Fig. 5a). The frequency distributions of the scale of effect values were unimodal and the interquartile intervals were less than 250 m (Supporting information). The variability of the scale of effect values increased with increasing average foraging distance of the Scale of effect for the observed bee species For ten species, we selected the Poisson distribution, and for the six remaining species the negative Binomial distribution (Table 1). Among all suitable models, we selected a single model per species to identify the scale of effect (Table 1) but alternative models were also analysed (Supporting informa tion). For two species (H. simplex, L. pallens), no models appeared suitable for scale of effect analysis (AICcModel > AICcNull Model and ∆AICcinter-scale < 4, respectively), thus these species were removed from the analysis (Table 1). Based on the selected models, we detected significant spatial autocor relation in three species and therefore removed the sampling sites responsible for the autocorrelation (one sampling site for A. nitida and H. scabiosae and five sampling sites for L. malachurum). Although we detected significant landscape effects for all species, for some (e.g. L. pauxillum), we noticed low deviance explained (Table 1) with AICc close to the null models (Supporting information). λ distances and relationship with foraging distance for the modelled bees For each modelled bee, the analyses of the percentage change in visitation rates in the target fields (%ΔVisitation) accord ing to the increasing radius of the circular input maps revealed contrasted patterns depending on the landscape context of the fields (Fig. 4a). As expected, for each modelled bee, the maxi mum percentage change in visitation rates (%ΔVisitationMAX) decreased with increasing radius of the circular input maps (Fig. 4a). Therefore, for each modelled bee, we identified the radius, abbreviated λ1%, representing the distance beyond which landscape composition is responsible for less than 1% change in the visitation rates in the target sunflower fields. We showed that λ1% distances increased significantly with increasing average foraging distance of the modelled bees (p-value < 0.001). The λ1% distances were linearly related to the average foraging distances (Fig. 4b) through the following equation: λ1% = 5.4α + 253 (rpearson = 0.99, R2 = 0.99). Using multi-scale analyses (Fig. 1), based on AICc com parisons across buffer radii, we detected the scale of effect for each bee species (Fig. 5b). The scale of effect increased significantly with increasing typical homing distance (p-value = 0.036). The linear relationship between the typical homing distance (THD) and the scale of effect (S) is given by the following equation: S = 1.8 THD + 162 (rpearson = 0.56, R2 = 0.32). The results of the jackknife subsampling revealed that removing two observations drastically changed the scale of effect (Fig. 5b). For four species (A. haemorrhoa-1, H. sca biosae-8, L. pauxillum-14, L. puncticolle-15), we found a mul timodal distribution in the frequency distribution of the scale of effect values, with modes separated by more than 1000 m (Supporting information).f Sensitivity analysis The sensitivity analysis on floral and nesting resource parameters revealed negligible variation in scale of effect (Supporting information). Discussion Improving our ability to estimate, a priori, the scales at which species interact with their environment is funda mental to understand the patterns and processes underly ing species–habitat relationships (Thornton and Fletcher 2014). Based on simulations using the InVEST pollina tion model plus empirical data, we showed that the aver age foraging distance (and the typical homing distance for observed bees) is positively correlated with the scale of effect 7 7 Figure 4. Left-hand plot (a), example for three modelled bees (α = 50, 100, 150 m) of the relationships between the percentage change in visitation rates in the centre of the target sunflower fields according to the radius of the circular input maps. For the modelled bee with an average foraging distance of 50 m, the grey lines represent the percentage change in visitation rates for the 50 target sunflower fields. The black lines represent the maximum percentage change in visitation rates for three modelled bees. The horizontal dashed line is the 1% threshold and the vertical dashed lines the λ1% distances associated with the modelled bees. Right-hand plot (b), linear relationship between the average foraging distance of the modelled bees (α), and their λ1% distance. The grey dashed line represents the average foraging distance (x = y equation). Figure 4. Left-hand plot (a), example for three modelled bees (α = 50, 100, 150 m) of the relationships between the percentage change in visitation rates in the centre of the target sunflower fields according to the radius of the circular input maps. For the modelled bee with an average foraging distance of 50 m, the grey lines represent the percentage change in visitation rates for the 50 target sunflower fields. The black lines represent the maximum percentage change in visitation rates for three modelled bees. The horizontal dashed line is the 1% threshold and the vertical dashed lines the λ1% distances associated with the modelled bees. Right-hand plot (b), linear relationship between the average foraging distance of the modelled bees (α), and their λ1% distance. The grey dashed line represents the average foraging distance (x = y equation). with bees’ foraging distances. Our simulation results are vali dated with empirical data as we observed a similarity between the linear equations for the modelled bees (S* = 1.8α − 35) and observed bee species (S* = 1.8THD + 162). Discussion Our results support the hypothesis that allometric relationships related to species mobility can predict their scale of effect (Wiens 1989, Jackson and Fahrig 2012, Ricci et al. 2013). for wild bees. We defined and estimated a novel metric, the maximum distance of effect, noted λ1%, representing the distance beyond which landscape composition is respon sible for less than 1% change in the simulated visitation rates of bees. We demonstrated that λ1% is linearly related to the average foraging distance (α) of the modelled bees (λ1% = 5.4α + 253) and that this index represents an upper limit distance beyond which the scale of effect for wild bees is unlikely to occur. The scale of effect for 13 out of the 14 observed bee species was below λ1%. Therefore, our study is the first successful attempt to refine the general guideline proposed by Jackson and Fahrig (2012) and to validate it with empirical data. g Compared to our simulation results there was much vari ability in the estimated scale of effect for a given bee spe cies, which hints that there are other factors at play. A first source of scale of effect variability is directly related to its definition. The scale of effect is defined as the single geo graphical extent exhibiting the best relationship between the landscape variables and the biological response studied (Jackson and Fahrig 2012). Focusing on a single scale occults that the related scales also explain the biological response (Miguet et al. 2016). The concept of scale of effect should rather be understood as a ‘scale domain’ of effect (Wiens 1989). Using resampling methods or sensitivity analyses, as we did (Moraga et al. 2019), helps in revealing these scale domains. Here, randomly removing a few sampling sites caused drastic shifts in the scale of effect for the observed bee species. Although other multi-scale studies used similar sized samples (Steffan-Dewenter et al. 2002, Krauss et al. 2003, Meyer et al. 2009, Moraga et al. 2019), scale of effect varia tions is probably due to our small sampling size relative to the biological variability in species abundance. Factors affecting the scale of effect The size of the dots represents the occurrence frequency of the scale of effect values. The grey dashed line rep resents the x = y equation. frequency distribution of the scale of effect, which is con sistent with recent studies (Moraga et al. 2019, Stuber and Fontaine 2019). Multimodality may reflect artefacts related to the heterogeneous distribution of habitats (Dormann and Seppelt 2007) or that species abundance is shaped by dif ferent ecological processes acting at different spatial (and temporal) scales (e.g. foraging versus breeding activities or individual versus population processes; Stuber and Fontaine 2019, Desaegher et al. 2021). The multimodality creates ambiguity when investigating the scale of effect of a species, especially when results depend on the explanatory variables studied (Moraga et al. 2019, Stuber and Fontaine 2019, Moll et al. 2020) or on the statistical model selected. In our case, for the species L. malachurum (11), supplementary analyses performed on alternative models revealed drastically different results with a scale of effects at 200 m, compared to 1500 m with the selected model (Supporting information). Future studies should account for this intra-species scale of effect variability, when aiming to identify factors that affect the scale of effect (Miguet et al. 2016, Stuber and Fontaine 2019). multi-scale analyses while being conservative regarding scale of effect variability. h l b d d h h ff f l d It has recently been recommended that the effects of land scape on biological responses should be investigated using distance weighting functions instead of a step function as is implicitly assumed in multi-scale analyses (Chandler and Hepinstall-Cymerman 2016, Miguet et al. 2017, Moll et al. 2020). The InVEST pollination model and the λ index do that as they are based on negative exponential weighting functions that account for the decreasing influence of land scape composition on pollinator supply and visitation rate with increasing distance (Sharp et al. 2018). The use of dif ferent weighting function probably accounts for the quantita tive discrepancy with Jackson and Fahrig (2012) results (S* ≈ 4–9 times median dispersal distance). InVEST pollination model does not take into account the order of the seasons or phenological gaps in floral resources when predicting visitation rates (Sharp et al. 2018). Nevertheless, this should only have a limited impact on the estimation of the λ index and the scale of effect for the mod elled bees. Factors affecting the scale of effect A common explanation for the scale of effect is that it reflects the characteristic distance at which organisms interact with their environment (Holland et al. 2004). However, using a model that assumes that bee diversity is proportional to the size of bee habitats, Dormann and Seppelt (2007) showed that the scale of effect for bee diversity may simply reflects the distribution of habitats in the landscape and does not nec essarily reflect a characteristic distance of biological interac tion. The origin of the scale of effect has also been called into question because of the absence of empirical validation of the effect of species mobility on the scale of effect (Miguet et al. 2016, Stuber et al. 2018, Moll et al. 2020). Here, using a spa tially explicit pollination model, our simulation results pro vide theoretical evidence that the scale of effect is correlated A second source of scale of effect variability is the pos sible existence of multiple scale domains of effect. For four observed bee species, we found multimodality in the 8 Figure 5. Left-hand plot (a), relationship between the scale of effect for the modelled bees and their average foraging distances (α). Right- hand plot (b), relationship between the scale of effect for the observed bee species and the prediction of their typical homing distance based on the intertegular distance according to Greenleaf et al. (2007). The numbers represent the species identity (Table 1). In both plots, the black circles represent the scale of effect based on the complete sample size, and the blue dots represent the scale of effect values obtained with jackknife subsampling. The size of the dots represents the occurrence frequency of the scale of effect values. The grey dashed line rep resents the x = y equation. Figure 5. Left-hand plot (a), relationship between the scale of effect for the modelled bees and their average foraging distances (α). Right- hand plot (b), relationship between the scale of effect for the observed bee species and the prediction of their typical homing distance based on the intertegular distance according to Greenleaf et al. (2007). The numbers represent the species identity (Table 1). In both plots, the black circles represent the scale of effect based on the complete sample size, and the blue dots represent the scale of effect values obtained with jackknife subsampling. Factors affecting the scale of effect Indeed, there is no reason to think of a spatial bias in the distribution of these underestimated resources relative to our sampling sites. More generally, the same argument holds if we underestimate or overestimate the importance of some land use categories by attributing the floral or nesting resource availability and is supported by our sensitivity analysis.h Supporting information The supporting information associated with this article is available from the online version. Data availability statement Data are available from the Dryad Digital Repository: <https:// doi.org/10.5061/dryad.0rxwdbs2k> (Desaegher et al. 2022). Strength and limits of the λ index Our simulation results concerning maximum distance of effect λ1%, revealed that landscape effects can still be observed far beyond the scale of effect, and probably further than it is commonly acknowledged. The strength of this index, is that it restricts the upper limit of the range of candidate scales in The main limitation of our study is related to the vali dation dataset, as we have a small sampling size, sometimes 9 9 Ouin: Conceptualization (supporting); Data curation (sup porting); Formal analysis (supporting); Funding acqui sition (lead); Investigation (supporting); Methodology (supporting); Project administration (supporting); Resources (supporting); Software (supporting); Supervision (lead); Validation (supporting); Visualization (supporting); Writing – original draft (supporting); Writing – review and editing (supporting). David Sheeren: Conceptualization (support ing); Data curation (supporting); Formal analysis (support ing); Funding acquisition (lead); Investigation (supporting); Methodology (supporting); Project administration (lead); Resources (equal); Software (equal); Supervision (lead); Validation (supporting); Visualization (supporting); Writing – original draft (supporting); Writing – review and editing (supporting). low explanatory power, and only included 16 ground- nesting bee species (three genera). Also we did not account that bee abundance can depend on floral resources available before the sampling month or more generally on the phe nological continuity along the year as is growingly acknowl edged (Timberlake et al. 2019, Guezen and Forrest 2021). Therefore, we call for future studies to test our equation with other species, in different seasons and accounting for other factors, for instance the aggregation of habitats that has been shown to affect the scale of effect (Ricci et al. 2013). However it should be reassuring that the model we used already showed its ability to explain the field estimates of bee abundance (Lonsdorf et al. 2009, Groff et al. 2016, Desaegher et al. 2021). References Anselin, L. 1995. Local indicators of spatial association – LISA. – Geogr. Anal. 27: 93–115. g Barton, K. 2022. MuMIn: multi-model inference. R package ver. g Barton, K. 2022. MuMIn: multi-model inference. R package ver. 1.46.0. – <https://CRAN.R-project.org/package=MuMIn>. p p j g p g Baude, M. et al. 2016. Historical nectar assessment reveals the fall and rise of floral resources in Britain Nature 530: 85 Baude, M. et al. 2016. Historical nectar assessment reveals the fall and rise of floral resources in Britain. – Nature 530: 85. Acknowledgements – This study was supported by a grant from Occitanie region. We thank Fabien Laroche, Marc Deconchat, Emilie Andrieu, Francois Calatayud and Yousra Hamrouni for their help and insightful comments. We are grateful to the farmers for allowing us to work in their fields and to everyone involved in the field surveys and species identification. l Cane, J. H. 1991. Soils of ground-nesting bees (Hymenoptera: Apoidea): texture, moisture, cell depth and climate. – J. Kansas Entomol. Soc. 64: 406–413. Carrié, R. et al. 2017. Relationships among ecological traits of wild bee communities along gradients of habitat amount and frag mentation. – Ecography 40: 85–97.il ii Funding – Field work was part of the Sebioref project funded by the Occitanie region and the French National Research Institute for Agriculture, Food and Environment (INRAE). Carrié, R. et al. 2018. 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For example, when applying the equation to the wild bee community observed in our study area, we obtained a λ1% = 2.5 km (THD weighted mean = 420 m). A safe distance between sites would be two times λ1% (non-overlapping buffer radius of λ1%). Landscape sampling design is, of course, a tradeoff between the distanc ing and number of sites and the extent of the study area. Finally, as a quick estimate of the maximum distance of landscape effects on wild bees, λ index could help landscape managers and conservation biologists, in scaling or designing nature reserves. The peer review history for this article is available at <https:// publons.com/publon/10.1111/ecog.05758>. Author contributions Foraging ranges of solitary bees. – J. Anim. Ecol. 71: 757–764. Ouin, A. et al. 2022. Chapter Two – Building a shared vision of the future for multifunctional agricultural landscapes. Lessons from a long term socio-ecological research site in south-western France. – In: Bohan, D. A. et al. 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https://openalex.org/W4297982575	https://www.researchsquare.com/article/rs-1800985/latest.pdf	English	null	Assessment of awareness and knowledge of schistosomiasis among school-aged children (6–13 years) in the Okavango Delta, Botswana	Global health research and policy	2,022	cc-by	7,100	Background Schistosomiasis is a Neglected Tropical Disease (NTD) with negative effects on public health. Endemic in 78 tropical and sub- tropical countries, schistosomiasis afflicts approximately 250 million people worldwide, with more than 90% cases occurring in Sub- Saharan Africa [1–3]. An estimated 20,000-200,000 people die of schistosomiasis each year [4]. The disease is ranked among the top ten causes of disability in six African countries [5] with an estimated 3, 7900 Years of Life with Disability (YLDs) in 2012 attributed to schistosomiasis. The highest prevalence of the disease in Africa is seen in Nigeria (29 million) followed by Tanzania (19 million) and Ghana and the Democratic Republic of Congo both at 15 million [6]. The disease is ranked among the top ten causes of disability in six African countries [5]. School-Aged Children (SAC) bear most of the burden of this disease in Africa [6, 7]. Botswana bears an overall prevalence of 10.2% for Schistosoma. Haematobium with the Okavango region carrying the highest burden of the disease Ministry of Health and Wellness [8]. The integrated control program, implemented between 1985 and 1993 managed to greatly reduce the prevalence and severity of the diseases within the country. Schistosomiasis is a parasitic disease caused by blood flukes of the genius schistosome [4, 9]. Humans are usually infected by five species of Schistosoma mansoni, Schistosoma japonicum, Schistosoma mekongi, and Schistosoma intercalatum causing intestinal infection and haematobium responsible for urinary infection [10]. The sub-Saharan African infection is caused by S. mansoni and S. haematobium. The disease is often acquired during domestic, occupational, and livelihood activities which involve contact with water infested with cercariae from the intermediate host-the snail (6)]. Climate change, ecological changes such as dam construction, and socio-economic factors have been crucial factors in the continuous transmission of schistosomiasis in endemic areas [6, 10]. The World Health Assembly (WHA) has made several resolutions [11] and specified control and elimination measures of schistosomiasis. These include snail control, mass treatment, improved sanitation, provision of potable water, and health education. Disease control programs recommended for schools include Mass Drug Administration (MDA), sanitary methods, and health education. The WHO guidelines recommend every-other-year praziquantel treatment of all School-Aged Children (SAC) in communities having moderate risk (prevalence between 10% and 49%), and annual SAC treatment for communities having high risk (prevalence ≥ 50%) [12]. Abstract Background: Schistosomiasis is a global health problem affecting 250 million people, with 90% in Sub-Saharan Africa. In Botswana, the burden is high in the Okavango delta because of the water channels. WHO recommends integrated measures, including access to clean water, sanitation, health education, and drugs to control and eliminate schistosomiasis. Gauging knowledge and awareness of schistosomiasis for School-Aged Children (SAC) is crucial. Our study aimed at assessing knowledge and awareness of schistosomiasis among SAC in the Okavango delta. Methods: A cross-sectional survey assessing awareness and knowledge of schistosomiasis in schools was conducted. 480 questionnaires were administered to gather demographic profiles, awareness, and knowledge of risky behaviors. Chi-square and descriptive analysis determined the differences in SAC`s awareness and knowledge levels based on localities, gender, age, and health education. Results: The results showed a low awareness level, with only (42%) of respondents having heard about the disease and (52%) knowing its local name. Younger children from Sekondomboro (83%) and Samchima lacked awareness, while children from Mohembo (77%) and those who had health education (70%) demonstrated significant awareness levels (p≤0.001). Seventy-two percent (72%) lacked knowledge of the cause and (95%) did not know the disease life-cycle. Children from Xakao (91%), (85%) Sepopa, and (75%) of younger children did not know haematuria is a symptom of the disease. Older and SAC with health education were more likely to know that swimming is a risk factor (p≤0.001) and (p≤0.05) respectively. Conclusions and recommendations: Although respondents from four schools demonstrated some level of awareness of the disease, and knowledge of risky behaviors, the study showed a lack of in-depth knowledge on the life-cycle and cause of the diseases. We, therefore, recommend the implementation of an integrated approach to health education and improvement in access to clean water and sanitation in all study areas. Research Article Page 1/14 Background Re-infection with Schistosoma haematobium among SAC is of global concern especially with low (34.6%) MDA program coverage and low implementation of preventive therapy (40.8%) in the African region [13]. Several studies [14–16], Page 2/14 Page 2/14 indicated that often after treatment new infection cases resurfaces to the point where baseline prevalence is reached within a short period. Recognizing the intense studies on the contribution of the aforementioned risk factors on the rate of infection with schistosomiasis, it is assumed that the complexity of the socio-economic, behavioral, and knowledge factors vary considerably at global, regional, and local levels. Despite the huge success in the national control program, Botswana still battles with transmission interruption and complete elimination of the disease. The Okavango region presents a major threat of schistosomiasis because of its potential for a resurgence due to flood recession farming and the predicted increase in the seasonal flow of the delta [17, 18]. In their research, Appleton, and Madsen [19] indicated that hydrological factors seem to play an important role in disease transmission than livelihoods activities. The authors [19] noted that the population living near the river and children attending schools near the river in Botswana are at an increased risk of repeated epidemics. Despite the significant prevalence of 10% of schistosomiasis in Botswana [8] its impairing morbidity and the resultant complications especially among the SAC has been undervalued in the past three decades, thus qualifying it as a neglected disease. Moreover, the scarcity of data showing risks factors and rates of re-infections in Botswana poses scientific and ethical challenges in addressing the burden of this disease. The 65.21 World Health Assembly resolutions [9] required the inclusion of all high-risk groups to eliminate schistosomiasis. Primary school-aged children in endemic foci are considered a high-risk group but there is a scarcity of data in Botswana showing the dynamics of re-infections among school children to inform current and future control strategies. Also, the Botswana schistosomiasis control program has heavily relied on the test and treats strategy, where cases detected through the Kato- Katz method are treated with 40mg/kg of praziquantel [20]. Communities in the Okavango have low levels of knowledge and awareness of the disease [21]. Adequate knowledge and adoption of correct and consistent preventive behavior in endemic areas are necessary for an effective and sustainable control program owing to the elimination of the disease. Background Although health promotion through community education is an important aspect of the elimination of any disease, a review of literature in this area for Botswana SAC revealed a paucity of empirical data owing to decal neglect of the disease. There is insufficient knowledge in Botswana which mapped the knowledge and awareness of schistosomiasis and its risk factors. This calls for assessment knowledge of key risk factors for infections within endemic communities. This study aimed at assessing risk factors for schistosomiasis among SAC in the Okavango delta. The specific objectives are to i) determine the SAC‘s i) Awareness of the disease, ii) general knowledge, and ii) knowledge of risk behaviors of schistosomiasis among SAC in the Okavango Delta. Methodology Study Design. This is a cross-sectional survey that assessed students’ knowledge and awareness of schistosomiasis haematobium and its risk factors. Study setting. The study was conducted in the Ngamiland district in the North-western part of Botswana with a population of 175,631 [22]. The site is part of the Okavango Delta which is one of the world`s largest wetlands. The delta comprises permanent, seasonal, and swampy areas which originate from the upstreams of Angola and Namibia. The Cubango River from Namibia enters Botswana and travels along with the stream passing into upper villages of the panhandle. As it travels along these villages it gives way to seasonal swamps. The flood is often at its peak at Mohembo between mid-March and mid- May immediately after the summer rains. The delta is residence to a multi-ethnic group of people who include: Bayeyi, Hambukushu, San/Khokhoi, and Batawana. More than 900 people live in six villages along the Okavango River where the study was conducted (Fig 1) [23]. Six schools in villages along the Okavango watercourse were purposively sampled as sites for the study. Each village has one primary school. The proximity of the villages to the wetland and water contact livelihoods which often involve schoolchildren provided a good opportunity for studying schistosomiasis. The selected villages are proximal to the seasonal flood plains/permanent water channels status. The upstream schools of Samchima, Mohembo, Skondomboro, Ngarange, Xakao, and Sepopa were included in the study. Sepopa is the one among selected villages that is further at the mid-upper delta or where the Panhandle spread into the alluvial Delta. Data analysis Data were exported from the REDCap and entered into Statistical Package for Social Scientist (SPSS) version 25.0 for management and analysis. Descriptive statistics (proportions and means values) were used to determine the frequency of responses. Chi-squared tests of independence were used to determine the associations between demographic variables and variables of interest. Inferences using p ≤ .05 to test for statistical significance were made on the relationship between variables. All quantitative analyses were done at the 5 % level of significance. Data Collection. Data was collected on January 18-28, 2020 using the questionnaire directly administered by the researchers. Community research assistants received training to assist with the data collection. Mini-sized cellular tablets which had the Research Electronic Data Capture (REDCap) software installed in them were used for data collection. The researchers were onsite to ensure appropriate data capturing and to address any technical issues. Two data collectors and the principal investigator used 4 electronic tablets to collect data across the six study sites. Sampling Since each village had only one primary school, all schools within the selected villages participated in the study with required samples calculated for every school. SAC was enrolled in the study if (i) parents/guardians had consented, (ii) pupils had assented, (iii) pupils were 6-13 years and were not cognitively impaired. The sample for the study consists population of children in standard 1 to 6 (age 6-13). Survey Questionnaire A structured questionnaire was developed to assess the knowledge and awareness of schistosomiasis. Knowledge was measured through a set of items that did not comprise a score but assessed if the respondents possess correct information and knowledge about the disease. The questionnaire consisted of three main sections. The first section covered socio-demographic information. The second section covered participants’ awareness and knowledge of the disease including the signs and symptoms, lifecycle the effect of the disease on children. The last section comprised questions on the respondents’ knowledge of key risk factors. Ethical Consideration The study design and protocol were reviewed and approved by the Human Research Development Committee at the Botswana Ministry of Health and Wellness (letter referenced HPDME: 13/18/1) and the University of Botswana Institutional Review Board (referenced UBR/RES/IRB/BIO/154). Parental consent was sought and children were asked for assent at the beginning of the interview. Study Population. The population for the study constituted school-aged children from standard one to six (6-13 years), within the selected schools. Sampling ≤insert figure 1≥ Page 3/14 Page 3/14 Page 3/14 Socio- demographics profile of respondents A total of 480 respondents 6 to 13 years were enrolled in the study, of which 245(51%) were females and 235(41%) males. The mean age for the participants was 9.13 years. Respondents were further divided into two age groups: 6-9 who formed the majority 282(59%) and 10-13 years who were 198(41%). The majority 101(21%) of respondents were from Mohembo and Xakao villages. The majority 97(20%) of the respondents were in the fifth grade and the lowest number 48(10%) of respondents were from grade one (Table 1). Respondents 183(38%) reported having received health education about the disease from any source, while the majority 297(62%) had not, while 183(38%) had received it. ≤insert table 1≥ ≤insert table 1≥ Page 4/14 Page 4/14 ≤Insert Table 2≥ ≤Insert Table 2≥ General Knowledge of Schistosomiasis among Respondents The majority 343(72%) did not know that schistosomiasis was caused by parasites released during snail bite when one comes in contact with infected water. In terms of participants’ knowledge of the effects of schistosomiasis on one’s health, a very small proportion 83(17%) and 108(23%) mentioned slow growth and poor concentration respectively. An overwhelming majority, 454(95%) of respondents did not know the life cycle of schistosomiasis in both humans and snails. Concerning participants’ knowledge of signs and symptoms of schistosomiasis, younger participants 211(75%) did not know that blood in the urine (haematuria) is the main symptom of schistosomiasis compared with older children. The majority of participants from Xakao 91(91%) and Sepopa 47(85%) and those with no health education 218(73%) did not know that haematuria is a symptom of schistosomiasis (see Table 3). The knowledge of fever as a symptom of schistosomiasis was very low <20% across all the socio-demographics of the respondents. The results on fever as a sign were not significant between the age groups (refer to table 3), of children who had and had no health education (p=0.935). An overwhelming majority of older participants 172(87%) than younger children 235(58%) did not know that lower abdominal pain is a symptom of schistosomiasis. Similarly, respondents from Xakao 99(99%), Sepopa 53(96%), and Mohembo 91(90%) didn’t know that abdominal pain was a symptom of the disease. A chi- square test for the difference in knowledge of abdominal pains showed that children from the three villages were more likely (p≤0.001) to demonstrate a lack of knowledge of abdominal pain as a symptom than children from other villages. The results for difference in knowledge of abdominal pain as symptom between age groups (p=0.288), gender (p=0.749) and children with no or some health education (p=0.965) were not significant. ≤Insert table 3≥ Awareness of Schistosomiasis among Respondents Older participants (10-13 years) 101(51%) were aware of Bilharzia compared to younger ones (6-9 years) 99(35%), and an almost equal proportion of females 103(42%) and males 97(41%) have heard about the disease. A higher proportion of SAC in Sekondomboro 50(79%) and Samchima 64(69%) villages had not heard about the disease. The majority, 260(93%), of children who had no health education about the disease indicated that they had not heard about the disease. A chi-square test conducted to determine if there were any significant differences among age groups and between those who received health education and those who did not, showed that older participants and those with health education were more likely to have heard about the disease (p≤0.001) and (p≤0.001) respectively. A relatively higher proportion of respondents in the age group 10-13, 112(57%), and males 122(57%) knew the local name of the disease to be “bilharzia”. The majority of respondents who had health education 128(70%) knew the local name of the diseases than those with no health education 121(41%). Most respondents from Mohembo village 78(77%) demonstrated awareness of the local name of the disease than those from other villages. A chi-square test indicated no significant differences in awareness of the local name of bilharzia between males and females in the four villages, (p=0.085) and the age groups (p=0.986). Respondents who had health education (p≤0.001) were more likely to be aware of the local name of the disease. Similarly, respondents from Mohembo were more likely to be aware of the local name of the disease (p≤0.001) than those from other villages. The majority of younger respondents 210(75%), Sekondomboro 52(83%), and Samchima 74(80%) villages indicated that children from their schools lacked awareness of the disease. Similarly, respondents who had no health education about the disease 262(88%) were more likely to mention that other children from their schools lacked awareness of the disease than those who had health education. An almost equal proportion of respondents in the gender category indicate that other children from their school lacked awareness of the disease. Younger children (p≤0.001), those who have no health education (p≤0.001), and those from Sekondomboro and Samchima (p≤0.001), were more likely to indicate that children from their schools lacked awareness of the disease. The results on the difference between males and females on this fact were not significant (p=0.830), (refer to table 2). Discussion Active participation of local populations in prevention and control programs in endemic areas is highly dependent on adequate knowledge and awareness of the disease especially among children who are often the target of control programs. Our study findings show low levels of awareness of the disease, where only (42%) of the respondents had heard about the disease and just above half (52%) knew the local name of schistosomiasis. These findings corroborate those of other studies [21]. Respondents who had health education at least knew that haematuria was a sign of the disease. However, having health education did not influence knowledge of signs and symptoms of the disease because our study records low levels of knowledge of the signs and symptoms. It is also important to note that 30% of respondents who had heard about the disease got information from school, compared to 5% who got it from the clinic. This may be attributed to the decadal neglect of the disease since 1993 when the national control program was terminated. A high proportion (62%) of respondents had no health education on the disease. This shows that health education as a control strategy has not been implemented over two decades. Our results are similar to a Yemen study [24], which reported a lower knowledge of signs and symptoms but a higher level of awareness. A systematic review of the knowledge, attitude, and practices on schistosomiasis found that knowledge of signs and symptoms is often low [25]. This poses challenges and threats to the uptake of most prevention and control strategies. However, our study found that males and those who had health education, significantly (p≤0.001) knew haematuria as a symptom compared to females and those without health education. This could be explained by the fact that the universal local name for urinary schistosomiasis in Botswana is “thutisamadi" which means blood in the urine. This finding is similar to that obtained in Swaziland [2] where 74% mentioned haematuria as a symptom. Again [26] in their study, recorded that among respondents who had prior knowledge of the disease, 80% mentioned haematuria as a symptom. With regards to males possessing more knowledge than females, a systematic review in sub-Saharan Africa [25], found eight out of twelve studies that reported high levels of knowledge among males compared to females. Knowledge of Risky Behaviors for the Disease among respondents Page 5/14 Page 5/14 The majority 199(71%) of respondents aged 6 to 9 years knew that swimming is a risk factor for schistosomiasis, with 149(81%) of those who had health education possessing knowledge. An equal proportion of females 186(76%) and males 180(77%) knew that swimming poses a risk for the disease. One hundred and seventy 170(85%) of the respondents who had heard about The majority 199(71%) of respondents aged 6 to 9 years knew that swimming is a risk factor for schistosomiasis, with 149(81%) of those who had health education possessing knowledge. An equal proportion of females 186(76%) and males 180(77%) knew that swimming poses a risk for the disease. One hundred and seventy 170(85%) of the respondents who had heard about schistosomiasis, knew that swimming was a risk for the disease. The majority 75(81%) of respondents from Samchima and Xakao 79(79%) villages knew swimming as a risk factor for Bilharzia. A chi-square test showed that older participants and those with health education were more likely to know that swimming is a risk factor (p≤0.001) and (p=0.037) respectively. Similarly, children who have heard about schistosomiasis were more likely to know swimming as a risk factor for the disease (refer to table 4). There were no significant differences within villages between those who know that swimming is a risk factor and those who did not know (p=0.115). ≤ Insert table 4≥ Children aged -10-13 years 140(71%) and those who received health education 136(74%) knew that walking barefooted in water poses a risk for schistosomiasis. Females 176(72%) were more knowledgeable of this fact than males 153(65%). Children who had received health education were more likely to know that walking in water barefooted is a risk factor for schistosomiasis (p=0.032). There was no significant difference in age and gender (refer to table 5) between those who did not know and those who know that walking barefooted in water is a risk factor for schistosomiasis. ≤ insert table 5≥ The majority of female respondents 186(76%) considered drinking contaminated water as a risk for schistosomiasis compared to males 169(72%). Of the 198 respondents aged 10-13, 154 (78%) mentioned that drinking contaminated water as a risk for the disease compared to 200(71%) of those aged 6 to 9 years. Respondents from Samchima 55(59%) and Sekondomboro 38(60%) did not mention this factor as a risk for the disease. Respondents who had health education 151(83%) on the diseases mentioned drinking contaminated water as a risk factor than those who had no education 204(69%) of the disease. Health education (p=0.01), and village (p=0.001) were significantly associated with mentioning drinking contaminated water as a risk for the disease. Discussion The authors of a review [25] attributed better knowledge among males to health education provided to fishermen and farmers because of their exposure to risky water practices. The difference in review and the Cameroon study [26] and our study is that our respondents were SAC while the two studies focused on adults. Page 6/14 Page 6/14 Our study also revealed that respondents do not know the cause and the life cycle of schistosomiasis. This finding is similar to what Swaziland's [2] study obtained. However, [27] reported a higher proportion (77.6%) of respondents who knew the cause of schistosomiasis. The difference may be that we asked closed-ended questions which limited exploration of possible responses which could have unearthed the real causes of the disease. Part of these differences might have been related to the fact that these studies used a different study method (that is, non-probability purposive sampling technique) than ours [28]. Regarding the mode of transmission of the disease, which is indicated as a proxy from the question on drinking contaminated water, our study found some misconceptions from the respondents. A significant proportion of pupils across all villages mentioned that drinking contaminated water poses a risk of contracting the disease. This can be attributed to the fact that residence in the Okavango area often depends on contaminated river water for domestic purposes [29]. Consequently, outbreaks of water-borne disease (diarrhea) are common especially during the dry season [30]. In addition to this, it is worth noting that the recurring unavailability and unreliability of water due to nonfunctioning taps and pumps and leaks experienced by villages in the Okavango delta, often prompt utilization of river water for domestic use even in schools [31]. This may have prompted the respondents in our study to believe that schistosomiasis may be caused by drinking contaminated water. Moreover, this could suggest the lack of integrative approaches towards schistosomiasis and STH programming. Of greater concern is how these fears translate into preventive practices. Regarding the knowledge of risk factors for schistosomiasis, our study found that younger pupils (aged 6-9 years) were more likely to know that swimming (p≤0.001) is a risk for the disease. This may be the influence of the teaching of content on schistosomiasis which is done at lower than upper school grades. Moreover, the health education in schools during MDA for STH in the past two years has contributed to this finding. Limitations of the study Notwithstanding the important findings from our study, we acknowledge the following limitations encountered during the implementation of the study; firstly, recall bias. The respondents were required to recall knowledge and awareness of the disease. This could in some way explain the low levels of knowledge obtained in the study. Secondly, there was an issue with the subjectivity of responses. The self-reported data obtained from respondents lacked validation. This study did not provide information about reasons for not having Knowledge about schistosomiasis. This calls for future research to probe the reasons for the lack of knowledge and awareness. There is a need, therefore, for a future qualitative study for this purpose. Discussion Other studies [25] concurred that programs such as MDA improve awareness, knowledge, and uptake of preventive chemotherapy. Conclusions And Recommendations This study found that most school children in the Okavango delta lack awareness and knowledge of schistosomiasis with the majority not knowing the life cycle and most of the symptoms of the disease. This provides a window of opportunity to re-instating prevention and control measures of the disease within the area. SAC demonstrated a fairly average knowledge of water contact risky behaviors of swimming and walking barefooted in infested water. Health education has impacted positively on the children’s awareness and knowledge, but other socio-demographics of age and gender did not. This is a signal of the importance of health education as a key component of the control program for schistosomiasis. There is thus the need for intense integration of school- based health education to improve the level of knowledge and awareness to reduce children’s water contact behaviors. We recommend the integration of health education components to both community and school-based MDA programs often conducted for NTDs within the district. Further research focused on unearthing reasons for low levels of awareness and knowledge is required to inform policy on elimination. More consideration and appreciation of the socio-cultural and cognitive behaviors of children than a focus on their age, gender, and place of residence which are found to have very little influence on their knowledge of risk factors for schistosomiasis prevention in the present study. List Of Abbreviations List Of Abbreviations Page 7/14 MDA Mass Drug Administration NTD Neglected Tropical Disease SAC School-Aged Children STH Soil-Transmitted Helminths WHA World Health Assembly WHO World Health Organization MDA Mass Drug Administration NTD Neglected Tropical Disease SAC School-Aged Children STH Soil-Transmitted Helminths WHA World Health Assembly WHO World Health Organization Acknowledgments g The authors are grateful to the National Institute for Health Research (NIHR) Global Health Research Programme (16/136/33) for giving the first author scholarship using UK aid from UK Government. Our sincere thanks to the school children who volunteered to participate in the study. Author`s Information: GPK is the corresponding author. Author`s Information: GPK is the corresponding author. Authors` Contributions GPK conceptualized the study, obtained ethical clearance, did the formal analysis, and wrote the draft manuscript. PN acquired funding and together with PLED offered supervision and editing while TTO helped with the writing. All authors read and approve the manuscript. GPK conceptualized the study, obtained ethical clearance, did the formal analysis, and wrote the draft manuscript. PN acquired funding and together with PLED offered supervision and editing while TTO helped with the writing. All authors read and approve the manuscript. Declarations Ethical Approval and Consent: The study was reviewed and approved by the Human Research Development Committee at the Botswana Ministry of Health and Wellness (letter referenced HPDME: 13/18/1) and the University of Botswana Institutional Review Board (referenced UBR/RES/IRB/BIO/154). Parental consent was sought and children were asked for assent to their participation in the study. Consent for Publication: The regional (Okavango) education office, parent and children consented to participate in the study. Consent, assent forms and letters are available from the corresponding author upon request. Consent for Publication: The regional (Okavango) education office, parent and children consented to participate in the study. Consent, assent forms and letters are available from the corresponding author upon request. vailable from the corresponding author upon request. Availability of Data and Materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Availability of Data and Materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing Interest: PLED is Deputy Editors-in-Chief for the Journal of Global Health Research and Policy and supervisor for GPK`s PhD studies. Competing Interest: PLED is Deputy Editors-in-Chief for the Journal of Global Health Research and Policy and supervisor for GPK`s PhD studies. Funding: This work was supported by National Institute for Health Research (NIHR) Global Health Research program (16/136/33). The corresponding author is ultimately responsible for the decision of submission and publication. Funding: This work was supported by National Institute for Health Research (NIHR) Global Health Research program (16/136/33). The corresponding author is ultimately responsible for the decision of submission and publication. References 1. Donohue RE, Mashoto KO, Mubyazi GM, Madon S, Malecela MN, Michael E. Biosocial determinants of persistent schistosomiasis among schoolchildren in Tanzania despite repeated treatment. Tropical medicine and infectious disease. 2017;2(4):61. 1. 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Hydrometeorology and flood pulse dynamics drive diarrheal disease outbreaks and increase vulnerability to climate change in surface-water-dependent populations: A retrospective analysis. PLoS medicine. 2018;15(11):e1002688. Tables pondents` Socio-demographic characteristics Variable Category Frequency (n) Percentage (%) Gender Female 245 51 Male 235 49 Age Group 6-9 282 59 10-13 198 41 Grade 1 48 10 2 83 17 3 85 18 4 92 19 5 97 20 6 75 16 School Mohembo 101 21 Ngarange 68 14 Samchima 93 19 Sekondomboro 63 13 Sepopa 55 12 Xakao 100 21 Health Education about the disease No Health Education 297 62 Had Health education 183 38 Table 1: Respondents` Socio-demographic characteristics Page 8/14 Communicating environment in the Okavango Delta, Botswana: an exploratory assessment of the sources, channels, and approaches used among the Delta Communities. Science Communication. 2012;34(6):776–802. 24. Sady H, Al-Mekhlafi HM, Atroosh WM, Al-Delaimy AK, Nasr NA, Dawaki S, et al. 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Attitude and Practice among primary school children in Mtera dam area, Mpwapwa district. 2012. Page 9/14 Page 9/14 28. Acharya S Developing novel ways of studying motility in Schistosoma mansoni and its potential contribution towards inhibiting Schistosomiasis. 2019. 28. Acharya S Developing novel ways of studying motility in Schistosoma mansoni and its potential contribution towards inhibiting Schistosomiasis. 2019. 28. Acharya S Developing novel ways of studying motility in Schistosoma mansoni and its potential contribution towards inhibiting Schistosomiasis. 2019. 29. Tubatsi G, Bonyongo M, Gondwe M. Water use practices, water quality, and households’ diarrheal encounters in communities along the Boro-Thamalakane-Boteti river system, Northern Botswana. Journal of Health, Population and Nutrition. 2015;33(1):1–12. 29. Tubatsi G, Bonyongo M, Gondwe M. Water use practices, water quality, and households’ diarrheal encounters in communities along the Boro-Thamalakane-Boteti river system, Northern Botswana. Journal of Health, Population and Nutrition. 2015 33(1) 1 12 29. Tubatsi G, Bonyongo M, Gondwe M. Water use practices, water quality, and households’ diarrheal encounters in communities along the Boro-Thamalakane-Boteti river system, Northern Botswana. Journal of Health, Population and Nutrition. 2015;33(1):1–12. ; ( ) 30. Alexander KA, Heaney AK, Shaman J. Hydrometeorology and flood pulse dynamics drive diarrheal disease outbreaks and increase vulnerability to climate change in surface-water-dependent populations: A retrospective analysis. PLoS medicine. 2018;15(11):e1002688. 31. Ngwenya B, Thakadu O, Phaladze N, Bolaane B. Table 3: Knowledge of signs and symptoms of schistosomiasis by demographic characteristics able 3: Knowledge of signs and symptoms of schistosomiasis by demographic characteristics Table 4: Knowledge of Swimming as a Risk Factor by Socio-Demographics Table 1: Respondents` Socio-demographic characteristics Table 2: Awareness of Schistosomiasis by demographic characteristics Table 2: Awareness of Schistosomiasis by demographic characteristics Page 10/14 Page 10/14 Page 10/14 Demographics Frequency (%) Heard about Bilharzia P- value Know the local name of the disease P- Value Think other children are aware of the disease P- Value No Yes Don’t Know Bilharzia No Yes Age 6-9 282 (59) 183 (65) 99 (35) 0.001 145 (51) 137 (49) 0.085 210 (75) 72 (25) ≤0.001 10-13 198(41) 97 (49) 101 (51) 86 (43) 112 (57) 117 (59) 81 (41) Gender Females 245(59) 142(58) 103 (42) 0.001 118 (48) 127 (49) 0.986 168 (69) 77 (31) 0.830 Males 235(41) 138 (59) 97 (41) 113 (48) 122 (52) 159 (68) 76 (32) Village Mohembo 101(21) 49 (49) 52 (51) 0.000 23 (23) 78 (77) 0.000 67 (66) 34 (34) ≤0.001 Ngarange 68 (14) 32 (47) 36 (53) 32 (47) 36 (53) 35 (52) 33 (48) Samchima 93 (19) 64 (69) 29 (31) 46 (50) 47 (50) 74 (80) 19 (20) Sekondomboro 63 (13) 50 (79) 13 (21) 42 (67) 21 (33) 52 (83) 11 (18) Sepopa 55 (11) 26 (47) 29 (53) 35 (64) 20 (36) 26 (47) 29 (53) Xakao 99 (21) 58 (59) 41 (41) 52 (53) 47 (47) 72 (73) 27 (27) Health Education Status No Health Education 297 (62) 260 (93) 37 (19) 0.000 176 (59) 121 (41) 0.000 262 (88) 35 (12) ≤0.001 Had Health Education 183 (38) 20 (11) 163 (89) 55 (30) 128 (70) 65 (35) 118 (65) Table 3: Knowledge of signs and symptoms of schistosomiasis by demographic characteristics Page 11/14 Page 11/14 Socio Demographics Frequency (%) Fever P- value Blood in Urine P- Value Lower Abdominal Pain P- Value No Yes No Yes No Yes Age 6-9 282 (59) 248(88) 34 (12) 0.084 211 (75) 71 (25) 0.114 235 (58) 47 (17) 0.288 10-13 198(41) 163 (82) 35 (18) 106 (54) 92 (46) 172 (87) 26 (13) Gender Female 245(59) 208 (85) 37 (15) 0.084 170 (69) 75 (31) 0.000 209 (85) 36 (15) 0.749 Male 235(41) 203 (86) 32 (14) 147 (63) 88 (37) 198 (84) 37 (15) Village Mohembo 101(21) 83 (81) 13 (13) 0.018 76 (75) 25 (25) 0.000 91 (90) 10 (10) ≤0.001 Ngarange 68 (14) 56(82) 12 (18) 32 (47) 36 (53) 59 (87) 9 (13) Samchima 93 (19) 70 (75) 23 (25) 33 (35) 60 (65) 57 (61) 36 (39) Sekondomboro 63 (13) 55 (87) 8(13) 38 (60) 25 (40) 48 (76) 15 (24) Sepopa 55 (11) 51 (93) 8(13) 47 (85) 8 (15) 53 (96) 2 (4) Xakao 99 (21) 91 (91) 9 (9) 91 (91) 9 (9) 99 (99) 1 (1) Health Education Status No Health Education 297 (62) 254 (86) 43 (14) 0.935 218 (73) 79 (27) 0.000 252 (85) 45 (15) 0.965 Had Health Education 183 (38) 157(86) 26 (14) 99 (55) 84 (46) 155 (85) 28 (15) Table 4: Knowledge of Swimming as a Risk Factor by Socio-Demographics Table 4: Knowledge of Swimming as a Risk Factor by Socio-Demographics Page 12/14 Page 12/14 Factor Variable Frequency (%) Swimming P-Value No (%) Yes (%) Age 6-9 282 (59) 83 (29) 199 (71) ≤0.001 10-13 198 (41) 31 (16) 167 (84) Gender Females 245 (51) 59(24) 186 (76) 0.862 Males 235 (49) 55(23) 180 (77) Village Mohembo 101 (21) 31 (31) 70 (69) 0.115 Ngarange 68 (14) 10 (15) 58 (85) Samchima 93 (19) 18 (19) 75 (81) Sekondomboro 63 (13) 18 (29) 45 (71) Sepopa 55 (12) 16 (29) 39 (71) Xakao 100 (21) 21 (21) 79 (79) Health Education No education 297 (62) 80 (27) 217 (73) 0.037 Had Education 183 (38) 34 (19) 149 (81) Heard about Disease Not Heard 280 (58) 84 (30) 196 (70) ≤0.001 Heard 200 (42) 30 (15) 170 (85) Table 5: Knowledge Walking in Water Barefooted by Socio-Demographics Factor Variable Frequency (%) Walking In Water Barefooted P-value No (%) Yes (%) Age 6-9 282 (59) 93 (33) 189 (67) 0.392 10-13 198 (41) 58 (29) 140 (71) Gender Females 245 (51) 69 (35) 176 (72) 0.112 Males 235 (41) 82 (35) 153 (65) Health Education No education 297 (62) 104 (35) 193 (65) 0.032 Had Education 183 (38) 47 (26) 136 (74) Table 5: Knowledge Walking in Water Barefooted by Socio-Demographics Figures Page 13/14 Page 13/14 Page 13/14 Page 13/14 Figure 1 Map of the Okavango Delta and Study Sites ODIS (2019); “Okavango Delta Information System”, University of Botswana, Okavango Research Institute from https://www.odis.ub.bw/portal/home Figure 1 Map of the Okavango Delta and Study Sites ODIS (2019); “Okavango Delta Information System”, University of Botswana, Okavango Research Institute from https://www.odis.ub.bw/portal/home ODIS (2019); “Okavango Delta Information System”, University of Botswana, Okavango Research Institute from https://www.odis.ub.bw/portal/home Page 14/14
https://openalex.org/W3137303899	https://link.springer.com/content/pdf/10.1007/s10994-020-05939-8.pdf	English	null	Dealing with multiple experts and non-stationarity in inverse reinforcement learning: an application to real-life problems	Machine learning	2,021	cc-by	18,548	Machine Learning (2021) 110:2541–2576 https://doi.org/10.1007/s10994-020-05939-8 Machine Learning (2021) 110:2541–2576 https://doi.org/10.1007/s10994-020-05939-8 * Amarildo Likmeta amarildo.likmeta2@unibo.it Abstract In real-world applications, inferring the intentions of expert agents (e.g., human operators) can be fundamental to understand how possibly conflicting objectives are managed, help- ing to interpret the demonstrated behavior. In this paper, we discuss how inverse reinforce- ment learning (IRL) can be employed to retrieve the reward function implicitly optimized by expert agents acting in real applications. Scaling IRL to real-world cases has proved challenging as typically only a fixed dataset of demonstrations is available and further interactions with the environment are not allowed. For this reason, we resort to a class of truly batch model-free IRL algorithms and we present three application scenarios: (1) the high-level decision-making problem in the highway driving scenario, and (2) inferring the user preferences in a social network (Twitter), and (3) the management of the water release in the Como Lake. For each of these scenarios, we provide formalization, experiments and a discussion to interpret the obtained results. Keywords Inverse reinforcement learning · Model-free IRL · Truly batch IRL · IRL for real life · Multiple experts IRL · Non-stationary IRL Dealing with multiple experts and non‑stationarity in inverse reinforcement learning: an application to real‑life problems Amarildo Likmeta1,2 · Alberto Maria Metelli1 · Giorgia Ramponi1 · Andrea Tirinzoni1 · Matteo Giuliani1 · Marcello Restelli1 Received: 15 March 2020 / Revised: 17 September 2020 / Accepted: 22 December 2020 / Published online: 14 March 2021 © The Author(s) 2021 1 Politecnico di Milano, Milan, Italy Editors: Yuxi Li, Alborz Geramifard, Lihong Li, Csaba Szepesvari, Tao Wang. 2 Università di Bologna, Bologna, Italy 1 Introduction Reinforcement learning (RL, Sutton and Barto 2018) is nowadays an established approach to address a variety of real-world sequential decision making problems. Successful results have been achieved in numerous fields such as robotics (eg., Kober et al. 2013; Levine et al. 2016), recommender systems (eg., Shani et al. 2005; Warlop et al. 2018), financial trading (eg., Dempster and Romahi 2002; Nevmyvaka et al. 2006; Buehler et al. 2019), and autonomous driving (eg., Kiran et al. 2020). 56789) 3 56789) 3 Machine Learning (2021) 110:2541–2576 2542 The crucial component of any application of RL is the definition of the reward function, which evaluates the quality of the agent’s action in each state. In real-world scenarios, it is often difficult to design a suitable reward function, able to induce the desired behavior. This is because the reward function is a succinct representation of the task (Sutton and Barto 2018), more abstract and connected to “what” objectives (or intentions) the agent is optimizing rather than “how”. Indeed, it is typically easier to observe the behavior of an expert agent, possibly a human operator, who plays an optimal policy w.r.t. an unknown reward function. The goal of Inverse reinforcement learning (IRL, Ng and Russell 2000a; Abbeel and Ng 2004) is to recover a reward function that explains the expert’s behavior. IRL can be of enormous importance in real-world applications as it might help justify and interpret the expert’s choices and identify some trade-offs that a hypothetical human opera- tor makes, even implicitly. Even more than RL, IRL roots its natural motivations in real-life applications. Indeed, the experts are usually humans and the demonstrations come from observing the human who is performing the task. While imitating human behavior is rela- tively simple, interpreting its decisions is a rather complex task, also considering that for a human being to communicate precisely these motivations might be hard. IRL belongs to the broader class of Imitation Learning (IL, Osa et al. 2018) algorithms, whose high-level purpose is to “learn from demonstrations”. While IRL has the goal of producing a reward function, other techniques, such as Behavioral Cloning (BC, Argall et al. 2009), are meant to output an imitating policy, i.e., a policy that performs actions similarly, in some metric sense, to those demonstrated by the expert. 2 Preliminaries In this section, we introduce the basic concepts about sequential decision-making problems (Sect. 2.1), we formalize the RL and IRL problems (Sect. 2.2), and we introduce the spe- cific parametric setting we will employ (Sect. 2.3). Given a set X , we denote with P(X) the set of all probability distributions over X. 1 Introduction Then, we move to a case study in which we tackle the non-stationarity of the expert’s objectives. This application, presented in Sect. 7, consists in recovering the intentions of a human operator in charge of controlling the water release of the Como Lake dam. Finally, we present in Sect. 8 a discussion of the obtained results, highlighting the strengths and weaknesses of our approach and possible open questions. In this paper, we present three case studies of IRL in real-world scenarios. We employ 훴 -GIRL (Ramponi et al. 2020), a newly introduced batch model-free IRL approach that is based on the policy gradient and extends GIRL (Pirotta and Restelli 2016), taking into account the uncertainty of the gradient estimates, that is presented in Sect. 3.1. Then, we introduce two extensions of 훴-GIRL: the first one for dealing with the multiple-intention setting (MI-훴-GIRL, Sect. 3.2) that was already introduced in Ramponi et al. (2020) and the second one to address the non-stationarity of the reward function (NS-훴-GIRL, Sect. 3.3), which is a novel algorithmic contribution of this work. The subsequent sections are devoted to the illustration of the case studies. For each of them, we present the setting, the modelization, the design of the reward function class, the experimental results and their interpretation. We start with two scenarios in which we address the problem of IRL from multiple experts. In Sect. 5, we aim at inferring the intentions of humans driving along the highway; while in Sect. 6, we consider multiple Twitter users that act in the social network by reposting tweets. Then, we move to a case study in which we tackle the non-stationarity of the expert’s objectives. This application, presented in Sect. 7, consists in recovering the intentions of a human operator in charge of controlling the water release of the Como Lake dam. Finally, we present in Sect. 8 a discussion of the obtained results, highlighting the strengths and weaknesses of our approach and possible open questions. 1 Introduction Although BC is typi- cally simpler and can be cast into a supervised learning problem, the produced policy is typically non-transferable to different environments. Instead, the reward function generated by an IRL method encodes the general expert’s intentions and, therefore, can be employed even under shifts in the environment dynamics. Thus, contrary to the imitating policy, such a reward function can be employed to perform forward RL in the original environment, transferred to different domains, or used in simulation. For a detailed review of the state of the art in IL refer to the recent survey (Osa et al. 2018).i Despite its potential benefits, scaling IRL to real-world applications has historically demonstrated to be more challenging than RL. The most widespread applications are limited to the domains where the environment can be accessed or based on simulation, such as robotics (eg., Ratliff et al. 2006), path planning (eg., Ziebart et al. 2008; Boularias et al. 2011), or simulated car driving (eg., Abbeel and Ng 2004). The fundamental reasons behind this slower development can be ascribed to the peculiar requirements needed for applying IRL to real-world scenarios, which are frequently not met by common IRL algo- rithms. Those requirements can be summarized as follows: – Batch setting. When dealing with a real application, we cannot always assume to have access to the environment. Thus, we must account for the fact that only a batch of dem- onstrations collected by observing the expert is available. Further interaction with the environment might be impossible, even for just collecting additional data. – Model-Free setting. In addition to the batch requirement, in real-world applications, no model of the environment dynamics is usually available (even if available it might be overly simplified to be used effectively) and no interaction is allowed to learn it implic- itly or explicitly. – Model-Free setting. In addition to the batch requirement, in real-world applications, no model of the environment dynamics is usually available (even if available it might be overly simplified to be used effectively) and no interaction is allowed to learn it implic- itly or explicitly. Consequently, the range of IRL algorithms that can be actually employed for these applications is rather small, which we refer to as truly batch model-free. 1 Introduction At the best of our knowledge, they are limited to two categories: the ones that make use of structured 1 3 Machine Learning (2021) 110:2541–2576 2543 classification (Klein et al. 2012, 2013) and those based on the policy gradient (Pirotta and Restelli 2016; Metelli et al. 2017; Tateo et al. 2017; Ramponi et al. 2020). The previous requirements are necessary for most real-world scenarios; however, there may be additional challenges: classification (Klein et al. 2012, 2013) and those based on the policy gradient (Pirotta and Restelli 2016; Metelli et al. 2017; Tateo et al. 2017; Ramponi et al. 2020). The previous requirements are necessary for most real-world scenarios; however, there may be additional challenges: – Multiple experts. The available data might come from different experts (e.g., differ- ent human operators), possibly by playing different policies and/or optimizing different objectives. Therefore, the IRL algorithm should be able to group/cluster agents based on the demonstrated intentions. – Non-Stationarity. The environment in which the data collection process is carried out might change over time as well as the policy demonstrated by the expert. Thus, a viable IRL method must identify the time points at which the agent’s intention changes and deal with them appropriately. In this paper, we present three case studies of IRL in real-world scenarios. We employ 훴 -GIRL (Ramponi et al. 2020), a newly introduced batch model-free IRL approach that is based on the policy gradient and extends GIRL (Pirotta and Restelli 2016), taking into account the uncertainty of the gradient estimates, that is presented in Sect. 3.1. Then, we introduce two extensions of 훴-GIRL: the first one for dealing with the multiple-intention setting (MI-훴-GIRL, Sect. 3.2) that was already introduced in Ramponi et al. (2020) and the second one to address the non-stationarity of the reward function (NS-훴-GIRL, Sect. 3.3), which is a novel algorithmic contribution of this work. The subsequent sections are devoted to the illustration of the case studies. For each of them, we present the setting, the modelization, the design of the reward function class, the experimental results and their interpretation. We start with two scenarios in which we address the problem of IRL from multiple experts. In Sect. 5, we aim at inferring the intentions of humans driving along the highway; while in Sect. 6, we consider multiple Twitter users that act in the social network by reposting tweets. 2.2 Reinforcement learning and inverse reinforcement learning We now focus on the formalization of the reinforcement learning (RL, Sutton and Barto 2018) and the inverse reinforcement learning (IRL, Ng and Russell 2000a) problems. Let M ⧵R be an MDP without reward function, given a policy 휋∈훱 and a reward function R ∈R , we define the expected return JM(휋, R) as the expected discounted sum of the rewards collected by executing 휋 in the environment: (1) JM(휋, R) = 피 S0 ∼휇 At ∼휋(⋅\|St) St+1 ∼P(⋅\|St, At) [+∞ ∑ t=0 훾tR(St, At) ] , (1) where we made the unusual choice of making explicit the dependence on the reward func- tion R, which will turn useful in the following. For a fixed reward function R ∈R , we can look at the expected return JM(휋, R) as an index of the performance of a policy 휋∈훱 in the MDP M . This viewpoint directly leads to the standard formulation of the RL problem. where we made the unusual choice of making explicit the dependence on the reward func- tion R, which will turn useful in the following. For a fixed reward function R ∈R , we can look at the expected return JM(휋, R) as an index of the performance of a policy 휋∈훱 in the MDP M . This viewpoint directly leads to the standard formulation of the RL problem. (RL Problem) Let M ⧵R be an MDP without reward function and let RE ∈R be a reward function. The RL problem consists in finding an optimal policy, i.e., any policy 휋∗ RE ∈훱 maximizing the expected return JM(휋, RE): (2) 휋∗ RE ∈arg max 휋∈훱 JM(휋, RE). (2) We made explicit the dependence of the optimal policy 휋∗ RE on the reward function RE since different reward functions may induce different optimal policies. The problem presented above admits, in general, multiple solutions (Sutton and Barto 2018), although a determin- istic Markovian stationary optimal policy always exists (Puterman 1994). Typically, when tackling the RL problem, we are interested in finding just one optimal policy and not the whole set of optimal policies.f In the IRL setting, however, we take a different perspective. We are given an expert’s policy 휋E , i.e., the policy of an agent who behaves optimally w.r.t. some unknown reward function RE . 2.1 Sequential decision‑making We model the agent-environment interaction by means of a Markov Decision Pro- cess (MDP, Puterman 1994). An MDP is a 6-tuple M = (S, A, P, R, 훾, 휇) , where S and A are the state space and the action space respectively, P ∶S × A →P(S) is the transi- tion model that for each state-action pair (s, a) ∈S × A provides the probability distri- bution of the next state P(⋅\|s, a) , R ∶S × A →ℝ is the reward function that provides 1 3 Machine Learning (2021) 110:2541–2576 2544 the reward R(s, a) collected by the agent when performing an action a ∈A in state s ∈S , 훾∈[0, 1] is the discount factor, and 휇∈P(S) is the probability distribution of the initial state. We denote with M ⧵R the MDP devoid of the reward function and with R = {R ∶S × A →ℝ} being the set of all reward functions for a given MDP M. The behavior of an agent acting in an MDP M is modeled by means of a Marko- vian stationary policy 휋∶S →P(A) that provides for each state s ∈S the probability distribution of the action played by the agent 휋(⋅\|s) . A policy 휋 is deterministic if it prescribes a single action for each state. We denote with 훱 the set of all Markovian sta- tionary policies. The execution of a policy 휋∈훱 in an MDP M generates a sequence of state-action pairs denoted by 휏= (S0, A0, … , ST−1, AT−1, ST) and called trajectory such that S0 ∼휇 , At ∼휋(⋅\|St) , St+1 ∼P(⋅\|St, At) for all t ∈{0, … , T −1} and T denotes the trajectory length. 2.2 Reinforcement learning and inverse reinforcement learning Our goal consists in finding a reward function, not necessarily equal to RE , such that 휋E turns out to be an optimal policy. We will refer to these reward func- tions as compatible. 1 3 1 3 Machine Learning (2021) 110:2541–2576 2545 (IRL Problem) Let M ⧵R be an MDP without reward function and let 휋E ∈훱 be an expert policy. The IRL problem consists in finding a compatible reward function, i.e., any reward function R∗ 휋E ∈R that makes the expert’s policy 휋E optimal: (3) R∗ 휋E ∈ { R ∈R ∶휋E ∈arg max 휋∈훱 JM(휋, R) } . (3) Like the RL problem, the IRL problem admits multiple solutions. However, in the IRL setting the multiplicity of solutions is more critical, leading to the well-known ambi- guity problem (Ng and Russell 2000a, b). For instance, the constant reward function R(s, a) = c ∈ℝ for all (s, a) ∈S × A makes any policy (so also 휋E ) an optimal policy. Clearly, among all the possible reward functions that make 휋E optimal, not all of them have the same ability to “discriminate”, i.e., to capture that variations of 휋E must be subopti- mal.1 As a consequence, assessing the quality of a reward function is a challenging task, especially when, as in real-world scenarios, it is not possible to use the recovered reward to perform forward learning. In practice, however, the RL problem cannot be solved exactly as the dynamics of the environment modeled by P and the reward function R are unknown. Thus, interaction with the environment is necessary to learn the optimal policy. Similarly, in the IRL setting the expert’s policy 휋E is unknown, but a set of demonstrated trajectories D = {휏i}n i=1 generated by running 휋E in the environment M is usually available. 3 For any 𝛼∈ℝ>0 , the reward functions R and 훼R induce the same optimal policies. 1 This problem has been partially formalized in the notion of policy rank (Metelli et al. 2017). 2f 2 The differentiability requirement will be necessary for employing policy gradient methods (Sutton et 2000; Peters and Schaal 2008). 2.3 Parametric setting with linear reward In many real-world scenarios, especially when dealing with continuous state spaces (and possibly continuous action spaces), it is convenient to resort to a parametric representation of the policy space (Deisenroth et al. 2013). More formally, a policy 휋휽 belongs to a space of parametric differentiable policies, defined as:2 (4) 𝛱𝛩= {𝜋휽∶S →P(A), 휽∈𝛩⊆ℝd}, (4) where 훩 is the policy parameter space. As in Pirotta and Restelli (2016), we restrict our treatment of IRL to the case in which the expert’s policy 휋E can be represented within 훱훩 , i.e., there exists 휽E such that 휋E(⋅\|s) = 휋휽E(⋅\|s) almost surely for all s ∈S. Similarly, we model the reward function as a parametric mapping R흎 , and we enforce the additional constraint of being a linear mapping defined in terms of a feature function 흓 . More formally, we define a space of linear reward functions as: (5) R = R흎= 흎T흓∶흎∈ℝq ≥0, ‖흎‖1 = 1 , (5) where 흓∶S × A →ℝq is a (state-action) feature function. The simplex constraint on the reward weights 흎 (i.e., 흎∈ℝq ≥0 and ‖흎‖1 = 1 ) allows to avoid the ambiguity of rescaling rewards by a constant (Pirotta and Restelli 2016).3 1 3 2546 Machine Learning (2021) 110:2541–2576 In this setting, we abbreviate the expected return JM(휋휽, R흎) as JM(휽, 흎) , highlighting the dependence on the policy parameters 휽 and on the reward parameters 흎 . Exploiting the linearity of the reward function, the expected return decomposes as: (6) JM(휽, 흎) = 피 S0 ∼휇 At ∼휋휽(⋅\|St) St+1 ∼P(⋅\|St, At) [+∞ ∑ t=0 훾tR흎(St, At) ] = 흎T흍(휽), (6) where 흍(휽) denotes the feature expectations (Abbeel and Ng 2004), which are defined in terms of the feature function 흓 and on the played policy 휋휽 as: where 흍(휽) denotes the feature expectations (Abbeel and Ng 2004), which are defined in terms of the feature function 흓 and on the played policy 휋휽 as: (7) 흍(휽) = 피 S0 ∼휇 At ∼휋휽(⋅\|St) St+1 ∼P(⋅\|St, At) [+∞ ∑ t=0 훾t흓(St, At) ] . (7) Thus, the expected return is a linear combination, through the weights 흎 , of the feature expectations. This view allows JM(휽, 흎) to be interpreted as a linear scalarization of a multi-objective problem, in which the different objectives (or intentions in the IRL jargon) are represented by 흍(휽). 3 Gradient‑based inverse reinforcement learning In this section, we revise the class of IRL algorithms, named truly batch model-free, which employ techniques based on the policy gradient (Sutton et al. 2000; Peters and Schaal 2008) to recover the reward function optimized by the expert (eg., Pirotta and Restelli 2016; Metelli et al. 2017; Tateo et al. 2017; Ramponi et al. 2020). The main advantage of these approaches that make them suitable for tackling real-world scenarios is that they do not need to have access to the environment (or to a model of it) and are able to output a reward function using only a dataset of trajectories generated by the expert’s policy. Unlike widely known IRL methods, they do not need to solve the forward RL problem in order to assess the quality of each candidate reward function (thus saving a lot of computational time, especially in complex and high-dimensional RL problems) and no interaction is nec- essary to collect additional data. If 휋휽∈훱훩 is differentiable w.r.t. to its parameters 휽 , the policy gradient can be expressed as (Sutton et al. 2000; Peters and Schaal 2008): ∇휽J(휽, 흎) = 피 S0 ∼휇, At ∼휋휽(⋅\|St), St+1 ∼P(⋅\|St, At) [ +∞ ∑ t=0 훾tR흎(St, At) t∑ l=0 ∇휽log 휋휽(Al\|Sl) ] = ∇휽흍(휽)흎, 1 3 1 Machine Learning (2021) 110:2541–2576 2547 where ∇휽흍(휽) = (∇휽휓1(휽)\| … \|∇휽휓q(휽)) ∈ℝd×q is the Jacobian matrix of the feature expectations 흍(휽) w.r.t. to the policy parameters 휽 . When the expert’s policy 휋휽E ∈훱훩 is an optimal policy for the reward function R흎E , 휽E is a stationary point of the expected return J(휽, 흎E) = (흎E)T흍(휽) and, thus, the gradient of ∇휽J(휽E, 흎E) = ∇휽흍(휽E)흎E must vanish (first-order necessary conditions for optimality Nocedal and Wright 2006). In other words, the weight vector 흎E , associated to the reward function optimized by the expert, belongs to the null space of the Jacobian ∇휽흍(휽E) . This leads to the condition:4 (8) if 휽E ∈arg max 휽∈훩 JM(휽, 흎E) then 흎E ∈null(∇휽흍(휽E)). (8) We call all the 흎E reward vectors that satisfy the above equation weak compatible, respect to Eq. (3). 4 In principle, it is not guaranteed that the null space contains a unique vector even under the simplex con- straint (Eq. (5)). The multiplicity of the solutions is typically a symptom of a bad feature design. Indeed, it is always possible to remove one or multiple features to obtain a unique weight vector fulfilling the condi- tion. 3 Gradient‑based inverse reinforcement learning There are two problems that have to be addressed before applying this condition, both deriving from the fact that we have access neither to the explicit representation of the expert’s policy 휋E nor to the environment model, but just to a dataset D = {휏i}n i=1 of trajec- tories of length T generated by the expert’s policy 휋휽E : (1) Behavioral cloning. To compute the Jacobian ∇휽흍(휽E) it is necessary to have access to a parametric representation of the expert’s policy, to calculate the scores ∇휽log 휋휽E . Starting from the dataset of trajectories D = {휏i}n i=1 generated by 휋휽E , we can employ a Maximum Likelihood (ML) procedure to get an estimate ̂휽 E of the expert’s policy parameters 휽E (1) Behavioral cloning. To compute the Jacobian ∇휽흍(휽E) it is necessary to have access to a parametric representation of the expert’s policy, to calculate the scores ∇휽log 휋휽E . Starting from the dataset of trajectories D = {휏i}n i=1 generated by 휋휽E , we can employ a Maximum Likelihood (ML) procedure to get an estimate ̂휽 E of the expert’s policy parameters 휽E (9) ̂휽 E ∈arg max 휽∈훩 n ∑ i=1 T−1 ∑ t=0 log 휋휽(Ai,t\|Si,t). (9) The ML estimate is known to be consistent under mild assumptions, i.e., ̂휽 E →휽E as the number of trajectories n grows to infinity (Casella and Berger 2002). Other approaches based on Bayesian techniques (e.g., maximum a posteriori) are suitable when prior information on 휽E is available (Tateo et al. 2017). The ML estimate is known to be consistent under mild assumptions, i.e., ̂휽 E →휽E as the number of trajectories n grows to infinity (Casella and Berger 2002). Other approaches based on Bayesian techniques (e.g., maximum a posteriori) are suitable when prior information on 휽E is available (Tateo et al. 2017). (2) (2) Jacobian estimation. Given a policy parametrization 휽 , it is possible to get an unbiased estimate of the Jacobian matrix by resorting to sample-based estimators for standard policy gradient methods, such as REINFORCE (Williams 1992) and G(PO)MDP (Bax- ter and Bartlett 2001). For the sake of completeness, we report below the G(PO)MDP- like estimator, defined for all u ∈{1, … , d} and v ∈{1, … , q} as:5 (2) Jacobian estimation. 4 In principle, it is not guaranteed that the null space contains a unique vector even under the simplex con- straint (Eq. (5)). The multiplicity of the solutions is typically a symptom of a bad feature design. Indeed, it is always possible to remove one or multiple features to obtain a unique weight vector fulfilling the condi- tion. 5 The concentration properties of this estimator, being a straightforward extension, can be derived from those of G(PO)MDP (Papini et al. 2019). 5 The concentration properties of this estimator, being a straightforward extension, can be derived from those of G(PO)MDP (Papini et al. 2019). 3 Gradient‑based inverse reinforcement learning Given a policy parametrization 휽 , it is possible to get an unbiased estimate of the Jacobian matrix by resorting to sample-based estimators for standard policy gradient methods, such as REINFORCE (Williams 1992) and G(PO)MDP (Bax- ter and Bartlett 2001). For the sake of completeness, we report below the G(PO)MDP- like estimator, defined for all u ∈{1, … , d} and v ∈{1, … , q} as:5 5 The concentration properties of this estimator, being a straightforward extension, can be derived from those of G(PO)MDP (Papini et al. 2019). 3 Machine Learning (2021) 110:2541–2576 2548 (10) ̂∇휽흍uv(휽) = 1 n n ∑ i=1 T−1 ∑ t=0 ( t∑ l=0 ∇휽u log 휋휽(Ai,l\|Si,l) ) 훾t(흓v(Si,t, Ai,t) −buvt ), (10) where buvt is a baseline that can be employed to reduce the variance of the estimate and obtained extending the classical one employed in G(PO)MDP (Deisenroth et al. 2013 Equation 2.17) for the Jacobian: buvt = 피 ∑t−1 l=0 ∇휽u log 휋휽(AlSl) 2 훾t흓v(St, At) 피 ∑t−1 l=0 ∇휽u log 휋휽(AlSl) 2 , where the expectation is taken w.r.t. the randomness of the trajectories. Being an average of n independent trajectories, ̂∇휽흍(휽) concentrates around its true value ∇휽흍(휽) as n grows to infinity. Furthermore, thanks to the central limit theorem, its distribution is asymptoti- cally Gaussian (Casella and Berger 2002). E The approximations introduced by estimating the expert’s policy parameters 휽E via behavioral cloning and by using samples to compute ̂∇휽흍(휽) prevent the direct application of condition (8) for the determination of the expert’s weights. This is due to the fact that the estimated Jacobian ̂∇휽흍(휽) might result full rank even if the true Jacobian has a rank smaller than q, leading to a zero-dimensional null space. We will discuss in the following section how to deal with this problem. 6 Refer to Section 4 in Ramponi et al. (2020)) for the detailed derivation. 3.1 ˙‑gradient inverse reinforcement learning In this section, we revise the recently presented 훴-Gradient inverse reinforcement learn- ing (훴-GIRL, Ramponi et al. 2020), which is able to solve the IRL problem in a fully batch model-free setting, accounting also for the uncertainty on the Jacobian estimate. The basic idea is to look at the Jacobian estimate ̂∇휽흍(휽) as a noisy version of the true Jacobian ∇휽흍(휽) . For this purpose, we model ̂∇휽흍(휽) as a Gaussian random matrix N ( 퐌, 1 n휮 ) , which is justified by the central limit theorem, being the estimated Jacobian a sample mean.i i Since there exists a weight vector 흎E , which defines the reward function optimized by the expert, such that ̂∇휽흍(휽)흎E = 0 , whenever ̂∇휽흍(휽) is full rank, we are allowed to move its components in order to get a new estimate 퐌 having non-empty null space. Using the Gaussian likelihood model, we formulate the IRL problem as the problem of finding the weights 흎 and the new Jacobian 퐌 that jointly maximize the likelihood of the estimated Jacobian.6 This leads to the optimization problem: min 흎∈ℝq ≥0 ‖흎‖1 = 1 ∇휽흍(휽)흎 2 (흎⊗Id) T휮(흎⊗Id) −1, (𝛴-GIRL) where ⊗ denotes the Kronecker product and Id is the identity matrix of order d. Clearly, we need to specify the noise model encoded by the covariance matrix 휮 . In practice, the sample covariance matrix ̂휮 is often used in the experiments after applying some necessary where ⊗ denotes the Kronecker product and Id is the identity matrix of order d. Clearly, we need to specify the noise model encoded by the covariance matrix 휮 . In practice, the sample covariance matrix ̂휮 is often used in the experiments after applying some necessary 1 3 1 3 Machine Learning (2021) 110:2541–2576 2549 correction to enforce the well-conditioning (Ledoit and Wolf 2004). For a specific choice of 휮 , we reduce to the objective function of GIRL (Pirotta and Restelli 2016): correction to enforce the well-conditioning (Ledoit and Wolf 2004). For a specific choice of 휮 , we reduce to the objective function of GIRL (Pirotta and Restelli 2016): min 흎∈ℝq ≥0 ‖흎‖1 = 1 ̂∇휽흍(휽)흎 2 2. (GIRL) min 흎∈ℝq ≥0 ‖흎‖1 = 1 ̂∇휽흍(휽)흎 2 2. (GIRL) Finally, we can employ the Gaussian likelihood model to define the likelihood of dataset D, used to compute ̂∇휽흍(휽) , given the weight vector 흎 . 7 The notation is taken from (Barratt 2018). 3.1 ˙‑gradient inverse reinforcement learning We will denote this quantity as p(D\|흎):7 (11) p(D흎) = max 퐌∈ℝd×q 퐌휔= 0 √ n √ (2휋)dq det(휮) e −n 2 vec ̂∇휽흍(휽)−퐌 2 휮−1 , (11) (11) where vec denotes the vectorization operator, that, given a matrix, recovers a vector obtained by stacking its columns. where vec denotes the vectorization operator, that, given a matrix, recovers a vector obtained by stacking its columns. Remark 1 (On the Suboptimality of the Expert) In principle, if no knowledge about the reward function optimized by the expert is available, we are unable to detect whether the expert is suboptimal. This is because, we can always design a reward function in which the demonstrated behavior is optimal (unless the expert’s contradicts itself). Instead, if we assume that the reward function optimized by the expert lies in our class of reward func- tions, i.e., it is linear, and we are unable to find a weight vector making the gradient van- ish, we can conclude that the expert is suboptimal. In such a case, similarly to Pirotta and Restelli (2016), instead of looking at the null space of the Jacobian, we will recover the reward that induces the minimum change in the policy parameters, i.e., the reward that bet- ter explains the expert demonstrated behavior. 3.2 Dealing with multiple experts and intentions 3.2 Dealing with multiple experts and intentions In several applications, we have access to demonstrations generated by multiple experts who possibly optimize different objectives (i.e., different reward functions). The corre- sponding IRL problem, which consists in recovering the reward function optimized by each 1 3 Machine Learning (2021) 110:2541–2576 2550 expert, is commonly refered to as IRL about multiple intentions (MI-IRL, Babes et al. (2011)). Formally, suppose we have a set {E1, … , Em} of m experts, each of which demon- strates a policy 휋Ei ∈훱훩 by means of ni trajectories, Di = {휏1, … , 휏ni} . Furthermore, there exist k ≤m unknown reward functions {R흎1, … , R흎k} such that the i-th expert optimizes R흎ri , where ri ∈{1, … , k} are the unknown expert-intention assignments. The goal is to recover the set of k rewards together with the corresponding assignments (Fig. 1). In the remaining, we assume that we know the identity of the expert who generates each trajec- tory and the number of intentions k. W i h h b R i l (2020) hi h d h 훴GIRL Fig. 1 Plate notation of the probabilistic model employed for the clustering procedure. 훼j with j ∈{1, … , k} are the prior probabilities on the cluster assignment Fig. 1 Plate notation of the probabilistic model employed for the clustering procedure. 훼j with j ∈{1, … , k} are the prior probabilities on the cluster assignment Fig. 1 Plate notation of the probabilistic model employed for the clustering procedure. 훼j with j ∈{1, … , k} are the prior probabilities on the cluster assignment expert, is commonly refered to as IRL about multiple intentions (MI-IRL, Babes et al. (2011)). Formally, suppose we have a set {E1, … , Em} of m experts, each of which demon- strates a policy 휋Ei ∈훱훩 by means of ni trajectories, Di = {휏1, … , 휏ni} . Furthermore, there exist k ≤m unknown reward functions {R흎1, … , R흎k} such that the i-th expert optimizes R흎ri , where ri ∈{1, … , k} are the unknown expert-intention assignments. The goal is to recover the set of k rewards together with the corresponding assignments (Fig. 1). In the remaining, we assume that we know the identity of the expert who generates each trajec- tory and the number of intentions k. We now revise the approach by Ramponi et al. 3.2 Dealing with multiple experts and intentions (2020), which extends the 훴-GIRL algorithm to the MI-IRL setting. We note that a simple solution would be to run 훴-GIRL (or any other IRL algorithm) independently on the sets of trajectories demonstrated by each different expert. However, this solution is likely to yield poor performance when each expert provides very small amounts of data, as is common in real-world scenarios. A more data-efficient solution is to cluster the given trajectories (or equivalently the experts) according to their underlying intention (Babes et al. 2011) so that it is possible to run the IRL algorithm on larger datasets (the clusters). Ramponi et al. (2020) build exactly on top of this idea. Since computing the clusters requires the intentions to be known and vice versa, the authors propose an expectation-maximization (EM) frame- work that maximizes the total likelihood of the data. In the E-step, the algorithm uses the current estimates of the reward weights to compute the probabilities zij that the i-th expert optimizes the j-th estimated reward. In the M-step, the algorithm uses the current probabilities zij to update the reward weights. This can be done, for each reward weight, by solving a weighted version of the 훴-GIRL objective: (12) min 흎j ∈ℝq ≥0 ‖‖‖흎j‖‖‖1 = 1 m ∑ i=1 zijni‖‖‖∇휽흍i(휽)흎j‖‖‖ 2 [(흎j⊗Id)휮i(흎j⊗Id)T]−1, j ∈{1, … , k}. (12) The two steps are then repeated until convergence. The final output of the algorithm are the estimated reward weights together with the corresponding “soft” expert assignments (i.e., the probabilities zij ). Refer to Algorithm 1 for the pseudocode of Multiple-Intention 훴 -GIRL (MI-훴-GIRL). Machine Learning (2021) 110:2541–2576 Machine Learning (2021) 110:2541–2576 Fig. 2 Plate notation of the probabilistic model employed for the change-point detection procedure in the expert’s intentions. In this section, we formalize the problem of IRL with a non-sta- tionary expert’s reward function. Our setting assumes that we have access to a lifelong tra- jectory 흉= (휏1\| … \|휏T) obtained from the concatenation of T trajectories D = {휏i}T i=1.8 Within the lifelong trajectory 흉 the expert displays a non-stationary behavior since it opti- mizes k ≤T reward functions R = (R흎1, … , R흎k) , where k is referred to as number of regimes. In particular, there exists a set of indexes T = {t0, t1, … , tk} with 1 = t0 < t1 < ⋯< tk−1 < tk = T , inducing the intervals Ij = {tj−1, … , tj −1} , such that for each j ∈{1, … , k} the set of trajectories Dj = {휏i}i∈Ij , made of nj = tj −tj−1 + 1 trajecto- ries, are generated by the expert who optimizes the same reward function R흎j . We assume to know the number of regimes k, the subdivision of the lifelong trajectory 흉 in the T trajec- tories D = {휏i}T i=1 , but not the set of indexes T , nor the reward functions R . Clearly, we expect that for different intervals Ij not only the reward function changes, but also the pol- icy performed by the expert (Fig. 2). A naïve solution would be to treat this problem as a multiple-intention IRL problem in which each expert Ei generated the dataset consisting of a single trajectory Di = {휏i} for i ∈{1, … , T} . However, this approach has at least two drawbacks. First, the estimate of the reward function will likely be very noisy since only one trajectory is available for each expert. Second, we are totally disregarding that the expert’s intention changes sequentially. Thus, it would be unrealistic to cluster non-contiguous intervals. For these reasons, we take inspiration from the change-point detection algo- rithms (Aminikhanghahi and Cook 2017) and we adapt it to the non-stationary IRL setting. Given a dataset D of trajectories and a reward weight 흎 we employ the likelihood function p(D\|흎) defined in Eq. 8 The granularity of the subdivision of the lifelong trajectory to the T sub-trajectories is a design choice, based on the knowledge of the environment. For instance, in the Como Lake case study, given the cycle- stationarity of the environment, each sub-trajectory is associated to one year of data. The length of the sub- trajectories determines the agent’s planning horizon employed in the IRL process. 3.3 Dealing with non‑stationary experts In many real-world scenarios, the expert who controls a system (e.g., a human operator) might modify its behavior over time. This is because its objectives might change or the environment might evolve. We can interpret this phenomenon as a form of non-stationarity 1 3 2551 4 Related works In recent years, there have been several successful applications of imitation learning meth- ods to real-world problems. Robotics is perhaps the most common example (Osa et al. 2018). In this setting, learning policies on real robots is often prohibitive due to both sam- ple-complexity and safety reasons, while expert demonstrations are fairly simple to obtain. Due to their simplicity, behavioral cloning (BC) methods have received considerable atten- tion. Kober and Peters (2009) trained a robotic arm to hit a ball in the table-tennis game. The arm was guided by a human expert to generate trajectories and the ball-hitting pol- icy was learned directly via BC on these demonstrations. Englert et al. (2013) addressed the same problem but with an under-actuated robot using a model-based BC technique. Abbeel et al. (2010) trained policies to drive an RC helicopter from human-teleoperation trajectories. A similar problem was considered by Ross et al. (2013), who trained a control- ler for an unmanned aerial vehicle capable of avoiding obstacles (e.g., trees in a forest). Zhang et al. (2018) trained policies for several robotic manipulation tasks (e.g., grasping or pushing objects) directly from images, with demonstrations generated in virtual reality. Finn et al. (2017) used meta-learning to train image-based controllers that adapt to several manipulation tasks using only a single visual demonstration. For a thorough discussion of the applications of BC methods, we refer to the recent surveys by Hussein et al. (2017) and Osa et al. (2018). Although IRL methods have also enjoyed many success stories in complex robotics problems, their application in this context is considerably more difficult than BC. In fact, as mentioned in the introduction, in this kind of problems, a model of the environment is hardly ever available in practice, it is difficult or unsafe to interact with the real system, and expert demonstrations are often very limited. Therefore, many traditional IRL tech- niques are not applicable and model-free and data-efficient (e.g., batch) methods are typi- cally preferred. Among the notable applications, Boularias et al. (2011) used a model-free variant of MaxEnt IRL to learn the “ball-in-a-cup” task, in which a robot must swing a ball connected to a rope into a cup. The task was demonstrated by a human expert only a very small number of times and the resulting controller was shown successfully on a real robot. Machine Learning (2021) 110:2541–2576 (11), we define the likelihood of the lifelong trajectory 흉 as the prod- uct of the likelihoods of the individual trajectories 휏i: (13) L(훺\|흉) = p(흉\|훺) = T ∏ i=1 k∑ j=1 p(휏i\|흎j)ퟙ{i∈Ij}, (13) where 훺= (흎1, … , 흎k, t1, … , tk−1) is the concatenation of the parameters. Now we can derive the objective function that we seek to optimize for the parameters 훺: where 훺= (흎1, … , 흎k, t1, … , tk−1) is the concatenation of the parameters. Now we can derive the objective function that we seek to optimize for the parameters 훺: 3 Machine Learning (2021) 110:2541–2576 2552 Q(훺) = log L(훺\|흉) = k∑ j=1 T ∑ i=1 ퟙ{i∈Ij} log p(휏i\|흎j) = k∑ j=1 ∑ i∈Ij log p(휏i\|흎j) = k∑ j=1 log p(Dj\|흎j), where we recall that Dj = {휏i}i∈Ij . In order to optimize the objective function Q(훺) we adapt the change-point detection algorithm Opt which employs a dynamic programming approach to determine the optimal solution to the identification of the change points T (Bellman 1958; Aminikhanghahi and Cook 2017; Truong et al. 2020). The adaptation of this algorithm to our non-stationary IRL problem is reported in Algorithm 2, which we name Non-Stationary 훴-GIRL (NS-훴-GIRL). It is worth noting that the optimization of such objective consists in solving O(T2) IRL problems, one for each 1 ≤u < v ≤T: (14) min 흎uv ∈ℝq ≥0 ‖‖흎uv‖‖1 = 1 (v −u) v−1 ∑ i=u ‖‖‖∇휽흍i(휽)흎uv‖‖‖ 2 [(흎uv⊗Id)휮i(흎uv⊗Id)T] −1. (14) 3 1 Machine Learning (2021) 110:2541–2576 2553 4 Related works Bogert and Doshi (2014) proposed an IRL method for a real patrolling problem in which a robot must penetrate the perimeter patrolled by other robots inferring their intentions. Finn et al. (2016) learned about house-keeping tasks (such as moving dishes and pour- ing liquids) using a model-free IRL algorithm with non-linear reward functions and visual demonstrations.i Autonomous driving is another field where the application of imitation learning (and, in particular, IRL) techniques has received increasing interest. This setting presents even more complications than robotics problems and, thus, the focus is typically on learning policies in simulation. However, there have been many attempts to integrate real-world driving demonstrations and to deploy the resulting controllers to real cars. Several BC approaches have been proposed for learning end-to-end car-driving policies (which map raw sensor data to actions) directly from expert demonstrations. These approaches provide significant evidence of the capabilities of neural network-based controllers but are typically difficult to deploy on real cars due to safety and interpretability reasons. Codevilla et al. (2018) and Dosovitskiy et al. (2017) trained end-to-end image-based policies for complex urban driving domains. The trained models were evaluated in the real-world using a toy car. Similarly, Pan et al. (2017) adopted an end-to-end imitation learning method for off- road autonomous driving that was successfully tested using toy cars. 1 3 Machine Learning (2021) 110:2541–2576 2554 The application of IRL methods is typically on specific driving problems. Ziebart et al. (2008) considered the problem of predicting driving behavior and route preferences. The authors applied their MaxEnt IRL algorithm to a large dataset of real GPS data from dif- ferent taxi cabs and showed that it was able to capture the route choices of the drivers. A similar problem was considered by Wulfmeier et al. (2017) who extended the approach of Ziebart et al. (2008) to learn non-linear reward functions. Silver et al. (2013) proposed a maximum-margin method to learn driving maneuvers from human demonstrations and successfully tested the resulting controller on a real-world vehicle. Kuderer et al. (2015) employed the MaxEnt IRL algorithm to learn driving styles from demonstrations in order to optimize the comfort perceived by passengers in the autonomous vehicle. The approach uses data obtained by recording real drivers with different driving styles. 9 We use the right-hand traffic rules. 10 Our method uses trajectories collected by human experts in simulation, but we never interact with the simulator to perform forward RL. Thus, the presence of the simulator is less critical for our approach. 5.1 IRL from multiple experts Highway driving is a widely employed benchmark for RL and IRL algorithms, thanks to the potentially simple representation of the environment and the “few-constrained” possibility in choosing the action. Here we focus on the problem of high-level control, where the main decision the agent has to make is when to change lanes. This is a quite common scenario, close to the setting considered in real autonomous vehicles, where the presence of a low-level controller, which assures compliance with the safety dis- tance with other vehicles, managing the speed accordingly and maintaining the center of the lane, is assumed. Therefore, the lane-change problem consists in controlling the ego vehicle on the highway and deciding when it is convenient to perform a lane change on the left to overtake, or a lane change on the right, to occupy the rightmost free lane.9 Driver agents in this setting typically aim at proceeding along the highway as fast as possible, while displaying a “natural” behavior, respectful of driving rules. In this case study, we consider a mixed real/simulated setting. The demonstrations are collected by human drivers, but the environment in which humans operate is simu- lated. We employ SUMO simulator, an open-source, highly portable, microscopic and continuous road traffic simulation package designed to handle large road networks (Kra- jzewicz et al. 2012). SUMO focuses on high-level control of the car, integrating an internal system that controls the vehicle dynamics. This mimics the low-level controller of autonomous vehicles. Slight changes have been made to the simulator to ensure that the car-follower models employed in the simulator are aligned with low-level controllers used in real autonomous driving systems. For this reason, we believe that our setting is not significantly different from the fully real environment. Furthermore, since we are interested in recovering a reward function which is a transferable element, rather than an imitating policy, the simulated environment is less critical than in the BC case. In this kind of driving problems, the use of classical IRL algorithms is extremely chal- lenging, since any interaction with the environment (e.g., to learn the optimal policy for a candidate reward function) must be performed in simulation and must account for the 1 3 Machine Learning (2021) 110:2541–2576 2555 Fig. 3 Range in which the car in front is considered for front distances, highlighted in blue. The value recorded is highlighted in yellow (Color figure online) Fig. 5.2 System modeling We present here details about the state representation considered in the lane change sce- nario. We focus on three-lane highways, but the state space can be generalized to an arbi- trary number of lanes. The state is composed of 25 high-level features extracted by the observations of the environment. For two vehicles in each lane, one at the front and one at the rear of the ego vehicle, we record the distance from the ego vehicle. The distance considered is the one from the front bumper of the following vehicle to the rear bumper of the leading vehicle, as shown in Fig. 3. A vehicle is considered in front of the ego vehicle as long as its front bumper is in front of the rear bumper of the ego vehicle. We also record their speeds and their lateral positions inside the corresponding lanes, to know if they are making lane changes. The variables that represent the state of the ego vehicle are its speed, its position over the lanes, a flag indicating whether the ego vehicle is changing lanes, and two flags that check whether the ego vehicle has the free-left or the free-right. The free-left and the free-right are evaluated only for vehicles visible by the sensors, therefore within the visibility range, otherwise, they are true. The position of the ego vehicle in the lanes is rep- resented as occupancy weights. For each lane of the highway, we record in what percentage the ego vehicle is in each lane, considering the offset between the front bumper of the vehi- cle and the center of the lane. Figure 4 shows an example of a lane change from the third lane to the second lane (lanes ordered from right to left), together with the corresponding occupancy arrays. In Fig. 4a the offset between the front bumper of the car (highlighted in blue) and the center of the lane (highlighted in yellow), is highlighted in green. In this case, the vehicle is 60% in the third lane and 40 % in the second lane. The action space in the lane-change highway scenario consists of three actions, car_fol- lowing, lane_change_right and lane_change_left . The car_following action leaves con- trol of the car to the low-level controller which follows the planned route, cornering when necessary, but does not make lane changes. 5.2 System modeling Furthermore, it controls the vehicle speed to avoid collisions and maintains a safe distance with the vehicle in front. The controller sets the safety speed considering the vehicles that are in sight of the sensors only and adjusts it respecting the maximum practicable deceleration and acceleration. The remaining two actions are the lane changes, left or right. These maneuvers are non-interruptible, once issued, they cannot be reverted. For more details on the environment modelling see Lik- meta et al. (2020). 5.1 IRL from multiple experts 4 The ego vehicle lane occupancy during a lane change (Color figure online) Fig. 3 Range in which the car in front is considered for front distances, highlighted in blue. The value recorded is highlighted in yellow (Color figure online) Fig. 3 Range in which the car in front is considered for front distances, highlighted in blue. The value recorded is highlighted in yellow (Color figure online) Fig. 4 The ego vehicle lane occupancy during a lane change (Color figure online) Fig. 3 Range in which the car in front is considered for front distances, highlighted in blue. The value recorded is highlighted in yellow (Color figure online) Fig. 4 The ego vehicle lane occupancy during a lane change (Color figure online) Fig. 4 The ego vehicle lane occupancy during a lane change (Color figure online) differences with the real vehicle.10 훴-GIRL, on the other hand, requires only agents’ dem- onstrations and can identify the reward function that the expert optimizes without interact- ing with the environment. More specifically, we consider the case where we have interac- tions from multiple agents and we can identify which agent each of our demonstrations belongs to. The goal is to cluster agents based on their intentions.i The immediate application of the results of IRL in this scenario is in the field of autono- mous driving. Specifically, we can exploit the clustering of agents based on their intentions to identify agents that demonstrate unwanted behaviors. This allows removing from the dataset demonstrations that would result in an imitation policy showing these unwanted behaviors, such as unsafe driving or non-compliance with driving rules, with a possible benefit in subsequent BC applications. Furthermore, and most importantly, we can use the 1 1 3 Machine Learning (2021) 110:2541–2576 2556 identified reward functions to understand the different trade-offs performed by human driv- ers. Consequently, those rewards can be employed to train an autonomous controller that replicates (and possibly improves) the human behavior, only using the demonstrations of agents that optimize a “safe” reward function. In the following sections, we will refer to the controlled vehicle as the ego vehicle. 5.3 Reward design The lane change scenario is a classic example of a multi-objective task. Humans consider several objectives, corresponding to the reward features, while driving along highways, including: (1) going as fast as possible, (2) occupying the rightmost free lane, (3) avoiding useless lane changes, and (4) keep safety distances with other vehicles. To encode these 1 3 Machine Learning (2021) 110:2541–2576 2557 objectives we employ three reward features. All the features are meant as punishment, so they have negative values: – Free-right ( 휙R ): to encode the objective of occupying the rightmost lane we use a binary feature, activated during the timesteps when the agent could perform a lane change on the right. – Lane-change ( 휙L ): a binary feature is used to encode the objective of avoiding too many lane changes Since the lane change is non interruptable and lasts 3s (30 timesteps) Fig. 5 3D inteface used to collect the human demonstrations Fig. 6 Feature expectations of the human agents in the highway task Fig. 5 3D inteface used to collect the human demonstrations Fig. 5 3D inteface used to collect the human demonstrations Fig. 5 3D inteface used to collect the human demonstrations Fig. 5 3D inteface used to collect the human demonstrations Fig. 6 Feature expectations of the human agents in the highway task Fig. 6 Feature expectations of the human agents in the highway task objectives we employ three reward features. All the features are meant as punishment, so they have negative values: objectives we employ three reward features. All the features are meant as punishment, so they have negative values: – Free-right ( 휙R ): to encode the objective of occupying the rightmost lane we use a binary feature, activated during the timesteps when the agent could perform a lane change on the right. – Free-right ( 휙R ): to encode the objective of occupying the rightmost lane we use a binary feature, activated during the timesteps when the agent could perform a lane change on the right. – Lane-change ( 휙L ): a binary feature is used to encode the objective of avoiding too many lane changes. Since the lane change is non-interruptable and lasts 3s (30 timesteps), this punishment is given entirely at the beginning of the lane change and has a high value (30). 1 3 Machine Learning (2021) 110:2541–2576 2558 Fig. 7 Distributions of lane changes for each agent. 5.3 Reward design The y-axis reports the fraction of demonstrated actions where either a lane change to the left (in blue) or one to the right (in orange) was performed (Color figure online) Fig. 7 Distributions of lane changes for each agent. The y-axis reports the fraction of demonstrated actions where either a lane change to the left (in blue) or one to the right (in orange) was performed (Color figure online) – Distance front ( 휙D ): a feature to encode both the safety objective and the maintenance of the high-speed profile. This is a feature that incorporates the distance of the ego vehicle from the vehicle in front of it. It grows linearly with the distance to the front vehicle, the higher the distance of the ego vehicle from the vehicle in front the higher is its value. It has the highest value (0) when there is no vehicle in front. This objective also encodes the high-speed objective, since it is the low-level controller that regulates the speed of the ego vehicle when it is about to violate safety distances. Without any vehicle ahead, the ego vehicle continues to accelerate until it reaches the maximum allowed road speed. 5.4 Data description We want to identify the intentions that drive human agents in changing lanes while driving, but most of the actions in the dataset are car-following. Figure 7 shows the distri- butions of lane changes for each agent. Finally, it is worth noting that the distribution of the actions in the dataset is highly unbal- anced. We want to identify the intentions that drive human agents in changing lanes while driving, but most of the actions in the dataset are car-following. Figure 7 shows the distri- butions of lane changes for each agent. 5.4 Data description In the SUMO simulator, we model scenarios with different road topologies and traf- fic intensities, randomizing the flow of vehicles, to ensure the generation of sufficiently general and realistic situations. We set the control frequency to 10 Hz for all our experi- ments, which means that we choose an action to be performed every 100 ms. During the simulation, SUMO provides information about the other vehicles around the ego vehicle. More specifically, we can query SUMO for the positions and velocities of all the cars in the simulation. This information is also available for the decision-making module in a real car, being provided by the sensing module. To collect the dataset we built a 3D-interface on top of the SUMO traffic simulator, connected to the traffic simulator. The 3D interface, shown in Fig. 5, was used by human drivers to collect trajectories. The dataset consists of demonstrations provided by 10 different drivers. Each set of demonstrations consists of 50 trajectories each of 400 steps, recorded at 10 Hz, resulting in trajectories equal to 40 s of driving time, for a total of 5.5 hours of driving.ff The agents show different behaviors. To grasp an initial understanding of the differ- ences, we show in Fig. 6 the feature expectations for all the agents considered. In Appen- dix A.1, we also show some 2D visualizations of the trajectories of some of the experts. 1 3 Machine Learning (2021) 110:2541–2576 2559 Fig. 8 Accuracy (fraction of correctly-predicted actions) of the BC models in the AD task (Color figure online) Fig. 8 Accuracy (fraction of correctly-predicted actions) of the BC models in the AD task (Color figure online) uracy (fraction of correctly-predicted actions) of the BC models in the AD task (Color figure Finally, it is worth noting that the distribution of the actions in the dataset is highly unbal- anced. We want to identify the intentions that drive human agents in changing lanes while driving, but most of the actions in the dataset are car-following. Figure 7 shows the distri- butions of lane changes for each agent. Fig. 9 IRL loss (Eq. 12) in the Highway domain as a function of the number of clusters Fig. 9 IRL loss (Eq. 12) in the Highway domain as a function of the number of clusters Finally, it is worth noting that the distribution of the actions in the dataset is highly unbal- anced. 5.5 Results The BC phase is performed by means of a one-layer neural network, with 8 hidden units and a Boltzmann output layer to represent the policy model for the AD task. Different architectures were explored, but the simpler models were unable to accurately predict the agents’ behaviors and more complex models did not offer substantial improvements. We recall that in this task, the BC dataset is highly unbalanced, with most of the actions in the dataset being car-following (NOP) and only a small portion being lane changes. To deal with this problem, we employed oversampling over the minority classes. Figure 8 shows the accuracy of all agents’ policies derived via BC, for each action separately. We can notice that the BC models generally predict the agents’ behaviors well, except for the 4th agent, which seems to have a more non-deterministic response to the state. For agents Craig and Judy, the column corresponding to the lane-change left is not represented since the respective agents never performed that action. 1 3 Machine Learning (2021) 110:2541–2576 2560 Table 1 The reward weights learned by 훴-GIRL in the AD task Reward features N. agents Free-right Lane-change Distance front Cluster 1 0.76 0.00 0.24 3 Cluster 2 0.09 0.00 0.91 5 Cluster 3 1.00 0.00 0.00 1 Cluster 4 0.19 0.81 0.00 1 Table 2 Cluster assignment made by 훴-GIRL in the AD task Agents Cluster 1 Eve, Grace, Alice Cluster 2 Carol, Erin, Bob, Dan, Chuck Cluster 3 Craig Cluster 4 Judy (a) (b) (c) Fig. 10 Visualization of the weights of the clusters (Color figure online) Table 1 The reward weights learned by 훴-GIRL in the AD task task Free-right Lane-change Distance front Cluster 1 0.76 0.00 0.24 3 Cluster 2 0.09 0.00 0.91 5 Cluster 3 1.00 0.00 0.00 1 Cluster 4 0.19 0.81 0.00 1 Table 2 Cluster assignment made by 훴-GIRL in the AD task Agents Cluster 1 Eve, Grace, Alice Cluster 2 Carol, Erin, Bob, Dan, Chuck Cluster 3 Craig Cluster 4 Judy (a) (b) (c) Fig. 10 Visualization of the weights of the clusters (Color figure online) Fig. 11 Intra-cluster BC evaluations (Color figure online) (a) (b) (c) Fig. 10 Visualization of the weights of the clusters (Color figure online) (b) (c) (c) (b) Fig. 10 Visualization of the weights of the clusters (Color figure online) Fig. 11 Intra-cluster BC evaluations (Color figure online) Fig. 5.5 Results 11 Intra-cluster BC evaluations (Color figure online) Fig. 11 Intra-cluster BC evaluations (Color figure online) 1 3 Machine Learning (2021) 110:2541–2576 2561 −80 −60 −40 −20 0 Averege return Cluster 1 −80 −60 −40 −20 0 Averege return Cluster 2 −80 −60 −40 −20 0 Averege return Cluster 3 Trained policy Cluster agents Out of cluster agents −80 −60 −40 −20 0 Averege return Cluster 4 Fig. 12 Average return of the policy trained with the reward function of each cluster and mean of the aver- age return of the experts divided based on whether they have been assigned to the cluster (Color figure online) −80 −60 −40 −20 0 Averege return Cluster 3 Trained policy Cluster agents Out of cluster agents −80 −60 −40 −20 0 Averege return Cluster 4 Fig. 12 Average return of the policy trained with the reward function of each cluster and mean of the aver- age return of the experts divided based on whether they have been assigned to the cluster (Color figure online) Clustering results We employ Multiple-Intention 훴-GIRL, as described in Sect. 3.2, with k = 4 clusters. The results are summarized in Tables 1 and 2, with a visualization of the reward weights in Fig. 10a. We can identify four clear clusters. The first cluster includes three agents showing the “best” behavior. These agents keep the right lane while overtaking slow vehicles. This translates into a high weight for the free-right objective, since it is a binary feature activated when we could perform a lane change to the right, and some weight for the distance-front objective. As we mentioned earlier, the distance-front objective is related to maintaining high speed, as the low-level controller starts to deceler- ate to maintain safety distances when the front vehicles are too close. In fact, they start overtaking vehicles only when the low-level controller starts to slow down. It is also inter- esting to note that the change-lane objective is not given any weight, since changing lanes without motivation is already suboptimal because it decreases speed while changing lane and creates unnecessary free-rights. The next cluster contains agents who rarely occupy the right lane but focus on maintaining a high-speed profile that provides most of the weight to the front-distance feature. 5.5 Results Again, useless lane changes are implicitly optimized, because they affect the speed of the ego vehicle and are advantageous only when you employ them to overtake a slow vehicle. Finally, we have two clusters composed of single agents. One of them tends to keep the right lane, but changes lanes more rarely and takes longer time to decide to change lanes, while the last agent focuses only on the free right features and changes lane to the right immediately when given the possibility. To investigate the robustness of clustering through 훴-GIRL, we increase the number of clusters. By construction, the clustering loss function will always separate clusters (Fig. 9). The remarkable behavior of MI-훴-GIRL in this problem is that an overestimation of the number of clusters can be easily detected since the weights of the separated clusters will not differ much from the original one. This can be seen in Fig. 10b and c where the newly added clusters are close to the existing ones. 1 3 Machine Learning (2021) 110:2541–2576 2562 Bob Alice Carol Chuck Craig Dan Erin Eve Grace Judy −200 −100 0 Average return Cluster 1 Bob Alice Carol Chuck Craig Dan Erin Eve Grace Judy −100 −50 0 Average return Cluster 2 Trained policy Cluster agents Out of cluster agents Bob Alice Carol Chuck Craig Dan Erin Eve Grace Judy −300 −200 −100 0 Average return Cluster 3 Bob Alice Carol Chuck Craig Dan Erin Eve Grace Judy −150 −100 −50 0 Average return Cluster 4 Fig. 13 Average return of the policy trained with the reward function of each cluster and average return of each expert evaluated with the reward function of each cluster (Color figure online) Fig. 13 Average return of the policy trained with the reward function of each cluster and average return of each expert evaluated with the reward function of each cluster (Color figure online) BC fails to generalize We investigate how transferable the BC policies are between agents belonging to the same cluster. To this purpose, we took the policies trained to imi- tate each agent separately and tested them on all demonstrations of their identified clusters (excluding their own). Figure 11 shows the accuracies of the BC policies of each agent. We only show 8 agents as the other two are alone in their respective clusters. 5.5 Results We can clearly see that BC may easily fail to generalize across agents who might show slightly different behaviors while optimizing the same reward functions. Recall, in fact, that for any reward function, there might exist multiple optimal policies. Hence, agents with the same intent 1 3 Machine Learning (2021) 110:2541–2576 2563 Table 3 Cluster assignment based on the feature expectations Agents Cluster 1 Bob, Alice, Dan, Eve, Grace Cluster 2 Carol, Erin Cluster 3 Craig, Judy Cluster 4 Chuck can demonstrate different policies, and BC tends to “overfit” to the specific behavior. IRL methods, on the other hand, can correctly group different behaviors into the same intent.i y g pf Next we investigate how “good” the recovered reward functions fit the behaviour of the agents assigned to them. Unfortunately, since we do not have the “true” rewards it is hard to do a quantitative evaluation of the results since we cannot compute the error on the recovered weights. Nonetheless, given the presence of the simulator, we can train agents that optimize the recovered reward functions. We used the methods described in Likmeta et al. (2020), which use parametrized rule-based policies to represent the agent’s policy, and have shown good performance in this Highway environment. We trained one agent for each cluster and we compare the expected return of these trained policies, with the average return of each agent in that specific reward function. For details on the training procedure see Appendix A.2. The results are shown in Fig. 12. Here we show for each of the 4 clus- ters, the return of the policy trained in the environment with the reward function of the cluster in pink, compared to the average return of the agent assigned to that cluster under the reward function of the cluster in green and the return of the other agents (assigned to other clusters) under the same reward function, in blue. So every subplot of the figure shows performances of different policies all evaluated with the same reward which means that their performances are comparable (comparisons accross clusters cannot be made since they have different reward functions). As can be expected, in all clusters, the agents not assigned to that cluster perform poorly. The best performing policy is generally the policy explicitly optimizing that reward function, except in Cluster 2 where the RL training procedure failed to recover a good policy. 5.5 Results And finally the returns of the policies assigned to the cluster, have a policy close to the policies which explicitly optimize that return. The results of Cluster 2, where the trained policy is the one performing the worst, show also the general difficulty of evaluating the reward functions recovered from our algorithms, since we are not guaranteed that an agent trained with the given reward functions will achieve the optimal performcance. Figure 13 shows in detail the performances of each agent in each cluster. Clustering via feature expectations Finally, we compare the clustering performed using 훴-GIRL, with a clustering based on the features expectations of the agents. To this end, we cluster the feature expectations of the agents using a K-means with k = 4 clusters. Table 3 shows the results of this clustering. Compared to the clustering done by IRL, we can see that the first cluster contains agents that show very heterogeneous behaviors. This cluster contains the three “good” agents, identified by the IRL clustering, together with two agents that show a greater preference for keeping a high-speed profile (Bob and Dan). It also sep- arates agent Chuck from the other agents who show a preference towards maintaining a high-speed and the left lane, because her feature expectations are “far” from the others, even though the objectives are the same. 1 3 Machine Learning (2021) 110:2541–2576 2564 6 Case study 2: Twitter IRL from multiple experts Social networks like Twitter and Facebook are actively used by millions of users every day. Inferring the users’ interests and intentions is a relevant problem in this context with a vari- ety of possible applications (Piao and Breslin 2018). For instance, understanding why users perform certain actions, like posting a message or clicking on an ad, and what their pref- erences are, allows the system to provide personalized recommendations and, in general, improve the user experience. Similarly, inferred intentions might help detect and counter dangerous agents, such as bots or fake profiles, who could harm the system or its users. Several learning-based techniques have been designed for this problem (Saravia et al. 2017; Song et al. 2015; Xu et al. 2011; Sadri et al. 2019). We refer the reader to an inter- esting survey by Piao and Breslin (2018). To the best of our knowledge, the only previous work that has applied IRL to this problem is Das and Lavoie (2014), in which the authors presented an IRL-based algorithm to infer the intentions of Reddit users. Although almost no previous work has been proposed in this direction, we believe that IRL is a natural and relevant alternative to address this problem. In fact, common existing techniques typically focus on learning the users’ behavior, i.e., how users will respond to certain stimuli, in order to understand what their interests are. However, form our perspective, social network users are learning agents who act in order to maximize certain objectives, and inferring these objectives is what really informs us about their interests and behavior. Here we explore the adoption of IRL methods, precisely the MI-훴-GIRL algorithm, to the problem of inferring the users’ intentions on Twitter. In particular, we try to answer the fol- lowing questions: “Why does a user decide to retweet a post? What is her intention in deciding to post the tweet?” This problem poses several challenges from the IRL perspective. First, we do not have a simulator of the environment and the interaction with the social network might be time-prohibitive and, in some cases, illegal. Therefore, model-free and batch algorithms are required. Furthermore, although lots of data are available for free, collecting this data is very time-consuming and requires significant preprocessing (cleaning, filtering, anonymizing, etc.). Finally, the problem involves a huge amount of agents whose behaviors and intentions depend on those of other agents. 1 3 6.1 System modeling We now describe our simplified model of the user-Twitter interaction. Among the several actions that a user can perform on Twitter, we restrict our attention to the most common one: re-tweeting a post. In our model, a user observes a tweet (generated by another user) and has to decide whether to re-tweet it or not. Intuitively, this simple model allows us to capture most of the relevant interests and intentions of Twitter users. In fact, there exist several reasons why a user might decide to re-tweet a post or not. For instance, the user might be personally inter- ested in the content/topic of the post, or she might think that the post would be appreciated by other Twitter users, or she might simply intend to re-tweet everything (e.g., a spam bot). In each episode of interaction with the social network, the agent observes a sequence of tweets and must decide for each one whether to retweet it or not. The state encodes informa- tion about the last observed tweet and about the agent’s past behavior. It is modeled by three variables: the popularity of the tweet, the number of retweets recently performed by the agent, and the retweet time. The popularity score encodes the likelihood that the general community 1 3 Machine Learning (2021) 110:2541–2576 2565 Table 4 The reward weights learned by 훴-GIRL: popularity score of a retweet, number of retweets in a window T, and retweet time ( 훿time) Reward features N. agents Popularity N. retweets 훿time Cluster 1 0.56 0.00 0.44 4 Cluster 2 0.16 0.19 0.65 6 Cluster 3 0.78 0.03 0.19 4 Fig. 14 Twitter clustering statistics. Average number of followers (left), followings (center) and retweets (right) for each cluster Table 4 The reward weights learned by 훴-GIRL: popularity score of a retweet, number of retweets in a window T, and retweet time ( 훿time) Reward features N. agents Popularity N. retweets 훿time Cluster 1 0.56 0.00 0.44 4 Cluster 2 0.16 0.19 0.65 6 Cluster 3 0.78 0.03 0.19 4 Fig. 14 Twitter clustering statistics. Average number of followers (left), followings (center) and retweets (right) for each cluster Fig. 14 Twitter clustering statistics. Average number of followers (left), followings (center) and retweets (right) for each cluster will like the last observed tweet. 6.1 System modeling Note, however, that the popularity score of the retweet might, in fact, depend on the past actions since, for example, agents that retweet interesting content might increase their number of followers and thus the popular- ity of their retweets. The retweet time is reset to zero if the agent performed a retweet in the current step or updated accordingly as described above if the agent did not retweet, and similarly for the number of retweets. 6.1 System modeling It is computed as the average of the number of likes to the tweet and the number of retweets, will like the last observed tweet. It is computed as the average of the number of likes to the tweet and the number of retweets, Popularity-score = Nlike + Nretweet 2 , and then normalized by the average popularity-score of the user’s tweets. The num- ber of retweets performed by the agent is computed on a retweet window of T = 10 steps, i.e., the last 10 observed tweets. Finally, the retweet time is a measure propor- tional to the time elapsed since the last retweet performed by the agent. It is computed as 훿time = 0.1 ⋅(t −t0) −1 , where t is the first time the agent receives a tweet that she decides to retweet after having retweeted at time t0 < t . State transitions work as follows. The next tweet does not depend on the current one or the agent’s actions since it is generated natu- rally by the environment (i.e., by other users). Note, however, that the popularity score of the retweet might, in fact, depend on the past actions since, for example, agents that retweet interesting content might increase their number of followers and thus the popular- ity of their retweets. The retweet time is reset to zero if the agent performed a retweet in the current step or updated accordingly as described above if the agent did not retweet, and similarly for the number of retweets. and then normalized by the average popularity-score of the user’s tweets. The num- ber of retweets performed by the agent is computed on a retweet window of T = 10 steps, i.e., the last 10 observed tweets. Finally, the retweet time is a measure propor- tional to the time elapsed since the last retweet performed by the agent. It is computed as 훿time = 0.1 ⋅(t −t0) −1 , where t is the first time the agent receives a tweet that she decides to retweet after having retweeted at time t0 < t . State transitions work as follows. The next tweet does not depend on the current one or the agent’s actions since it is generated natu- rally by the environment (i.e., by other users). 6.2 Reward design In this domain, the reward features are the same as the state ones, except that the Pop- ularity-score is set to zero whenever the agent does not re-tweet. Intuitively, these fea- tures allow us to capture different interesting intentions. For instance, users who want 3 3 Machine Learning (2021) 110:2541–2576 2566 Fig. 15 Map of the Lake Como basin Fig. 15 Map of the Lake Como basin to share content that is interesting to the community typically focus on the popularity score while keeping reasonable values for the other two features (so that they do not appear spammers). On the opposite side, users who want to spam every tweet focus on the number of retweets, ignoring their popularity. 6.3 Data collection and processing The dataset was collected using the tweepy API (http://docs.tweepy.org), a Python library for accessing the Twitter API. We selected 14 Twitter accounts, and we obtained all of their followed accounts (5745 in total), using the API. For every of these 5759 ( 14 + 5745 ) accounts we collected their tweets and re-tweets from November 2018 to the end of Janu- ary 2019 using a crawling process. We obtained a total number of 468304 tweets posted by these accounts on the Social Network. We assumed that each user only observes tweets from the accounts she follows, hence ignoring those coming from general (not followed) Twitter users. Furthermore, since a (human) Twitter user is very unlikely to view all the tweets from her followings while generating trajectory data we considered a probability of 0.01 that the agent sees each tweet. We used this process to split the tweet data for each agent into trajectories of 20 tweets, which were used directly to run MI-훴-GIRL. 6.4 Results We perform behavioral cloning on the agents’ demonstrations employing a two-layer neu- ral network (8 neurons each). Then, we divide the demonstrations in trajectories of size 20, which gives us exactly one retweet window in every trajectory. We apply MI-훴-GIRL with 1 3 1 Machine Learning (2021) 110:2541–2576 2567 k = 3 clusters. The results are shown in Table 4, while Fig. 14 reports some statistics of the three clusters found.i k = 3 clusters. The results are shown in Table 4, while Fig. 14 reports some statistics of the three clusters found.i We can observe that the users in the first cluster seem to be interested in retweeting posts with high popularity at a high frequency, i.e., they aim at maximizing the popular- ity score while minimizing the retweet time. This cluster can be interpreted as a grouping of standard Twitter users. This is also confirmed by Fig. 14, which shows that users in this cluster follow many other users while having fewer followers, the standard situation in the social network. The second cluster, on the other hand, groups users who do not aim at retweeting too often. These are users who do not frequently use the social network, as they have few retweets and follow a small number of people. The last cluster is perhaps the most interesting one: these agents tend to retweet all popular tweets. After inspecting the users assigned by the algorithm to this cluster, we found that they are mostly commer- cial accounts (e.g., bots, companies, or two HR managers). Not surprisingly, they show the intention to post popular tweets, but they are uninterested in following other accounts, as Fig. 14 highlights. For completeness, we show in Appendix B.1 the results of clustering based on feature expectations. 7 Case study 3: Como Lake Dam IRL from non‑stationary expert The same algorithm is also used to identify changes in the operations of an Alpine hydropower reservoir in response 3 3 Machine Learning (2021) 110:2541–2576 2568 Fig. 16 Lake level and flood thresholds (left); cyclostationary average inflow, release and demand (right). Shaded areas refer to the historical variability over the 1946–2010 period Fig. 16 Lake level and flood thresholds (left); cyclostationary average inflow, release and demand (right). Shaded areas refer to the historical variability over the 1946–2010 period to the transition from a regulated electric energy market to a free setting. In Mason et al. (2018), a multilateral negotiation process replaces IRL in the identification of the prefer- ence among multiple objectives. The method assumes that multiple virtual agents, which independently optimize different objectives, periodically negotiate a compromise policy for the operation of the system. The authors also model preference dynamics via periodic negotiations where the agents’ attitudes in each negotiation step are determined by the recent system performance. 7.1 System modeling The system is modeled as a discrete-time, periodic, nonlinear, stochastic MDP with a continuous state variable representing the water stored in the lake St , a continuous action that controls the water released at , a state-transition function affected by the sto- chastic lake inflow qt+1 to describe the mass balance equation of the lake storage, i.e. (15) (15) St+1 = St + qt+1 −rt+1(St, at, qt+1) St+1 = St + qt+1 −rt+1(St, at, qt+1) where St is the lake storage at time t; qt+1 the inflow in the time interval [t, t + 1) , rt+1 the water volume released in the same interval, which coincides with the action at corrected, where appropriate, with a non-linear release function determining the minimum and maxi- mum releases feasible for the time interval to respect physical and legal constraints (e.g., spills when the lake level exceeds the maximum capacity). In the adopted notation, the time subscript of a variable indicates the time instant when its value is deterministically known. The reservoir storage is known at time t by measuring the lake level ht and thus is denoted as St , while the net inflow is denoted as qt+1 because it can be known only at the end of the time interval. 7 Case study 3: Como Lake Dam IRL from non‑stationary expert Lake Como is a sub-alpine lake in northern Italy, characterized by an active storage capac- ity of 254 Mm3 fed by a 4552 km2 catchment (Fig. 15). The main tributary and only emis- sary of the lake is the Adda river, the fourth longest Italian river, whose sublacual part originates in the southeastern branch of the lake and feeds eight run-of-the-river hydroelec- tric power plants and serves a dense network of irrigation canals belonging to four irriga- tion districts, with a total irrigated area of 1400 km2 . The southwestern branch of the lake constitutes a dead-end exposed to flooding events, particularly in the city of Como which is the lowest point of the lake shoreline. The hydro-meteorological regime is characterized by scarce discharge in winter and summer, and peaks in late spring and autumn due to snowmelt and rainfall, respectively. Snowmelt from May to July is the most important con- tribution to the seasonal lake storage. The agricultural districts downstream prefer to store snowmelt in the lake to satisfy the peak summer water demands, when the natural inflow is insufficient to meet irrigation requirements. Yet storing such water increases the lake level and, consequently, the flood risk, which could instead be minimized by keeping the lake level as low as possible. The lake regulation has, therefore, to balance flood protection to the lake shores and water supply to downstream users. While the objectives that the human operator in charge of deciding the water release are known, their relative importance is unknown and it might change over time. In this setting, IRL can help in understanding the operator preferences. This knowledge could lead to the future development of artificial systems helping the operator by suggesting a suitable water release amount or even a fully automatic controller. Additionally, exploiting NS-훴-GIRL, we can capture the possible variations in the operator preferences over time. While RL is receiving growing attention in the water community, the application IRL is still in its infancy. In Mason (2018), the Cutting-Plane Inverse Reinforcement Learning algorithm (Pirotta 2016) is first tested in a synthetic case study; the experiments show that CPIRL is able to identify the specific tradeoff underlying a simulated control policy and to distinguish among different formulations of the same objective. 7.2 Reward design We model the competing interests of flood control and water supply using the following reward functions, with a specific feature accounting for intense drought events: 1 3 1 3 Machine Learning (2021) 110:2541–2576 2569 – Water supply deficit ( 휙D ): the daily water deficit between the lake release rt+1 and the water demand dt of the downstream system: Fig. 17 Graphical representation of the weights recovered for one (left) and five (right) regimes. The crosses use the same color coding as the intervals in Fig. 20 (Color figure online) Fig. 17 Graphical representation of the weights recovered for one (left) and five (right) regimes. The crosses use the same color coding as the intervals in Fig. 20 (Color figure online) – Water supply deficit ( 휙D ): the daily water deficit between the lake release rt+1 and the water demand dt of the downstream system: – Water supply deficit ( 휙D ): the daily water deficit between the lake release rt+1 and the water demand dt of the downstream system: Fig. 18 Comparison of the historical regulation (red square) with the set of Pareto optimal control poli- cies (gray circles) exploring the trade-off between flood control and water supply obtained in Giuliani et al. (2019). The historical regulation reveals a preference for reducing floods, as confirmed by our results in Fig. 17 (Color figure online) Fig. 18 Comparison of the historical regulation (red square) with the set of Pareto optimal control poli- cies (gray circles) exploring the trade-off between flood control and water supply obtained in Giuliani et al. (2019). The historical regulation reveals a preference for reducing floods, as confirmed by our results in Fig. 17 (Color figure online) (16) 휙D t = max(rt −dt, 0). (16) – Flood risk ( 휙F ): a penalization function that is large for small releases associated to high lake levels: – Flood risk ( 휙F ): a penalization function that is large for small releases associated to high lake levels: – Flood risk ( 휙F ): a penalization function that is large for small releases associated to high lake levels: (17) 𝜙F t = ⎧ ⎪ ⎨ ⎪⎩ 0 if rt+1 > rF − rF−rt+1 rF−rF 2 if rF ≤rt+1 ≤rF −1 otherwise , (17) with rF = 120 and rF = 5. with rF = 120 and rF = 5. with rF = 120 and rF = 5. 7.2 Reward design 1 3 Machine Learning (2021) 110:2541–2576 2570 Fig. 19 IRL and BC losses as a function of the number of regimes. The IRL loss is the one of Eq. 14. The BC loss is the cross-entropy of the same intervals computed by IRL Fig. 20 Identified year intervals for different number of regimes (Color figure online) Fig. 19 IRL and BC losses as a function of the number of regimes. The IRL loss is the one of Eq. 14. The BC loss is the cross-entropy of the same intervals computed by IRL Fig. 19 IRL and BC losses as a function of the number of regimes. The IRL loss is the one of Eq. 14. The BC loss is the cross-entropy of the same intervals computed by IRL Fig. 20 Identified year intervals for different number of regimes (Color figure online) Fig. 20 Identified year intervals for different number of regimes (Color figure online) – Drought risk ( 휙L ): a penalization that is large for large releases associated to low lake levels: – Drought risk ( 휙L ): a penalization that is large for large releases associated to low lake levels: = 500 and rL = 5: (18) 𝜙L t = ⎧ ⎪ ⎨ ⎪⎩ 0 if rt+1 < rL − rt+1−rL rL−rL 2 if rL ≤rt+1 ≤rL −1 otherwise , (18) 𝜙L t = ⎧ ⎪ ⎨ ⎪⎩ 0 if rt+1 < rL − rt+1−rL rL−rL 2 if rL ≤rt+1 ≤rL −1 otherwise , (18) with rL = 500 and rL = 5: Fig. 19 IRL and BC losses as a function of the number of regimes. The IRL loss is the one of Eq. 14. The BC loss is the cross-entropy of the same intervals computed by IRL 7.4 Results We employed as state representation the concatenation of the lake level ht , the inflow of the previous day qt , the demand of the current day dt and the actions of the previous two days at−1 and at−2: (19) st = (ht, qt, dt, at−1, at−2 ). (19) For the preliminary BC phase, we employed a Gaussian policy with fixed variance 휎2 = 1 and mean which is linear in the state st. The human operator has a set of preferences that are unknown. First of all, we focus on the IRL results without any time interval subdivision, i.e., when considering just one regime. The weights recovered in this case are shown in Fig. 17 left. We notice a slight predominance of the interest in controlling the floods, whose feature 휙F is weighted with 휔F = 0.47 , whereas the remaining weight is divided between the feature of the demand 휙D ( 휔D = 0.34 ) and the control of the drought events 휙L ( 휔L = 0.19 ). These preferences can be validated by comparing the historical data with the set of Pareto optimal control policies exploring the tradeoff between flood control and water supply obtained in Giuliani et al. (2019). The mapping of the historical regulation and of the Pareto optimal solutions in the space of water supply deficit and flood control illustrated in Fig. 18 shows how the opera- tor is almost Pareto efficient and the historical regulation attains very good performance in terms of flood control at the cost of high values of water supply deficit. li Since the available data span a time period of 65 years, we investigate whether the behavior of the lake operator displays a stationary intention or the underlying preferences change over time. As many environmental systems, the Lake Como is a non-stationary sys- tem that has undergone several alterations over time, which might have changed the prefer- ences of the regulator. For this purpose, we employ NS-훴-GIRL, described in Sect. 3.3, by considering a single trajectory the sequence of states and actions observed along a one year period. The results are shown in Fig. 19 and 20. First of all, looking at the IRL loss (Fig. 19) we observe a significant improvement moving from one regime to two, then the loss keeps reducing but with smaller benefits. 7.3 Data description The dataset is composed of the historical trajectory of lake levels, inflows, and releases over the period 1946–2010, which are illustrated in Fig. 16. All data are daily and were provided by Consorzio dell’Adda (www.addaconsorzio.it). In Fig. 16 we show the lake level, together with the inflow and release for a year, averaged over the considered time interval (1946–2010). 1 3 1 3 Machine Learning (2021) 110:2541–2576 2571 7.4 Results Our results capture this transition, with the period (2003–2010) that is associated with a new regime that assigns a high weight to reducing drought risk ( 휔L = 0.59 ) and supplying water demand ( 휔D = 0.41 ) while reducing the importance of flood control. l This analysis allows grasping a general overview of how the operator’s preferences, which are modeled via intentions, change over time. However, several questions remain open. First, the choice of a suitable number of regimes, by just observing the data is chal- lenging. The elbow analysis can provide some suggestions, but still, we needed the domain expert’s knowledge to understand whether a subdivision is reasonable. Second, the five regime setting allows us to provide some interpretation but displays limits as well. Spe- cifically, the subdivision is sometimes inaccurate. We may wonder why the fourth interval (the one in which the flooding control objective is predominant) begins in 1989 instead of 1988, being that the Como flooding event occurred in 1987. Third, the results are sig- nificantly dependent on the choice of the features. A suitable feature design is an iterative process that needs to account for both the domain peculiarities and the characteristics of the employed IRL algorithms. 7.4 Results From an elbow analysis, we can conclude that a number of regimes of 4 or 5 result suitable for the problem. Looking at the BC loss, we notice an overall reduction as well, although not monotonic. Concerning the interval subdivision, according to the properties of the real domain and the events that occurred, we believe that the most interpretable one is the case with five regimes. The first two time periods (1946–1949 and 1950–1959) can be seen as set-up periods in which the operator tries different policies and learns how to operate the dam, which was constructed in 1946. Overall, this period displays a preference towards reducing flood risk. Moving to the third period (1960-1988) we appreciate a notable change in the intentions trade-off. Indeed, the estimated weights increase the preference toward satisfy- ing the downstream water demand ( 휔D = 0.47 ) while reducing the interest in avoiding the floods ( 휔F = 0.33 ). The notable length of this period can be interpreted as an indicator of the fact that the human regulator has converged to a stable policy. However, starting from 1989 we notice a significant variation of trade-off that becomes largely driven by flood control. In the time interval 1989–2002, most of the weight (0.94) is given to the feature 휙F . This change of intention can be justified by the large flood event that occurred in 1987, registering the highest level in the historic records. This event was followed by other floods, which might have further consolidated this conservative behavior that mini- mizes flood risk. In recent years, the climatic conditions in the region have manifested a 1 1 3 3 Machine Learning (2021) 110:2541–2576 2572 drying trend inducing a further modification of the operator’s preferences. The summer of 2003–2005–2006 represent extreme, unprecedented drought events (Giuliani et al. 2016). Our results capture this transition, with the period (2003–2010) that is associated with a new regime that assigns a high weight to reducing drought risk ( 휔L = 0.59 ) and supplying water demand ( 휔D = 0.41 ) while reducing the importance of flood control. drying trend inducing a further modification of the operator’s preferences. The summer of 2003–2005–2006 represent extreme, unprecedented drought events (Giuliani et al. 2016). 1 3 Declarations Conflict of interest The authors declare that they have no conflict of interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 8 Discussion and conclusions ii As future work, we intend to extend our approach to deal with settings in which the action space of the demonstrations differs from the action space of the task in which 1 3 Machine Learning (2021) 110:2541–2576 2573 the reward functions will be applied. For instance, in the car driving problem, we might consider the case where demonstrations come from the low-level control of the vehi- cle, but the reward function will be applied for the high-level control. Furthermore, we might consider a more extreme scenario, when we do not observe the actions performed by the expert but only their effects on the state of the environment. the reward functions will be applied. For instance, in the car driving problem, we might consider the case where demonstrations come from the low-level control of the vehi- cle, but the reward function will be applied for the high-level control. Furthermore, we might consider a more extreme scenario, when we do not observe the actions performed by the expert but only their effects on the state of the environment. Supplementary Information The online version contains supplementary material available at https://d org/10.1007/s10994-020-05939-8. Author Contributions Not Applicable Author Contributions Not Applicable Funding Open access funding provided by Alma Mater Studiorum - Università di Bologna within the CRUI-CARE Agreement. Availability of data and material The data relative to the AD task and the Como d Availability of data and material The data relative to the AD task and the Como dam operation are available in the code repository. The data relative to the Twitter case study are available upon request because they need to be anonymized.Code availability The code of the experiments will be made available at https://github.com/amarildolikmeta/irl_real_ life. 8 Discussion and conclusions We tackled the problem of inferring the intentions of human operators in several real-world scenarios via IRL algorithms. In these settings, it is important to have algorithms that oper- ate in a model-free, batch manner, since, in most applications, the model of the environ- ment is not available and there is no possibility of interaction as well. We applied the MI-훴 -GIRL algorithm to the Twitter and AD tasks, identifying multiple clusters of agents with different reward functions. Furthermore, we proposed an extension to the 훴-GIRL algo- rithm to deal with non-stationary intentions of the expert and applied it to the real-world case of the Lake Como dam operation, identifying multiple operating regimes. We inter- preted these regimes with the evolution of the dam environment supported by historical data on the climatic events that occurred in the geographical area of the dam. Although we were able to employ these algorithms in real-life scenarios, it is worth not- ing that their application should not be seen as a black-box. Being in a fully-batch setting, without further interaction with the environment, these algorithms depend heavily on the system modeling phase. A bad design of the state, action, policy and most importantly, reward space can highly affect the final results. We witnessed this phenomenon in all our applications, and we believe that it is a price to pay when giving away the possibility to interact with the environment. Typically, a bad state, action and policy space design can be detected in the behavioral cloning phase, as usually the accuracy of the imitating policy is low. Reward design is a more delicate phase, as with most of the IRL algorithms. 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https://openalex.org/W3183248656	https://iris.unibas.it/bitstream/11563/149885/1/Cialdea2021_Article_TheScientificWorkOfVladimirMaz.pdf	English	null	The scientific work of Vladimir Maz’ya	Analysis and mathematical physics	2,021	cc-by	6,536	Analysis and Mathematical Physics (2021) 11:143 https://doi.org/10.1007/s13324-020-00421-z Analysis and Mathematical Physics (2021) 11:143 https://doi.org/10.1007/s13324-020-00421-z 1 Department of Mathematics, Computer Sciences and Economics, University of Basilicata, V.le dell’Ateneo Lucano, 10, 85100 Potenza, Italy Alberto Cialdea1 Received: 12 October 2020 / Revised: 22 October 2020 / Accepted: 23 October 2020 © The Author(s) 2020 B Alberto Cialdea cialdea@email.it cialdea@email.it 1 Department of Mathematics, Computer Sciences and Economics, University of Basilicata, V.le dell’Ateneo Lucano, 10, 85100 Potenza, Italy The scientiﬁc work of Vladimir Maz’ya Alberto Cialdea1 1 Introduction Vladimir Maz’ya is an outstanding mathematician whose work had a profound impact on the modern analysis. In more than 60 years of activity, he wrote over 500 papers and 40 monographs. Figure 1 gives an impressive glance of his books. The great depth of the results he has obtained, his fundamental new ideas, and his skilled technique characterize his papers. Among his most important results are the discovery of the equivalence between Sobolev and isoperimetric/isocapacitary inequalities (which we shall describe in Sect. 2), his counterexamples related to Hilbert’s 19th and 20th problems (see Sect. 3), his solution, together with Yuri Burago, of a problem in harmonic potential theory posed by Riesz and Nagy, his solution with Mikhail Shubin of a problem in the B Alberto Cialdea cialdea@email.it 143 Page 2 of 14 A. Cialdea Fig. 1 Maz’ya’s books spectral theory of the Schrödinger operator formulated by Gelfand, his solution with Victor Havin of a problem related to harmonic approximation posed by Mergelyan. He solved also Vladimir Arnol’d’s problem for the oblique derivative boundary value problem and Fritz John’s problem on the oscillations of a ﬂuid in the presence of an immersed body. He gave also seminal contributions to the development of the theory of capacities, nonlinear potential theory, the asymptotic and qualitative theories of arbi- trary order elliptic equations, the theory of ill-posed problems, the theory of boundary value problems in domains with piecewise smooth boundary. He introduced the con- cept of Approximate Approximation, which provides a completely new approach to fast numerical procedures. Fig. 1 Maz’ya’s books Fig. 1 Maz’ya’s books Fig. 1 Maz’ya’s books spectral theory of the Schrödinger operator formulated by Gelfand, his solution with Victor Havin of a problem related to harmonic approximation posed by Mergelyan. He solved also Vladimir Arnol’d’s problem for the oblique derivative boundary value problem and Fritz John’s problem on the oscillations of a ﬂuid in the presence of an immersed body. He gave also seminal contributions to the development of the theory of capacities, nonlinear potential theory, the asymptotic and qualitative theories of arbi- trary order elliptic equations, the theory of ill-posed problems, the theory of boundary value problems in domains with piecewise smooth boundary. He introduced the con- cept of Approximate Approximation, which provides a completely new approach to fast numerical procedures. 1 Introduction spectral theory of the Schrödinger operator formulated by Gelfand, his solution with Victor Havin of a problem related to harmonic approximation posed by Mergelyan. He solved also Vladimir Arnol’d’s problem for the oblique derivative boundary value problem and Fritz John’s problem on the oscillations of a ﬂuid in the presence of an immersed body. He gave also seminal contributions to the development of the theory of capacities, nonlinear potential theory, the asymptotic and qualitative theories of arbi- trary order elliptic equations, the theory of ill-posed problems, the theory of boundary value problems in domains with piecewise smooth boundary. He introduced the con- cept of Approximate Approximation, which provides a completely new approach to fast numerical procedures. He received several Honours and Awards: – 1962, Prize of the Leningrad Mathematical Society, Russia. – 1990, Doctor honoris causa of the University of Rostock, Germany. – 1999, Humboldt Research Prize. – 1999, Humboldt Research Prize. – 2001, Corresponding Fellow of the Royal Society of Edinburgh. – 2002, Member of the Royal Swedish Academy of Sciences. – 2003, Verdaguer Prize of the French Academy of Sciences. – 2004, The Celsius Gold Medal of the Royal Society of Sciences at Uppsala. – 2009, Senior Whitehead Prize of the London Mathematical Society. – 2012, Elected Fellow of the American Mathematical Society. – 2013, Foreign Member of the Georgian National Academy of Sciences. His reputation is attested by several Conferences which have been organized in his honour: His reputation is attested by several Conferences which have been organized in his honour: – Sobolev spaces and potential theory. Conference in honour of Vladimir Maz’ya, Kyoto, Japan, 1993. Page 3 of 14 The scientiﬁc work of Vladimir Maz’ya 143 – Functional Analysis, Partial Differential Equations and Applications. Conference in honour of Vladimir Maz’ya, Rostock, Germany, 1998. – Functional Analysis, Partial Differential Equations and Applications. Conference in honour of Vladimir Maz’ya, Rostock, Germany, 1998. – Analysis, PDEs and Applications. Conference on the occasion of the 70th birthday of Vladimir Maz’ya, Rome, Italy, 2008. – Analysis, PDEs and Applications. Conference on the occasion of the 70th birthday of Vladimir Maz’ya, Rome, Italy, 2008. – Nordic-Russian Symposium in honour of Vladimir Maz’ya on the occasion of his 70th birthday, Stockholm, Sweden, 2008. – Nordic-Russian Symposium in honour of Vladimir Maz’ya on the occasion of his 70th birthday, Stockholm, Sweden, 2008. 1 Introduction y – Analysis of Partial Differential Equations, Symposium in honour of Vladimir Maz’ya on the occasion of his 75th Birthday, Liverpool, UK, 2013. – Analysis of Partial Differential Equations, Symposium in honour of Vladimir Maz’ya on the occasion of his 75th Birthday, Liverpool, UK, 2013. – Sobolev Spaces and Partial Differential Equations, on the occasion of the 80th birthday of Vladimir Maz’ya, Accademia dei Lincei, Rome, Italy, 2018. – Sobolev Spaces and Partial Differential Equations, on the occasion of the 80th birthday of Vladimir Maz’ya, Accademia dei Lincei, Rome, Italy, 2018. y y y – Harmonic Analysis and PDE, International Conference in honor of Vladimir Maz’ya, Holon, Israel, 2019. – Harmonic Analysis and PDE, International Conference in honor of Vladimir Maz’ya, Holon, Israel, 2019. Laurent Schwartz and Vladimir Maz’ya (Paris, 1992) Laurent Schwartz and Vladimir Maz’ya (Paris, 1992) Several books and papers have been dedicated to him: – Two volumes of “The Maz’ya Anniversary Collection”, edited by Rossmann, J., Takaˇc, P., Wildenhain, Birkhäuser, 1999. – Two volumes of “The Maz’ya Anniversary Collection”, edited by Rossmann, J., Takaˇc, P., Wildenhain, Birkhäuser, 1999. – Mathematical Aspects of Boundary Element Methods, dedicated to Vladimir Maz’ya on the occasion of his 60th birthday, edited by M. Bonnet, A.M. Sändig and W. Wendland, Chapman & Hall/CRC Research Notes in Mathematics, London, 1999. – Mathematical Aspects of Boundary Element Methods, dedicated to Vladimir Maz’ya on the occasion of his 60th birthday, edited by M. Bonnet, A.M. Sändig and W. Wendland, Chapman & Hall/CRC Research Notes in Mathematics, London, 1999. – Perspectives in Partial Differential Equations, Harmonic Analysis and Applica- tions: A Volume in Honor of Vladimir G. Maz’ya’s 70th Birthday, edited by D. Mitrea and M. Mitrea, Proc. of Symposia in pure mathematics, Vol. 79, Amer. Math. Soc., Providence (R.I.), 2008. Analysis, Partial Differential Equations and Applications. The Vladimir Maz’ya AnniversaryVolume, editedbyA. Cialdea, F. Lanzara, P.E. Ricci, Operator Theory, Advances and Applications, Vol. 193, Birkhäuser, Berlin, 2009. Analysis, Partial Differential Equations and Applications. The Vladimir Maz’ya AnniversaryVolume, editedbyA. Cialdea, F. Lanzara, P.E. Ricci, Operator Theory, – D. Eidus et al, Mathematical work of Vladimir Maz’ya (on the occasion of his 60th birthday), Funct. Differ. Equ. 4 (1997), no. 1–2, pp. 3–11. – D. Eidus et al, Mathematical work of Vladimir Maz’ya (on the occasion of his 60th birthday), Funct. Differ. Equ. 4 (1997), no. 1–2, pp. 3–11. – M.S.Agranovich et al, Vladimir G. – M. S. Agranovich et al, Vladimir Gilelevich Maz’ya (on his 70th birthday), Russian Math. Surveys 63:1(2008), 189-196. 1 Introduction Eigenfunctions of the Fourier transform 26. Sobolev spaces in unrestricted domains Clearly, it is impossible to give a complete description of the scientiﬁc activity of Vladimir Maz’ya in one paper. I had to make some choices, also considering that certain topics will be discussed in other contributions by other participants of the meeting dedicated to his 80th anniversary. Vladimir Maz’ya has a reputation of being the solver of problems which are gen- erally considered as unsolvable. This is why Fichera once compared him with Santa Rita, the 14th century Italian nun who is the Patron Saint of Impossible Causes. About twenty years ago Vladimir Maz’ya and Sergej Nikol’skij met at ISAAC Conference in Berlin (2001). I remember how Nikol’skij told to Maz’ya that he could not sleep because of a problem he could not solve. Maz’ya asked what problem was. Nikol’skij’s answer was: I will not tell you, otherwise you will solve it ! From left to right: Ennio De Giorgi, Gaetano Fichera, Vladimir Maz’ya and Giorgio Salvini (President of Accademia dei Lincei) From left to right: Ennio De Giorgi, Gaetano Fichera, Vladimir Maz’ya and Giorgio Salvini (Preside of Accademia dei Lincei) 1 Introduction Maz’ya, On the occasion of his 65th birthday, Russian Journal of Mathematical Physics, Vol. 10, No. 3, 2003, pp. 239–244. – M.S.Agranovich et al, Vladimir G. Maz’ya, On the occasion of his 65th birthday, Russian Journal of Mathematical Physics, Vol. 10, No. 3, 2003, pp. 239–244. 143 Page 4 of 14 143 Page 4 of 14 143 A. Cialdea – M. S. Agranovich et al, Vladimir Gilelevich Maz’ya (on his 70th birthday), Russian Math. Surveys 63:1(2008), 189-196. – M. S. Agranovich et al, Vladimir Gilelevich Maz’ya (on his 70th birthday), Russian Math. Surveys 63:1(2008), 189-196. – M.V. Anolik et al, Vladimir Gilelevich Maz’ya (On the Occasion of his 70th Anniversary), Vestnik Sankt-Peterburgskogo Universiteta. Seriya 1. Matematika, Mekhanika, Astronomiya, no. 4 (2008), 3–6. – M.V. Anolik et al, Vladimir Gilelevich Maz’ya (On the Occasion of his 70th Anniversary), Vestnik Sankt-Peterburgskogo Universiteta. Seriya 1. Matematika, Mekhanika, Astronomiya, no. 4 (2008), 3–6. Vladimir Maz’ya and John Nash (Beijing, 2002) Vladimir Maz’ya and John Nash (Beijing, 2002) One of the characteristic of Maz’ya scientiﬁc activity is the great variety of topics. This is only a tentative list of the subjects of his research: 1. Equivalence of isoperimetric and integral inequalities 2. Theory of capacities and nonlinear potentials 3. Counterexamples related to the 19th and 20th Hilbert problems 4. Boundary behaviour of solutions to elliptic equations in general domains 5. Non-elliptic singular integral and pseudodifferential operators 6. Degenerating oblique derivative problem 7. Estimates for general differential operators 8. Boundary integral equations 9. Linear theory of surface waves 10. The Cauchy problem for the Laplace equation 11. Theory of multipliers in spaces of differentiable functions 12. Characteristic Cauchy problem for hyperbolic equations 13. Boundary value problems in domains with piecewise smooth boundaries 14. Asymptotic theory of differential and difference equations with operator coefﬁ- cients 15. Maximum modulus principle for elliptic and parabolic systems, contractivity of semigroups 16. Iterative procedures for solving ill-posed boundary value problems 17. Asymptotic theory of singularly perturbed boundary value problems 18. “Approximate approximations” and their applications Page 5 of 14 143 The scientiﬁc work of Vladimir Maz’ya 143 19. Wiener test for higher-order elliptic equations 20. Spectral theory of the Schrödinger operator 21. Navier-Stokes equations 22. History of Mathematics 23. Mesoscale asymptotic expansion 24. Criteria for the accretivity and form boundedness of second order elliptic equations 24. Criteria for the accretivity and form boundedness of second order elliptic equations 25. 2 Equivalence of isoperimetric/isocapacitary and integral inequalities Sobolev embedding theorems are well known. One of them asserts that if 1 < p < n, the following inequality holds ∥u∥L p∗() ≤C ∥∇u∥L p() + ∥u∥L p() (1) (1) for any u in the Sobolev space W 1,p(). Here is a domain in Rn and p∗= np/(n − p). This result was proved by Sobolev and later extended to the case p = 1 by 143 Page 6 of 14 Page 6 of 14 143 A. Cialdea Gagliardo and Nirenberg (1958). In these papers the domain satisﬁes the so-called cone property. This means that each point of the domain is the vertex of a spherical cone with ﬁxed height and angle which is situated inside the domain. Gagliardo and Nirenberg (1958). In these papers the domain satisﬁes the so-called cone property. This means that each point of the domain is the vertex of a spherical cone with ﬁxed height and angle which is situated inside the domain. The inequality (1) does not hold on an arbitrary domain. For example, it is easy to see that if = {(x, y) ∈R2 \| 0 < x < 1, 0 < y < x4}, the function u(x, y) = 1/x belongs to W 1, 3 2 (), but not to L6(). On the other hand there exist domains showing that cone property is not necessary for the validity of inequality (1). At a very young age, when he was still a fourth year undergraduate student, Vladimir Maz’ya discovered the equivalence between Sobolev embeddings like (1) and isoperi- metric and isocapacitary inequalities. The classical isoperimetric inequality states that for any planar domain with a rectiﬁable boundary of a ﬁxed lenght L we have 4π A ≤L2, (2) (2) where A istheareaof.Theequalityholdsifandonlyifisadisk.Then-dimensional generalization of (2) is (mesn g) n−1 n ≤CnHn−1(∂g), (3) (3) where g is a domain in Rn with smooth boundary ∂g and compact closure, and Hn−1 is the (n−1)-dimensional area. The constant Cn = n−1v−1/n n is such that (3) becomes equality for any ball (here vn denotes the volume of the unit ball). Maz’ya discovered that Sobolev inequality ∥u∥Ln/(n−1)(Rn) ≤Cn∥∇u∥L1(Rn), ∀u ∈C∞ 0 (Rn) holds with the same best constant of the isoperimetric inequality (3). Following this idea, Maz’ya was able to characterize more general inequalities like holds with the same best constant of the isoperimetric inequality (3). 2 Equivalence of isoperimetric/isocapacitary and integral inequalities p q y ( ) Following this idea, Maz’ya was able to characterize more general inequalities like Following this idea, Maz’ya was able to characterize more general inequalities like ∥u∥Lq(,μ) ≤C ∥∇u∥L p() + ∥u∥L p() , 1 ≤p ≤q where μ is a measure and where μ is a measure and ∥u∥Lq(,μ) = \|u\|qdμ 1/q . For example, he proved that the inequality For example, he proved that the inequality ∥u∥Lq(,μ) ≤C∥∇u∥L1(), ∀u ∈C∞ 0 () (4) ∥u∥Lq(,μ) ≤C∥∇u∥L1(), ∀u ∈C∞ 0 () (4) holds if and only if the following isoperimetric inequality is valid μ(g)1/q ≤CHn−1(∂g), ∀g ⋐, ∂g ∈C∞ (5) μ(g)1/q ≤CHn−1(∂g), ∀g ⋐, ∂g ∈C∞ (5) and the best constants in (4) and (5) coincide. and the best constants in (4) and (5) coincide. The scientiﬁc work of Vladimir Maz’ya Page 7 of 14 143 143 Let us consider the L p norm of the gradient (p > 1) instead of the L1 norm in (4), i Let us consider the L p norm of the gradient (p > 1) instead of the L1 norm in (4), i.e. ∥u∥Lq(,μ) ≤D∥∇u∥L p(), ∀u ∈C∞ 0 (), q ≥p ≥1. (6) Let us consider the L p norm of the gradient (p > 1) instead of the L1 norm in (4 i e Let us consider the L norm of the gradient (p > 1) instead of the L norm in (4), i.e. ∥u∥Lq(,μ) ≤D∥∇u∥L p(), ∀u ∈C∞ 0 (), q ≥p ≥1. (6) ∥u∥Lq(,μ) ≤D∥∇u∥L p(), ∀u ∈C∞ 0 (), q ≥p ≥1. (6) ∥u∥Lq(,μ) ≤D∥∇u∥L p(), ∀u ∈C∞ 0 (), q ≥p ≥1. (6 (6) Maz’ya proved that this inequality is equivalent to the “isocapacitary” inequality Maz’ya proved that this inequality is equivalent to the “isocapacitary” inequality μ(g)p/q ≤C capp g, g ⋐, ∂g ∈C∞, (7) (7) where the p-capacity capp is deﬁned as where the p-capacity capp is deﬁned as capp F = inf \|∇ϕ\|pdx : ϕ ∈C∞ 0 (), ϕ\|F ≥1 . More precisely, Maz’ya proved that More precisely, Maz’ya proved that – if (6) holds, then (7) is true and D ≥C1/p; – if (7) is valid, then (6) holds and D ≤p(p −1)(1−p)/pC1/p. – if (6) holds, then (7) is true and D ≥C1/p; – if (7) is valid, then (6) holds and D ≤p(p −1)(1−p)/pC1/p. Thisisonlythebeginningofthestory.Theequivalencebetweenintegralandisoperi- metric/isocapacitary inequalities proved to be extremely fruitful. 2 Equivalence of isoperimetric/isocapacitary and integral inequalities The Maz’ya book “Sobolev Spaces”, of which a second enlarged edition has been published in 2010, contains deep developments of this idea, which lead to several applications to the solvability of boundary value problems for elliptic equations and to theorems on the structure of the spectrum of the corresponding operators. 3 Counterexamples related to the 19th and 20th Hilbert problem The function u(x) = \|x\|a, where The function u(x) = \|x\|a, where The function u(x) = \|x\|a, where a = 2 −n 2 + n2 4 −(n −1)(κn + μ) ν + 2κ + μ , belongs to W 2,2 and is solution of the equation Au = 0 in B1. If κ = n(n −2), μ = n2, ν = (n −2)2 + ε (ε > 0) the strong ellipticity condition is satisﬁed and the exponent a is equal to belongs to W 2,2 and is solution of the equation Au = 0 in B1. belongs to W 2,2 and is solution of the equation Au = 0 in B1. belongs to W 2,2 and is solution of the equation Au = 0 in B1. If κ = n(n −2), μ = n2, ν = (n −2)2 + ε (ε > 0) the strong ellipticity condition i i ﬁd d h i l belongs to W , and is solution of the equation Au = 0 in B1. If κ = n(n −2), μ = n2, ν = (n −2)2 + ε (ε > 0) the strong ellipticity condition is satisﬁed and the exponent a is equal to belongs to W and is solution of the equation Au 0 in B1. If κ = n(n −2), μ = n2, ν = (n −2)2 + ε (ε > 0) the strong ellipticity condition is satisﬁed and the exponent a is equal to g q If κ = n(n −2), μ = n2, ν = (n −2)2 + ε (ε > 0) the strong ellipticity conditio is satisﬁed and the exponent a is equal to a(ε) = 2 −n 2 + n 2 ε 4(n −1)2 + ε . One can check that, if n > 4 and ε is sufﬁciently small, the solution \|x\|a(ε) is not bounded around the origin. This shows that a solution in W 2,2 of the equation Au = 0 does not need to be bounded. In the same paper Maz’ya shows how to construct similar counterexamples for elliptic equations of any order 2l and for quasilinear equations. This is how Maz’ya described the discovery of this counterexample in his book “Differential Equations of my young years” (p.180): Whatever I did to justify ana- lyticity of solutions of variational problems of higher order, nothing worked. 3 Counterexamples related to the 19th and 20th Hilbert problem The celebrated theorem proved indipendently by De Giorgi and Nash in 1957 states that every solution u ∈W 1,2 of the linear elliptic scalar equation ∂i(ai j(x)∂ju) = 0 with variable real valued bounded coefﬁcients satisﬁes Hölder’s condition. This result implies the inﬁnite differentiability (or the analyticity) of u. This has turned out to be essential for the complete solution of the 19th Hilbert’s problem. For many years peo- ple tried to extend this result to equations of higher order or to systems. It was a kind of shock when Maz’ya proved that the result does not hold for equations of higher order. Let us describe his counterexample which appeared in the paper Maz’ya, V. Examples of nonregular solutions of quasilinear elliptic equations with analytic coef- ﬁcients. Funkcional. Anal. i Priložen. 2:3, (1968) 53–57 (Russian). English translation: Functional Anal. Appl. 2 (1968), 230–234. with variable real valued bounded coefﬁcients satisﬁes Hölder’s condition. This result implies the inﬁnite differentiability (or the analyticity) of u. This has turned out to be essential for the complete solution of the 19th Hilbert’s problem. For many years peo- ple tried to extend this result to equations of higher order or to systems. It was a kind of shock when Maz’ya proved that the result does not hold for equations of higher order. Let us describe his counterexample which appeared in the paper Maz’ya, V. Examples of nonregular solutions of quasilinear elliptic equations with analytic coef- ﬁcients. Funkcional. Anal. i Priložen. 2:3, (1968) 53–57 (Russian). English translation: Functional Anal. Appl. 2 (1968), 230–234. pp Let A be the following fourth order differential operator Au ≡ν2 + κ xi x j \|x\|2 uxi x j + κ xi x j \|x\|2 u xi x j + μ xi x j xkxl \|x\|4 uxi x j xkxl Page 8 of 14 A. Cialdea 143 where the real constants ν, κ and μ are such that ν > 0, κ2 < μν. The last condition implies that the operator A is strongly elliptic. Consider the equation Au = 0 in B1 = {x ∈Rn \| \|x\| < 1}. This can be considered as Euler’s equation for the functional B1 ν(u)2 + 2κ xi x j \|x\|2 uxi x j u + μ xi x j \|x\|2 uxi x j 2 dx . 3 Counterexamples related to the 19th and 20th Hilbert problem I was stealing up to the problem in various ways, but the solution sneaked off. The problem did not want to be solved for the life of me! But one day, feeling desperate I decided to consider concrete examples in order to understand at least something, and almost at once found that the hypothesis of analyticity was wrong – this was not expected by anyone! Nina Nikolaevna Uraltseva was the ﬁrst whom I showed my counterexamples. She frowned saying “It’s impossible!”, but took my manuscript home and promised to check it. A week later she announced for all to hear at the Big Seminar that I was right. Later Maz’ya with Nazarov and Plamenevskii show that also scalar strongly ellip- tic second-order differential equations in divergence form with measurable bounded complex coefﬁcients in Rn (n > 4) can have generalized solutions which are not bounded in any neighborhood of an interior point of the domain (see Maz’ya, Nazarov, Plamenevskij: Asymptotic Theory of Elliptic Boundary Value Problems in Singularly Perturbed Domains. Vol. I, 2000, pp.391–393). While for n = 2 it is known that a generalized solution has to be Hölder continuous (see Morrey (1938), Trans. Am. Math. Soc.), the cases n = 3 and n = 4 are still unsolved. Page 9 of 14 The scientiﬁc work of Vladimir Maz’ya 143 This problem is contained in a very interesting collection of open problems which Vladimir Maz’ya recently published (Seventy Five (Thousand) Unsolved Problems in Analysis and Partial Differential Equations, Integral Equations and Operator Theory, 2018).Theyconcernfunctiontheory,functionalanalysis,theoryoflinearandnonlinear partial differential equations. 4 Multipliers in Sobolev spaces Cialdea The function u is said to be a solution of the generalized Dirichlet problem if ⎧ ⎪⎨ ⎪⎩ u ∈Wl p() Pu = f in u −g ∈Wl p() ∩˚W h p(), (8) (8) where f ∈Wl−2h p () and g ∈Wl p() are given. where f ∈Wl−2h p () and g ∈Wl p() are given. where f ∈Wl−2h p () and g ∈Wl p() are given. where f ∈Wl−2h p () and g ∈Wl p() are given. p p Let us suppose that the boundary of satisﬁes a condition expressed in terms o multipliers. Speciﬁcally, let us suppose that ∂ ∈Wl+1−h−1/p p if p(l −h) > n o l+1 h 1/ p p Let us suppose that the boundary of satisﬁes a condition expressed in terms of multipliers. Speciﬁcally, let us suppose that ∂ ∈Wl+1−h−1/p p if p(l −h) > n or ∂ ∈Ml+1−h−1/p p (δ) if p(l −h) ≤n. The latter means that for each point of the boundary there exists a neighborhood U and a Lipschitz function ϕ such that ∈Ml+1−h−1/p p (δ) if p(l −h) ≤n. The latter means that for each point of the oundary there exists a neighborhood U and a Lipschitz function ϕ such that U ∩ = {(x, y) ∈U \| x ∈Rn−1, y > ϕ(x)} (9) (9) and and ∥∇ϕ; Rn−1∥MWl−1−1/p p ≤δ . Here δ is a small constant and MW s p is the space of multipliers in W s p for s > 0 and L∞for s ≤0. Under these conditions on the boundary, Maz’ya and Shaposhnikova proved the following existence result: Given f ∈Wl−2h() g ∈Wl () the BVP (8) has one and only one solution Under these conditions on the boundary, Maz’ya and Shaposhnikova proved the following existence result: g Given f ∈Wl−2h p (), g ∈Wl p(), the BVP (8) has one and only one solution u ∈Wl p(). g Given f ∈Wl−2h p (), g ∈Wl p(), the BVP (8) has one and only one solutio u ∈Wl p(). p They show also that a simple sufﬁcient condition which implies their assumption on the boundary when p(l −h) ≤n is 1 0 [ωl−h(t)/t]pdt < ∞, where ωl−h is the modulus of continuity of the vector-function ∇l−hϕ, ϕ being the function in (9). 4 Multipliers in Sobolev spaces where ωl−h is the modulus of continuity of the vector-function ∇l−hϕ, ϕ being the function in (9). 4 Multipliers in Sobolev spaces Vladimir Maz’ya and Tatyana Shaposhnikova developed a deep theory of multipliers in spaces of differentiable functions. By a multiplier acting from one function space S1 into another S2, they mean a function which deﬁnes a bounded linear mapping of S1 into S2 by pointwise multiplication. In their theory, the role of the spaces S1 and S2 is played by Sobolev spaces, Bessel potential spaces, Besov spaces, and the like. In their book Theory of Sobolev Multipliers (the second very much enlarged edition appeared in 2009) they describe this theory by proving a lot of results, like characteriza- tion of multipliers, trace inequalities, relations between spaces of Sobolev multipliers and other function spaces, and so on. They provide also several applications to analysis, partial differential and integral equations. As they write in the Introduction of the book, they believe that the calculus of Sobolev multipliers provides an adequate language for future work in the theory of linear and nonlinear differential and pseudodifferential equations under minimal restrictions on the coefﬁcients, domains, and other data. Just to give an example of application of the theory of multiplers, let us consider elliptic boundary value problems in domains with “non-regular” boundaries. Let me introduce some notation. The space of multipliers acting from the Sobolev space W m p into Wl p (1 ≤p < ∞, m ≥l ≥0) is denoted by M(W m p →Wl p). Therefore saying that γ ∈M(W m p →Wl p) means that the pointwise moltiplication u →γ u deﬁnes a linear and continuos operator from W m p into Wl p. p p Let be an open subset of Rn and let P be the operator Pu = \|α\|,\|β\|≤h (−1)\|α\|Dα(aαβ(x) Dβu) where aαβ ∈Cl−h(), l ≥h. Let us suppose that the Gårding inequality Re \|α\|=\|β\|=h aαβ(x) DαuDβu dx ≥C ∥u∥2 W h 2 () holds for any u ∈C∞ 0 (). This implies that the equation Pu = f with f ∈W −h() is uniquely solvable in ˚W h(). holds for any u ∈C∞ 0 (). This implies that the equation Pu = f with f ∈W −h() is uniquely solvable in ˚W h(). 143 Page 10 of 14 143 A. 5 Lp-dissipativity of partial diﬀerential operators In a series of joint papers with Vladimir Maz’ya we have considered the problem of characterizing the L p-dissipativity of partial differential operators with complex coefﬁcients. I recall that a linear operator A deﬁned on D(A) ⊂L p() and with range in L p() is said to be L p-dissipative if Re ⟨Au, u⟩\|u\|p−2dx ≤0 r any u ∈D(A). Here is a domain in Rn and the functions u are complex valued. for any u ∈D(A). Here is a domain in Rn and the functions u are complex valued. Page 11 of 14 143 143 The scientiﬁc work of Vladimir Maz’ya One of our results gives a necessary and sufﬁcient condition for the L p-dissipativity of the partial differential operator with complex valued coefﬁcients Au = div(A ∇u) (10) (10) (ahk ∈L∞(), ⊂Rn, 1 < p < ∞). Speciﬁcally, under the assumption that Im A is symmetric (i.e. Im A t = Im A ), we have proved that A is L p-dissipative if and only if (ahk ∈L∞(), ⊂Rn, 1 < p < ∞). Speciﬁcally, under the assumption that Im A is symmetric (i.e. Im A t = Im A ), we have proved that A is L p-dissipative if and only if ), ⊂Rn, 1 < p < ∞). Speciﬁcally, under the assumption that Im A (i.e. Im A t = Im A ), we have proved that A is L p-dissipative if and \|p −2\| \|⟨Im A (x)ξ, ξ⟩\| ≤2 p −1 ⟨Re A (x)ξ, ξ⟩ (11) (11) for almost any x ∈, for any ξ ∈Rn. for almost any x ∈, for any ξ ∈Rn. for almost any x ∈, for any ξ ∈Rn. Examples show that if Im A is not symmetric or if there are lower order terms, this result is not true. In general condition (11) is only necessary. If Im A is symmetric, (11) is equivalent to the condition 4 p p′ ⟨Re A (x)ξ, ξ⟩+ ⟨Re A (x)η, η⟩−2(1 −2/p)⟨Im A (x)ξ, η⟩≥0 for almost any x ∈ and for any ξ, η ∈Rn. 5 Lp-dissipativity of partial diﬀerential operators (15) (15) The condition (15) is necessary for the L p-dissipativity of operator (14) in any dimension, even when the Poisson ratio is not constant. At the present it is not known if condition (15) is also sufﬁcient for the L p-dissipativity of elasticity operator for n > 2, in particular for n = 3. This is another open problem contained in the above mentioned collection “Seventy Five (Thousand) Unsolved Problems in Analysis and Partial Differential Equations”. Our results on L p-dissipativity can be found in the monograph Cialdea-Maz’ya, Semi-bounded Differential Operators, Contractive Semigroups and Beyond, Operator Theory: Advances and Applications, 243, Birkhäuser, Berlin (2014), where they are considered in the more general frame of semi-bounded operators. Very recently (Cialdea-Maz’ya, Criterion for the functional dissipativity of second order differential operators with complex coefﬁcients, to appear) we have introduced the more general concept of functional dissipativity of the operator (10) with respect to a certain function ϕ. Here ϕ is a positive function deﬁned on R+ such that s ϕ(s) is strictly increasing. Let us denote by the related Young function (t) = t 0 s ϕ(s) ds . If Re ⟨A ∇u, ∇(ϕ(\|u\|) u)⟩dx ≥0 for any u ∈ ˚H1() such that ϕ(\|u\|) u ∈ ˚H1(), we say that the operator A is functional dissipative or L-dissipative, in analogy with the terminology used when ϕ(t) = t p−2. for any u ∈ ˚H1() such that ϕ(\|u\|) u ∈ ˚H1(), we say that the operator A is functional dissipative or L-dissipative, in analogy with the terminology used when ϕ(t) = t p−2. ϕ We proved that, if Im A t = Im A , the operator A is L-dissipative if and only We proved that, if Im A t = Im A , the operator A is L-dissipative if and only if \|s ϕ′(s)\| \|⟨Im A (x) ξ, ξ⟩\| ≤2 ϕ(s) [s ϕ(s)]′ ⟨Re A (x) ξ, ξ⟩ (16) (16) for almost every x ∈ and for any s > 0, ξ ∈RN. for almost every x ∈ and for any s > 0, ξ ∈RN. As for L p-dissipativity, this condition leads to deﬁne a new class of operators, which we shall call -strongly elliptic. 5 Lp-dissipativity of partial diﬀerential operators More generally, if the matrix Im A is not symmetric, the condition 4 p p′ ⟨Re A (x)ξ, ξ⟩+⟨Re A (x)η, η⟩+2⟨(p−1 Im A (x)+ p′−1 Im A ∗(x))ξ, η⟩≥0 (12) for almost any x ∈ and for any ξ, η ∈Rn (p′ = p/(p −1)) is only sufﬁcient for the L p-dissipativity of A. 4 p p′ ⟨Re A (x)ξ, ξ⟩+⟨Re A (x)η, η⟩+2⟨(p−1 Im A (x)+ p′−1 Im A ∗(x))ξ, η⟩≥0 (12) for almost any x ∈ and for any ξ η ∈Rn (p′ = p/(p −1)) is only sufﬁcient for 4 p p′ ⟨Re A (x)ξ, ξ⟩+⟨Re A (x)η, η⟩+2⟨(p−1 Im A (x)+ p′−1 Im A ∗(x))ξ, η⟩≥0 (12) for almost any x ∈ and for any ξ, η ∈Rn (p′ = p/(p −1)) is only sufﬁcient for the L p-dissipativity of A. (12) Recently several authors have considered the class of operators such that the form (12) is not merely non-negative, but strictly positive, i.e. there exists κ > 0 such that 4 p p′ ⟨Re A (x)ξ, ξ⟩+ ⟨Re A (x)η, η⟩+ 2⟨(p−1 Im A (x) + p′−1 Im A ∗(x))ξ, η⟩ ≥κ(\|ξ\|2 + \|η\|2) (13) (13) for almost any x ∈ and for any ξ, η ∈Rn. These operators, which could be called p-strongly elliptic, are playing an increasingly important role in the study of differential operators with complex coefﬁcients, in particular in the study of boundary value problems with L p data. We remark that, if p = 2, condition (13) reduces to the classical strong ellipticity condition Re⟨A (x)ζ, ζ⟩≥κ\|ζ\|2 Re⟨A (x)ζ, ζ⟩≥κ\|ζ\|2 for almost any x ∈ and for any ζ ∈Cn. for almost any x ∈ and for any ζ ∈Cn. We have characterized the L p-dissipativity also for other classes of operators. Here I want just to mention a characterization of L p-dissipativity we have obtained for the the system of linear elasticity Eu = u + (1 −2ν)−1∇div u (14) (14) Page 12 of 14 143 A. Cialdea (ν being the Poisson ratio, ν > 1 or ν < 1/2). In the planar case we proved that the operator E is L p-dissipative if and only if (ν being the Poisson ratio, ν > 1 or ν < 1/2). In the planar case we proved that the operator E is L p-dissipative if and only if 1 2 −1 p 2 ≤2(ν −1)(2ν −1) (3 −4ν)2 . 6 History of mathematics In 1998 Vladimir Maz’ya and Tatyana Shaposhnikova published the huge volume “Jacques Hadamard. A Universal Mathematician”. It contains more than 500 pages and it was later translated in French and in Russian. It is both a biography and a description of Hadamard’s mathematics. The book is also full of photographs and pictures which make the reading extremely pleasant. The authors made an amazing job ﬁnding their sources. As Roger Cooke wrote in “Maz’ya’s work on the biography of Hadamard” (in Rossmann et al. (eds.), The Maz’ya anniversary collection (Birkhäuser Verlag, Basel, 1999)) although much of the relevantmaterialunfortunatelydisappearedduringWorldWarII,theywereabletoﬁnd a great deal of new material nevertheless. They located Hadamard’s grandson, Francis Picard and were able to look at the materials collected by Hadamard’s daughter Jacqueline, including her autobiography. They interviewed several mathematicians who had known Hadamard personally, and they submitted their drafts of the new and revised material to experts in a number of areas of mathematics and the history of mathematics,andthehistoryofFrenchmathematicsinparticular.Theresult,published in English this time, was a triumph of mathematical biography that one can only hope will be repeated for many other great twentieth-century mathematicians. …It will probably turn out to be the deﬁnitive biography of Hadamard. Another book written by Maz’ya and recently translated in russian, which gives an important contribution to the history of mathematics is “Differential Equations of My Young Years”. It is an autobiography which covers events from 1937 till 1968. The book is full of wonderful photographs. Reading this book means taking a dip in the past. It gives a precise description of the Soviet life in 40s-60s of the last century. Even if the book is full of mathematics, it can be read by anyone. As Havin writes in the foreword, the fact that mathematics appears on many of its pages in no way diminishes the book’s clarity of discourse and attraction to a variety of readers. y f y f In addition to more personal events from adolescence and early adulthood, we can read very interesting facts about the russian mathematics in that period. 5 Lp-dissipativity of partial diﬀerential operators They are the second order scalar operators such that [1 −2(t)]⟨Re A (x) ξ, ξ⟩+ ⟨Re A (x) η, η⟩ + [1 + (t)]⟨Im A (x) ξ, η⟩+ [1 −(t)]⟨Im A ∗(x) ξ, η⟩≥κ(\|ξ\|2 + \|η\|2) Page 13 of 14 The scientiﬁc work of Vladimir Maz’ya 143 for a certain κ > 0 and for almost every x ∈ and for any t > 0, ξ, η ∈RN. Here is the function deﬁned by the relation for a certain κ > 0 and for almost every x ∈ and for any t > 0, ξ, η ∈RN. Here is the function deﬁned by the relation s ϕ(s) = − s ϕ′(s) s ϕ′(s) + 2 ϕ(s) . 7 Other books Although Maz’ya’s books appearing in Fig. 2 do not concern his scientiﬁc activity, I think that they deserve a mention, because they are another proof of the great imagi- nation he has. 143 Page 14 of 14 143 Page 14 of 14 A. Cialdea A. Cialdea Fig. 2 Maz’ya’s fairy tales Fig. 2 Maz’ya’s fairy tales Fig. 2 Maz’ya’s fairy tales Fig. 2 Maz’ya’s fairy tales They are three books of fairy tales intended for middle school children. The heroes of these stories are animals and people, as well as space aliens and underground inhabitants. They are three books of fairy tales intended for middle school children. The heroes of these stories are animals and people, as well as space aliens and underground inhabitants. As Gohberg wrote in “Vladimir Maz’ya: friend and mathematician. Recollections.” (in Rossmann et al. (eds.), The Maz’ya anniversary collection (Birkhäuser Verlag, Basel, 1999)): whatever he writes is beautiful, his love for art, music and literature seeming to feed his mathematical aesthetic feeling. Funding Open access funding provided by Università degli Studi della Basilicata within the CRUI-CARE Agreement. Compliance with ethical standards Conﬂict of interest The author declares that there is no conﬂict of interest. Availability of data and materials Not applicable. The manuscript has no associated data. OpenAccess ThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published map and institutional afﬁliations.
https://openalex.org/W2096794521	https://europepmc.org/articles/pmc4077366?pdf=render	English	null	Atherton–Todd reaction: mechanism, scope and applications	Beilstein journal of organic chemistry	2,014	cc-by	17,048	Review Received: 04 January 2014 Accepted: 09 April 2014 Published: 21 May 2014 Keywords: amphiphiles; flame retardant; lipid conjugates; organophosphorus; phosphoramidate; phosphate © 2014 Le Corre et al; licensee Beilstein-Institut. License and terms: see end of document. Atherton–Todd reaction: mechanism, scope and applications Review Open Access Address: Université de Brest, Université Européenne de Bretagne, CEMCA, CNRS UMR 6521, SynNanoVect, IFR 148 ScInBIoS, 6 Avenue Le Gorgeu, 29238 Brest, France Email: Paul-Alain Jaffrès* - pjaffres@univ-brest.fr * Corresponding author Keywords: amphiphiles; flame retardant; lipid conjugates; organophosphorus; phosphoramidate; phosphate Beilstein J. Org. Chem. 2014, 10, 1166–1196. doi:10.3762/bjoc.10.117 Received: 04 January 2014 Accepted: 09 April 2014 Published: 21 May 2014 This article is part of the Thematic Series "Organophosphorus chemistry". Guest Editor: P. R. Hanson © 2014 Le Corre et al; licensee Beilstein-Institut. License and terms: see end of document. Abstract Initially, the Atherton–Todd (AT) reaction was applied for the synthesis of phosphoramidates by reacting dialkyl phosphite with a primary amine in the presence of carbon tetrachloride. These reaction conditions were subsequently modified with the aim to opti- mize them and the reaction was extended to different nucleophiles. The mechanism of this reaction led to controversial reports over the past years and is adequately discussed. We also present the scope of the AT reaction. Finally, we investigate the AT reaction by means of exemplary applications, which mainly concern three topics. First, we discuss the activation of a phenol group as a phos- phate which allows for subsequent transformations such as cross coupling and reduction. Next, we examine the AT reaction applied to produce fire retardant compounds. In the last section, we investigate the use of the AT reaction for the production of compounds employed for biological applications. The selected examples to illustrate the applications of the Atherton–Todd reaction mainly cover the past 15 years. Review 1. Introduction carbon tetrachloride by its treatment with an aqueous ammonia solution. They observed the formation of a precipitate that was identified as O,O-dibenzyl phosphoramidate. However, no reac- tion occurred when dibenzyl phosphite was mixed alone with carbon tetrachloride, whereas an exothermal reaction occurred if gaseous ammonia was passed through this solution or if this solution was shaken with an aqueous ammonia solution, with The reaction of dialkylphosphite with primary or secondary amines in the presence of a base in carbon tetrachloride produces phosphoramidates. This reaction, initially studied by F. R. Atherton, H. T. Openshawand, and A. R. Todd [1] in 1945 (Scheme 1) and now identified as the Atherton–Todd (AT) reaction, was actually discovered by chance. Indeed, these authors attempted to purify a solution of dibenzyl phosphite in 1166 1166 Beilstein J. Org. Chem. 2014, 10, 1166–1196. chlorophosphate 2 as a possible intermediate species. The pref- erence for mechanism 1 was justified by the existence of some similitude with the reactivity of carbon tetrachloride reported in literature (e.g., the synthesis of arylcarboxylate according to a Reimer–Tiemann reaction [3,4]) and because the second step (i.e., the nucleophilic substitution on the electrophilic phos- phorus) shares some characteristics with the reactivity of trichloroacetophenone (haloform reaction [5]). Moreover, the absence of the reactivity of alcohol when mixed with dialkyl phosphite and carbon tetrachloride in the presence of trialkyl- amine convinced the authors to prefer this first mechanism 1 (i). Nevertheless the same authors [2] revised their preference two years later and mentioned that the reaction may probably occur following mechanism 2 (Scheme 2-ii). This assumption was based on the observation that the replacement of carbon tetra- chloride by bromotrichloromethane enhanced the reaction rate. They explained this increase of reactivity by the easier nucleo- philic attack of the dialkylphosphite salt on the bromine atom of CBrCl3 when compared to the reaction with CCl4. It is also noteworthy, that the use of bromotrichloromethane allowed the phosphorylation of ethanol. The formation of tetrabenzyl pyrophosphate, observed by Atherton and Todd during the reac- tion that engaged benzyl phosphite, potassium hydroxide and carbon tetrachloride (or bromotrichloromethane), is also more easily explained by mechanism 2. Moreover, the impossibility to isolate the intermediate species in this reaction incited Atherton and Todd to prefer mechanism 2 (ii) because diethyl chlorophosphate 2 is a more reactive intermediate. chloroform and ammonium chloride as byproducts. Review 1. Introduction The replacement of ammonia with primary and secondary amines yielded N-substituted phosphoramidates (Scheme 1). It is note- worthy, that less nucleophilic amines like aniline can also be engaged in the AT reaction, but the expected phosphoramidates are only produced in modest yields if a tertiary amine is added to the reaction media. These findings were published in the initial works of Atherton and Todd and completed in 1947 [2]. Scheme 1: Pioneer works of Atherton, Openshaw and Todd reporting on the synthesis of phosphoramidate starting from dibenzylphosphite (adapted from [1]). Scheme 1: Pioneer works of Atherton, Openshaw and Todd reporting on the synthesis of phosphoramidate starting from dibenzylphosphite (adapted from [1]). To the best of our knowledge, we herein report the first compi- lation of the works which studied or used this reaction. First the question of the mechanism of the Atherton–Todd reaction, which has been widely discussed in literature, is addressed. Then, the scope of this reaction is presented. Finally selected applications of this reaction are discussed. This selection mainly covers the applications of the past 15 years. A special focus is put on the synthesis of flame-retardant materials and the design of phosphorus-based amphiphilic compounds. To the best of our knowledge, we herein report the first compi- lation of the works which studied or used this reaction. First the question of the mechanism of the Atherton–Todd reaction, which has been widely discussed in literature, is addressed. Then, the scope of this reaction is presented. Finally selected applications of this reaction are discussed. This selection mainly covers the applications of the past 15 years. A special focus is put on the synthesis of flame-retardant materials and the design of phosphorus-based amphiphilic compounds. After these pioneering works, the first investigation of the mechanism of the Atherton–Todd’s reaction was reported by Steinberg in 1950 [6]. In this work, the synthesis of dialkyl chlorophosphate 2 is reported by reacting dialkyl phosphite with carbon tetrachloride in the presence of 10 to 15 mol percent of trialkylamine acting as a catalyst. These authors proposed a more detailed mechanism for the formation of dialkyl chlorophosphate 2 with the suggestion of two distinct pathways (Scheme 3). One possibility included the nucleophilic attack of 2. Mechanism of the Atherton–Todd reaction [9] have reported that hexahy- droazepine (a secondary amine with a pKa of 11.1 (Scheme 3-iii), used as a base in the AT reaction) probably reacted first with carbontetrachloride to produce a charge- transfer complex (this type of interaction was confirmed by refractometric titration). However, in this reaction the trichloromethylphosphonate, which would result from the reac- tion of the anion CCl3− with chlorophosphate, was never observed because CCl3− probably reacted as a base in the pres- ence of dialkylphosphite (Scheme 3-ii). This experiment indi- cates that the basicity/nucleophilicity of the amine has an impact on the first step of the mechanism (a charge-transfer complex was not observed with a less basic amine like 2-aminopyridine) while the chlorophosphate 2 was assumed to be one common intermediate independent from the nature of the initial step. Almost 35 years after the work of Steinberg, Engel et al. [10] have re investigated the mechanism of the Atherton Todd reac Scheme 3: Two reaction pathways (i and ii) to produce chlorophos- phate 2. Charge-transfer complex observed when hexahydroazepine was used as a base (iii); adapted from [6] and [9]. attempts were never able to identify trace amounts of diethyl trichloromethylphosphonate (mechanism i, Scheme 2), thereby favoring mechanism 2 (Scheme 2). Additional experiments with the aim of discarding the involvement of a carbene species as an intermediate were carried out. The reactions achieved in cyclo- hexene (a solvent with the capacity to trap any traces of carbene) produced the same conversion rate. One exception to this last observation was found when the experimental condi- tions combined both the use of strict aprotic conditions (use of sodium hydride for the deprotonation of phosphite) and dimethyl phosphite as a substrate. This result might be rational- ized by the instability of trichloromethanide in aprotic media which in the presence of dimethyl phosphite or dimethyl chlorophosphate produced a significant amount of carbene. It must be mentioned that methyl esters of phosphate or phospho- nate have a particular reactivity since the methyl group can be easily removed by trimethylamine as illustrated by the dealkyla- tion of O,O-dimethyl phosphoramidate [11,12] or by the thermal sensitivity of dimethyl chlorophosphate as mentioned by Steinberg et al. [6]. However, no trace amount of carbene was detected when triethylamine was replaced with sodium hydride as a base, while all other parameters were identical. 2. Mechanism of the Atherton–Todd reaction 2. Mechanism of the Atherton–Todd reaction In their initial publication, Atherton and Todd have suggested two possible mechanisms to explain the formation of phosphor- amidate [1]. The first one (Scheme 2-i) was based on a two-step sequence with the formation of dialkyl trichloromethylphospho- nate 1 as the intermediate species. The second mechanism, which was not preferred at that time by these authors (Scheme 2-ii), was based on the formation of dialkyl Scheme 2: Mechanisms 1 (i) and 2 (ii) suggested by Atherton and Todd in 1945; adapted from [1]. Scheme 2: Mechanisms 1 (i) and 2 (ii) suggested by Atherton and Todd in 1945; adapted from [1]. 1167 1167 Beilstein J. Org. Chem. 2014, 10, 1166–1196. Scheme 3: Two reaction pathways (i and ii) to produce chlorophos- phate 2. Charge-transfer complex observed when hexahydroazepine was used as a base (iii); adapted from [6] and [9]. the deprotonated dialkyl phosphite on one chlorine atom of carbon tetrachloride (Scheme 3-i), whereas the second syn- thetic pathway (Scheme 3-ii) involved the nucleophilic attack of the base on carbon tetrachloride as a preliminary step. The kinetic data reported by Steinberg did not allow for discrimin- ation between these two possibilities. Beside the kinetic study, Steinberg accumulated further interesting information relative to the mechanism of the Atherton–Todd reaction. First, Stein- berg indicated that no reaction occurred when dialkyl trichloromethylphosphonate, prepared unambiguously by another method, was mixed with ammonia. This claim is in contradiction with one previous publication by Kamai [7,8], stipulating that phosphoramidate was produced by the reaction of dialkyl trichloromethylphosphonate and a selected amine. Steinberg has also shown that the structure of the amine has an influence on the rate of the reaction. Indeed, triethylamine is a much more efficient catalyst (1000 fold) than pyridine. Interest- ingly, tributylamine or tripentylamine catalyzed this reaction with the same rate than triethylamine. This last observation by Steinberg is in favor of the first mechanism (Scheme 3-i), since differences of the reaction rate should be expected for the nucleophilic addition of trialkylamine on carbon tetrachloride (Scheme 3-ii) depending on the structure of the alkyl chains. The works of Steinberg eliminated the possibility of a mecha- nism proceeding by radical processes. Indeed, the use of UV ir- radiation or radical initiators in the absence of trialkylamine was found to be unsuccessful to produce phosphoramidates. Recently, Krutikov et al. 2. Mechanism of the Atherton–Todd reaction These results indicated that the carbene pathway is unlikely to occur under the classical conditions of the Atherton–Todd reac- tion (dialkyl phosphite, trialkylamine, alkyl- or dialkylamine and carbon tetrachloride or bromotrichloromethane). Almost 35 years after the work of Steinberg, Engel et al. [10] have re-investigated the mechanism of the Atherton–Todd reac- tion, more specifically the first step (reaction of dialkyl phos- phite with carbon tetrachloride and a base) by using CPG as an analytical tool. Triethylamine or sodium hydride was used as base. With sodium hydride the deprotonation occurs in a preliminary step to produce sodium dialkyl phosphite. All these The use of dimethyl phosphite as a substrate in the AT reaction was also studied by Roundhill and co-workers in a series of arti- 1168 Beilstein J. Org. Chem. 2014, 10, 1166–1196. Beilstein J. Org. Chem. 2014, 10, 1166–1196. phite was considered in this study. It is indeed well-established that dimethyl phosphite has a singular reactivity when compared to others dialkyl phosphites [6,11]. Moreover, only unsubstituted amine (ammonia) and monosubstituted amine were considered in this theoretical study. cles. They investigated the role of the salt 3 (Scheme 4-i), which can be produced by reacting trialkylamine with dialkyl phosphite [13]. Indeed, it was previously reported that this salt can catalyze (2 mol percent) an AT reaction [14,15]. However, there is no evidence that this mechanism could have a general scope. Roundhill et al. [16] have first studied the consequences of the replacement of carbon tetrachloride by a member of the chlorofluorocarbon class of compounds. In this study, they observed that the introduction of a fluorine atom in place of a chlorine atom reduced the reactivity leading to the following relative reactivity: CCl4 > CFCl 3 > CF2Cl2 >> CHCl3. In this study, dimethyl phosphite was primarily used as a substrate, and cyclohexylamine as a nucleophilic amine. The formation of the salt 3 was observed when cyclohexylamine was added to dimethyl phosphite, thus pointing out the influence of the order of addition of the reactants. More recently, dialkyl trichloromethylphosphonate was again proposed as an intermediate species in a reaction producing dialkyl phosphate (Scheme 5-i) [18]. The authors postulated its formation on the basis of 31P NMR analysis. The 31P chemical shift reported for the intermediate in this study (−13 ppm) was not consistent with the chemical shift of dialkyl trichloromethylphosphonate reported in other studies (6.5 ppm [19,20]). 2. Mechanism of the Atherton–Todd reaction ntification of chloro- and bromophosphate as reaction intermediate (ii Scheme 5: Synthesis of dialkyl phosphate from dialkyl phosphite (i) and identification of chloro- and bromophosphate as reaction intermediate (ii) (adapted from [18] (i) and [22] (ii)). species present, which are water, ethanol and amine. Different experimental parameters listed below are in favor of the exis- tence of competitive reactions that could have crucial conse- quences on the issue of the reaction and on the nature of the intermediate species: i) the addition of the phosphorinane and trialkylamine was achieved at 0 °C (this should favor the forma- tion of the kinetic product). ii) Phosphorinane and trialkyl- amine were added dropwise on the primary amine (this is the best condition to have an excess of the nucleophilic amine versus the chlorophosphorinate intermediate and consequently to favor its addition on the chlorophosphate), iii) the low quan- tities of solvent used (this is also in favor of the addition of the nucleophilic amine, which is in competition with water and ethanol). reaction and ii) the direct use of chlorophosphate were evalu- ated for the derivatization of chiral amine (or alcohol) as reported in Scheme 6. For the AT reaction (i) [23] they used the conditions of Ji et al. [24], who have previously reported that the AT reaction can proceed in aqueous organic solvents (water/ ethanol or water/DMF). Accordingly, a mixture of phosphori- nane and CCl4 was added dropwise on a cooled (0 °C) solution of amino derivative and triethylamine in a water/ethanol mix- ture. For this reaction, the volume of the solvent mixture must be limited as mentioned in a review published by Feringa et al. [25]. The use of aqueous organic condition for the AT reaction led the authors to postulate trichloromethylphosphonate as an intermediate species because it is less sensitive to water when compared to chlorophosphate (Scheme 6) [26,27]. It must be noted that the intermediates were not characterized by NMR spectroscopy. Furthermore, we can hypothesize that when aqueous organic conditions are used, competitive reac- tions could take place on the chlorodioxaphosphorinane as an intermediate that would involve the different nucleophilic Moreover, these experimental details are also consistent with the reaction rate of chlorophosphate with nucleophiles reported by Corriu et al. [28]. 2. Mechanism of the Atherton–Todd reaction A 31P chemical shift at −13 ppm would be more likely attributed to a pyrophosphate (−12.6 ppm, R = Et) [1], whereas diethyl chlorophosphate is observed at 3.5 ppm [21]. The for- mation of pyrophosphate could be explained by the presence of trace amounts of water concomitantly with the absence of nucleophile species. Then, the addition of acetic acid produced a mixed anhydride as suggested by the authors (Scheme 5-i). In another recent study, chloro- and bromophosphate intermedi- ates were characterized by 31P NMR thus supporting the mech- anism 2 (Scheme 2). In this study, Döring et al. reported that the shielding effect of bromine was also correlated to its higher re- activity (Scheme 5-ii) [22]. Roundhill et al. [17] used computational chemistry to further investigate the mechanism of the Atherton–Todd reaction (HF- 6.31G* level of theory and Moller–Plesset (MP2) to correct correlation effects). They found that the calculation supported the mechanism shown in Scheme 4 that starts with the dealkyla- tion of dimethyl phosphite by an amine (Scheme 4-i). Then, this salt acted as a base to deprotonate dimethyl phosphite (Scheme 4-ii), which subsequently reacted with CCl4 to produce chlorophosphate 2 as an intermediate species. In the last step, the salt 3 is regenerated by deprotonation with trialkyl- amine (Scheme 4-v). Nevertheless, the scope of this theoretical study may be regarded as limited, since only dimethyl phos- Feringa et al. developed a methodology for the determination of the enantiomeric excess of a chiral amine based on the use of phosphorinanes derivatives. Two synthetic pathways i) the AT Scheme 4: Mechanism of the Atherton–Todd reaction with dimethylphosphite according to Roundhill et al. (adapted from [13] and [17]). Scheme 4: Mechanism of the Atherton–Todd reaction with dimethylphosphite according to Roundhill et al. (adapted from [13] and [17]). 1169 Beilstein J. Org. Chem. 2014, 10, 1166–1196. Scheme 5: Synthesis of dialkyl phosphate from dialkyl phosphite (i) and identification of chloro- and bromophosphate as reaction intermediate (ii) (adapted from [18] (i) and [22] (ii)). Scheme 5: Synthesis of dialkyl phosphate from dialkyl phosphite (i) and identification of chloro- and bromophosphate as reaction intermediate (ii) (adapted from [18] (i) and [22] (ii)). ynthesis of dialkyl phosphate from dialkyl phosphite (i) and identification of chloro- and bromophosphate as reaction intermediate (ii) m [18] (i) and [22] (ii)). 2. Mechanism of the Atherton–Todd reaction Indeed, they have shown that the second- order rate constants for the solvolysis of diethyl chlorophos- phate with different nucleophiles including water, ethanol, Scheme 6: Synthesis of chiral phosphoramidate with trichloromethylphosphonate as the suggested intermediate (i) and directly from a chlorophos- phate used as a substrate (ii) (adapted from [25-27]). Scheme 6: Synthesis of chiral phosphoramidate with trichloromethylphosphonate as the suggested intermediate (i) and directly from a chlorophos- phate used as a substrate (ii) (adapted from [25-27]). 1170 Beilstein J. Org. Chem. 2014, 10, 1166–1196. phenol and diethylamine were 0.35·10−4, 0.12·10−5, 0.38·10−3 and 0.28·10−1 L mol−1s−1, respectively. These data do not take into account the effect of an organic base present in the reac- tion media of an AT reaction. It reveals, however, that the sec- ondary amine is much more reactive than phenol, water and ethanol in a reaction with chlorophosphate. Consequently, the existence of the trichloromethylphosphonate as an intermediate species in the experiments of Feringa et al. must be considered with caution because several experimental details are in favor of the existence of a competitive process that could, actually, be in favor of the addition of the nucleophilic amine on chlorophos- phorinane as intermediate. propyl methylphosphonite 7-1 (Scheme 7-i) as a substrate. This substrate was purified by distillation with no racemization. However, rapid racemization was observed when 7-1 was treated with sodium methoxide in methanol. The use of 7-1 in the AT reaction in the presence of CCl4, tributylamine and aniline as a nucleophile produced 7-2 with full stereocontrol (note: due to the substitution of a hydrogen atom by an aniline moiety, the numbering used to determine the chirality at the phosphorus atom was changed; consequently, the (R) configur- ation is preserved in the course of this reaction). The authors also observed a racemization (Scheme 7-ii) when the same reac- tion was achieved without the addition of a nucleophile (aniline in the present case). The use of similar reaction conditions (CCl4, NEt3) led Mikolajczyk et al. [30] to observe the forma- tion of a mixture of diastereoisomers due to a racemization at the phosphorus atom. However, this racemization was not always observed which points out the different stability of diastereoisomeric species (cyclic phosphite in the present case). All the studies reported above shed some light on the mecha- nism of the AT reaction. None of these studies, however, focused on the chirality at the phosphorus atom. 2. Mechanism of the Atherton–Todd reaction The first investigation of the stereochemistry of the AT reaction was reported by Reiff and Aaron [29]. They used enantiopure O-iso- Scheme 7: Selection of results that address the question of the stereochemistry of the AT reaction (adapted from [29-32]). Scheme 7: Selection of results that address the question of the stereochemistry of the AT reaction (adapted from [29-32]). 1171 Beilstein J. Org. Chem. 2014, 10, 1166–1196. Other conditions of chlorination such as the use of N-chlorosuc- cinimide or, as reported more recently, CuCl2 produced, in the latter case, 7-6 with full stereocontrol (Scheme 7-iii) as unam- biguously determined by X-ray diffraction analysis [31]. The addition of nucleophilic species (amine, alcohol, alkyllithium, etc.) on 7-6 or other chlorophosphonites was stereocontrolled with the inversion of the configuration at the phosphorus atom. In another publication it is reported on the AT reaction starting from enantiopure (S)-phenyl tert-butyl phosphinous acid 7-5 (Scheme 7 iv) [32]. In that case the final product 7-7 was isolated with full stereocontrol. This result indicates that both steps (i.e., the chlorination and subsequently the substitution of the chlorine atom with the nucleophile) were stereocontrolled. The detailed mechanism of each step, which may involve penta- coordinated phosphorus intermediates [28], is not well estab- lished. From a stereochemical point of view, these results can be summarized as shown in Scheme 7-v. cases, the authors concluded that the first step (i.e., the forma- tion of a phosphoryl chloride intermediate) was achieved with retention of configuration (Scheme 7-v). Then, a subsequent attack of the nucleophilic substrate (amine, alcohol or thio- phenol) occurred at the opposite side of the phosphorus–chlo- rine bond to afford the substitution product with high stereo- specificity and an inversion of configuration. Zhao et al. have also investigated the stereochemistry of the AT reaction by using chiral valine hydrospirophosphorane as a sub- strate and phenol derivatives as nucleophiles [34]. The mecha- nism described in their publication suggested the formation of a chlorinated spirophosphorane species as an intermediate, which retains the configuration at the phosphorus center. This hypoth- esis is strongly supported by X-ray diffraction analysis of a single crystal structure of the P–Cl intermediate species (Scheme 8). Thereafter, the reaction with phenol proceeds with an inversion of the configuration after a nucleophilic attack at the opposite side of the phosphorus–chlorine bond. Other recent studies have reported stereocontrolled AT reac- tions. Han et al. 2. Mechanism of the Atherton–Todd reaction [33] described the preparation of optically active organophosphorus acid derivatives from menthyl-based H-phosphinates (or secondary phosphine oxides) and a nucleo- philic species (amines or alcohols) under AT reaction condi- tions. The reaction proceeded with a full stereoinversion at the phosphorus atom and led to the isolation of optically pure P–N coupling products with nearly quantitative yields (94%). The chemical structure and the stereochemistry of the products were unambiguously established by X-ray diffraction analysis. It is noteworthy that such coupling conditions have been extended to a wide variety of substrates including nitro-, methoxy-, trifluo- romethylphenol or thiophenol with an almost similar reactivity. However, an exception was made when considering the reac- tion with aliphatic thiols (e.g., n-alkylthiols) [33]. In all studied In conclusion, all studies which have investigated the mecha- nism of the Atherton–Todd reaction resulted in a summary of the likely reaction mechanism in Scheme 9. This mechanism seems to be general, but a different mechanism can take place when dimethyl phosphite is used due to its reaction with amine as shown in Scheme 4. On the basis of all these results, the hypothesis of the formation of dialkyl trichloromethylphospho- nate as an intermediate of the Atherton–Todd reaction must be discarded. 3. Scope and synthetic conditions CCl4 was the first solvent used in AT reactions. It plays a double role since it also acts as a halogenating agent that reacts Scheme 8: Synthesis of phenoxy spirophosphorane by the AT reaction (adapted from [34]). Scheme 8: Synthesis of phenoxy spirophosphorane by the AT reaction (adapted from [34]). 1172 Beilstein J. Org. Chem. 2014, 10, 1166–1196. Scheme 9: Suggested mechanism of the Atherton–Todd reaction, (i) and (ii) formation of chlorophosphate with a hindered or nucleophilic base; (iii) nucleophilic substitution at the phosphorus center with stereoinversion. Scheme 9: Suggested mechanism of the Atherton–Todd reaction, (i) and (ii) formation of chlorophosphate with a hindered or nucleophilic base; (iii) nucleophilic substitution at the phosphorus center with stereoinversion. is employed. However, there are also a few reports on AT reac- tions in water–organic solvent mixtures when reactive nucleo- philic species were used (primary amine) and under biphasic conditions with a phase-transfer agent as shown in Scheme 10 [38]. In this study, water and CCl4 were used to produce the biphasic system. NaOH is the base and benzyltriethylammo- nium bromide acted as a phase-transfer agent. This procedure was applied to the synthesis of N-arylphosphoramidate. It was observed that the classical AT reaction (CCl4, base, anhydrous solvent) applied to ortho-substituted anilines was not successful. However, the use of formanilide (R = H) or chloroacetanilide (R = CH2Cl) as a substrate (Scheme 10) produced the expected phosphoramidate by a mechanism which simultaneously involved deacetylation. The higher reactivity of formanilide and chloroacetanilide, when compared to aniline, was explained by the higher acidity of the remaining N–H bond. But the isolated yields were usually modest (50–70%) and only occasionally high (85%). With respect to the reactivity of chlorophosphate with water, Strawinski et al [21] reported that chlorophosphate can be placed in THF with 0.5 equiv of water without any trace of degradation but the addition of a base immediately gives rise to the formation of pyrophosphate (31P NMR around −12 ppm alkyl pyrophosphate and −25 ppm tetraaryl pyrophosphate) and with phosphite to produce chlorophosphate as an intermediate. However, many other solvents, including dichloromethane, chloroform, diethyl ether [35], THF [31], acetonitrile [34], DMF [36] and toluene [37] were also used. In that case a stoi- chiometric amount of the halogenating agent (CCl4, CBrCl3) is usually employed. Among these solvents, those that are not miscible with water are usually a good choice (e.g., CH2Cl2, CHCl3). 3. Scope and synthetic conditions At several occasions, alcohol (methanol, ethanol) was also used as a solvent even though it may compete with the nucleophilic species. This choice was made when the reagents were poorly soluble in other solvents (e.g., reagents like ammo- nium chloride salts). When this type of solvent is employed, the addition order of reagents can influence the result. Indeed, it is better to add CCl4 (or CBrCl3) and the tertiary amine at the end to be sure that the nucleophile (e.g., an amine) will be present when the chloro- or bromophosphate derivatives will be formed in situ. The mixture of reagents is usually achieved at 0–5 °C. Then, an additional stirring period of a few minutes to a few hours at rt (20 °C) is applied. The use of an anhydrous solvent is usually preferred in order to avoid the hydrolysis of chlorophos- phate to pyrophosphate or phosphate. It is also possible to add molecular sieves (4 Å) to the reaction medium to remove traces of water, which can be useful when a poor nucleophilic species Scheme 10: AT reaction in biphasic conditions (adapted from [38]). Scheme 10: AT reaction in biphasic conditions (adapted from [38]). 1173 Beilstein J. Org. Chem. 2014, 10, 1166–1196. to phosphate in the presence of additional water (31P NMR dialkyl phosphate 0.4 ppm and diaryl phosphate −9.6 ppm). With the use of a biphasic system it is likely that the reaction takes place in the CCl4 phase because all the reagents, except the hydroxide anion, are soluble in CCl4. The hydroxide anion is actually transferred to the organic phase thanks to the phase- transfer agent. [37]. As shown in Scheme 11, gaseous anhydrous ammonia was passed through a solution of diethyl phosphite and iodoform in toluene to produce the expected phosphoramidate in good yield (83%). In an independent experiment the authors have identi- fied the iodophosphate (31P at −41.0 ppm), a compound which decomposes when stored for a few hours at rt. With respect to the base used for the AT reaction, it can be noticed that a trialkylamine is generally the best choice. This amine is frequently triethylamine or diisopropylethylamine (Hünig’s base, DIPEA). 3. Scope and synthetic conditions With the AT reaction, the order of addition of reagents can impact its efficacy. Usually, the phosphite or the halide source (CBrCl3) is added dropwise to a mixture of the nucleophile (amine, phenol), trialkylamine (e.g., DIPEA) and a solvent cooled at −10 °C to 5 °C. According to this procedure, the chlorophosphate formed as an intermediate immediately reacts with the nucleophile already present in the reaction medium. It is noteworthy that another synthetic procedure consists to firstly prepare the chlorophosphate and then add the nucleophilic species (Scheme 13) [41]. Following this procedure, chlorophosphate was first prepared at a low temperature (−10 °C) by reacting dibenzyl phosphite with CCl4 in the presence of DIPEA and a catalytic amount of DMAP. Then, the nucleophile (in that case a phenol) is added dropwise to the chlorophosphate solution at −10 °C to produce, after purification, the triphosphate in 68% yield. Generally, the first protocol is preferred to avoid any hydrolysis of chlorophos- phate, which could be explained by the presence of trace amounts of water. aniline can be used. However, ortho-substituted aniline reacts with more difficulties according to Lukanov et al. [38]. Indeed the aniline must be activated as a sulfonamide (or acetamide) to produce the expected phosphoramidate [42]. The authors, who used a phase-transfer agent, postulated that this activation resulted from the increase of the acidity of the N–H bond despite the evident reduction of the nucleophilic character (Scheme 14). To further illustrate AT reactions with aniline, the work of Dumitrascu et al. [43] is worth mentioning. Recently, they have reported on the preparation of an aryl phosphoramidate with a styryl moiety from the corresponding aniline (Scheme 15). The phosphoramidate was isolated with an excellent yield (90%), and this product was subsequently used to prepare polymers. Other nitrogen-based nucleophiles were also engaged in AT reactions. Hydrazine is a substrate which produces phosphor- amidate in high yield. The first example, reported by Prokof’eva et al. [44], used arylhydrazine as a substrate. The reaction proceeded in CCl4 in the presence of triethylamine and produced phosphoramidates in 60–82% yield. Unsubstituted hydrazine can also be used as a nucleophile in the AT reaction [45-47]. The best synthetic conditions employed phase-transfer catalysis [48]. Accordingly, CCl4 was used as a solvent or co-solvent, triethylbenzylammonium chloride acts as a phase- transfer agent, and K2CO3 was used as a base. Hydrazine hydrate was the source of hydrazine. 3. Scope and synthetic conditions It must be noted that the use of this hindered base (DIPEA) could favor the mechanism detailed in Scheme 3 instead of a mechanism that would involve a nucleo- philic attack of the amine on the alkyl chain of the phosphite (Scheme 4). Some authors add catalytic quantities of dimethyl- aminopyridine (DMAP). For instance, this procedure was used to produce arylphosphate by reacting phenol with dibenzyl phosphite [40]. The presence of DMAP and limited excess of CCl4 (5 equiv) produced the expected aryl phosphate in excel- lent yield even at a low temperature (−10 °C). Interestingly, this study illustrates that the AT reaction is chemoselective since only the phenol reacts even in the presence of primary or sec- ondary alcohols (Scheme 12). Different halogenating agents were used to produce in situ chlorophosphate from phosphite. CCl4 was used systematically for a long period of time. However, Atherton and Todd [2] have shown that reaction rates were increased by using CBrCl3. This last halogenating agent is likely the reagent of choice when a stoichiometric amount or a slight excess is employed. Other halogenating agents were studied for the AT reaction. CBr4 [39] is also able to produce reactive bromophosphate as an inter- mediate species. However, it is more expensive compared to CBrCl3 and, to the best of our knowledge, better results with the use of CBr4 have not been published yet. The AT reaction was also reported with the use of trichloroacetonitrile as a halide source [35]. Accordingly, the chlorophosphate was formed in the presence of triethylamine. Iodoform (CHI3) is another reagent that can be used as a halide source for the AT reaction Scheme 11: AT reaction with iodoform as halide source (adapted from [37]). Scheme 11: AT reaction with iodoform as halide source (adapted from [37]). Scheme 12: AT reaction with phenol at low temperature in the presence of DMAP (adapted from [40]). Scheme 11: AT reaction with iodoform as halide source (adapted from [37]). Scheme 11: AT reaction with iodoform as halide source (adapted from [37]). me 11: AT reaction with iodoform as halide source (adapted from [37 Scheme 12: AT reaction with phenol at low temperature in the presence of DMAP (adapted from [40]). Scheme 12: AT reaction with phenol at low temperature in the presence of DMAP (adapted from [40]). 1174 Beilstein J. Org. Chem. 2014, 10, 1166–1196. 3. Scope and synthetic conditions Accordingly, a series of phosphory- lated O-alkylhydroxylamine was produced in medium to good yield (45–97%) [51]. The phosphorylation of phenol by the AT reaction was also reported under biphasic conditions in the presence of a phase- transfer agent. Interestingly, the study of Ilia et al. [57], reports the use of both liquid/liquid and liquid/solid biphasic systems. They observed that the second reaction conditions (i.e., the ones without the addition of water) produced better results with yields between 72 and 86%. In this reaction NaOH or K2CO3 was used as a base, tetrabutylammonium bromide as a phase- transfer agent and CCl4 as a reagent and solvent. Finally, enolate is another nucleophilic species that was engaged in the AT reaction as shown in Scheme 18. In this reaction, an allenylketone was mixed with NaH in THF at −10 °C. After deprotonation (30 min), diethyl phosphite in CCl4 was added dropwise. Treatment with acetic acid and purification on silica gel finally afforded the β-alkynyl-enolphosphate in 61–75% yield [58]. The use of alcohol as a nucleophile has been rarely reported indicating that its nucleophilicity is not good enough to produce the expected phosphate in good yield. This reaction was observed when CBrCl3 or CBr4 were used as a halide source according to the initial works of Atherton and Todd [1,2]. Nevertheless, this reaction is limited and successful only with an alcohol such as methanol or ethanol. In our group we have occasionally employed methanol as a solvent for selected reac- tions due to the low solubility of certain substrates (e.g., amino acid hydrochlorides). In that case, a noticeable amount of diethyl methyl phosphate was observed originating from the reaction of methanol on the chlorophosphate intermediate. Because the AT reaction is not efficient to produce alkylphos- phate from alkylalcohol, an alternative is the usage of chlorophosphate in the presence of a Lewis acid catalyst. For this purpose, Ti(t-BuO)4 was identified as an effective catalyst [52,53]. The use of phenol as a nucleophilic species resulted in better yields. Hence, the AT reaction applied to a substrate possessing both alcohol and phenol functional groups, produces the phosphate that engaged the phenol function as shown in Scheme 12. AT reactions with phenol are further illustrated by the work of Charette et al. 3. Scope and synthetic conditions The expected diethoxyphosphinylhydrazine was isolated in almost quantitat- ive yield (99%) after a purification step by liquid/liquid extrac- tion (Scheme 16). This procedure was also recently used by As indicated above, a tertiary amine is the preferred base for the AT reaction. However, primary or secondary amines can be also used when they simultaneously act as a nucleophile and a base. In that case, two equivalents of amine must be added (Scheme 11). The use of NaOH as a base was also reported (exemplified in Scheme 10) in a protocol involving phase- transfer catalysis. Initially, the nucleophile engaged in the AT reaction was a pri- mary or secondary alkylamine. However, the use of other nucleophilic species was reported. First, as illustrated above, Scheme 13: Synthesis of a triphosphate by the AT reaction starting with the preparation of chlorophosphate (adapted from [41]). Scheme 13: Synthesis of a triphosphate by the AT reaction starting with the preparation of chlorophosphate (adapted from [41]). Scheme 13: Synthesis of a triphosphate by the AT reaction starting with the preparation of chlorophosphate (adapted from [41]). Scheme 14: AT reaction with sulfonamide (adapted from [42]). Scheme 14: AT reaction with sulfonamide (adapted from [42]). 1175 Beilstein J. Org. Chem. 2014, 10, 1166–1196. Scheme 15: Synthesis of a styrylphosphoramidate starting from the corresponding aniline (adapted from [43]). Scheme 15: Synthesis of a styrylphosphoramidate starting from the corresponding aniline (adapted from [43]). Scheme 16: Use of hydrazine as nucleophile in AT reactions (adapted from [48]). Scheme 16: Use of hydrazine as nucleophile in AT reactions (adapted from [48]). [54]. Another interesting example, reported by Selikhov et al., involved an AT reaction between a functionalized coumarine and dioleyl phosphite [55]. In this reaction the authors used a mixture of a solvent (CH3CN/CHCl3) and an excess of CCl4. DIPEA and a catalytic amount of DMAP were also added in the reaction medium. The expected phosphate was isolated with 67% yield (Scheme 17-i). Better yields are usually obtained with phosphite possessing two shorter alkyl chains as exempli- fied by a reaction reported by Taylor et al (Scheme 17-ii) [56]. Matthews et al. to produce the same compound (diethoxyphos- phinylhydrazine) in a slightly lower yield (90%) [49]. O-Methoxyhydroxylamine is another nitrogen-based nucleo- phile engaged in AT reactions. The first example reported by Wadsworth et al. produced low yield [50]. The procedure was improved by the addition of a phase-transfer agent (triethylben- zylammonium chloride). 3. Scope and synthetic conditions who used this reaction in the first step of a multistep synthesis to produce chiral arylphospholane Azide, nitrile and thiocyanate were three other nucleophilic species used to produce pseudohalogenated phosphorus species by the AT reaction. Among them, the commercially available diphenyl phosphorazidate (DPPA) and diethyl phosphoro- cyanidate (DEPC) are widely employed as peptide coupling reagents [59-62]. DPPA was also used in many organic reac- 1176 Beilstein J. Org. Chem. 2014, 10, 1166–1196. Scheme 17: AT reaction with phenol as a nucleophilic species; synthesis of dioleyl phosphate-substituted coumarine derivative (i) and synthesis of diethyl aryl phosphate (ii) (adapted from [55] and [56]). Scheme 17: AT reaction with phenol as a nucleophilic species; synthesis of dioleyl phosphate-substituted coumarine derivative (i) and synthesis of diethyl aryl phosphate (ii) (adapted from [55] and [56]). Scheme 18: Synthesis of β-alkynyl-enolphosphate from allenylketone with AT reaction (adapted from [58]). phase-transfer catalysis. The catalyst was either TBAB (tetra- butylammonium bromide), TBAI (tetrabutylammonium iodide), TMAC (tetramethylammonium chloride) or 18-crown-6. The best result was obtained with TBAB and 18-crown-6 catalysts in a CH2Cl2/water or AcOEt/water mixture (yields included from 72 to 85%). Scheme 18: Synthesis of β-alkynyl-enolphosphate from allenylketone with AT reaction (adapted from [58]). Scheme 19: Synthesis of pseudohalide phosphate by using AT reac- tion (adapted from [67]). tions such as the Curtius rearrangement [59], the thiol ester syn- thesis [63], the azidation of alcohol and phenol [64], and the synthesis of phosphoramidate [65]. The most common way to prepare pseudo-halogenated phosphorus species involves a nucleophilic substitution on chlorophosphate with sodium salts (NaN3, NaCN or NaSCN) [66]. Due to the reactivity of chlorophosphate and its difficulty of storage, its production in situ by the AT reaction was studied by Shi et al. as depicted in Scheme 19 [67]. (RO)2P(O)Cl is generated from phosphite (RO)2P(O)H, CCl4 and a catalytic amount of Et3N. This chlorophosphate is immediately engaged in a reaction with a sodium salt NaX (X = N3, CN or NCS) to afford the pseudo- halide phosphorus species in satisfactory yields (77–88%). Interestingly, the same authors reported that pseudo-halide phosphate can also be obtained by using phase-transfer catal- ysis [68]. Accordingly, NaX (X = N3, CN or NCS) reacts with dialkyl phosphate in water–organic solvent mixtures under tions such as the Curtius rearrangement [59], the thiol ester syn- thesis [63], the azidation of alcohol and phenol [64], and the synthesis of phosphoramidate [65]. 3. Scope and synthetic conditions The most common way to prepare pseudo-halogenated phosphorus species involves a nucleophilic substitution on chlorophosphate with sodium salts (NaN3, NaCN or NaSCN) [66]. Due to the reactivity of chlorophosphate and its difficulty of storage, its production in situ by the AT reaction was studied by Shi et al. as depicted in Scheme 19 [67]. (RO)2P(O)Cl is generated from phosphite (RO)2P(O)H, CCl4 and a catalytic amount of Et3N. This chlorophosphate is immediately engaged in a reaction with a sodium salt NaX (X = N3, CN or NCS) to afford the pseudo- halide phosphorus species in satisfactory yields (77–88%). Interestingly, the same authors reported that pseudo-halide phosphate can also be obtained by using phase-transfer catal- ysis [68]. Accordingly, NaX (X = N3, CN or NCS) reacts with dialkyl phosphate in water–organic solvent mixtures under Scheme 19: Synthesis of pseudohalide phosphate by using AT reac- tion (adapted from [67]). The carbamate anion is another nucleophilic species recently engaged in the AT reaction with hydrospirophosphorane as sub- strate [69]. This nucleophilic species was generated in situ by reacting a secondary amine and CO2. Then, it reacted with chlorospirophosphorane produced in situ from hydrospirophos- phorane, CCl4 and Cs2CO3 (Scheme 20). This reaction can be viewed as an activation of CO2 by an amine to produce the nucleophilic carbamoyl moiety. The major product of this reac- tion corresponds to an inversion of the configuration at the phosphorus with diastereoisomeric excess included from 6.8 to 1177 Beilstein J. Org. Chem. 2014, 10, 1166–1196. Scheme 20: AT reaction with hydrospirophosphorane with insertion of CO2 in the product (adapted from [69]). Scheme 20: AT reaction with hydrospirophosphorane with insertion of CO2 in the product (adapted from [69]). Phosphinous acid (R2POH) was less extensively studied as a phosphorus-based substrate in AT reactions. However, one full study was reported by Bondarenko et al. [72]. These authors reported three different methods to produce phosphinic amides by reacting primary or secondary amines with phosphinous acid in the presence of CCl4 and aqueous NaOH. The first protocol was applied when methylamine or ammonia was used as a nucleophile (Scheme 23-i). In that case, a 50% aqueous solu- tion of NaOH was slowly added to a biphasic solution composed of aqueous methylamine, CCl4 and dichloromethane. 3. Scope and synthetic conditions e use of secondary phosphineselenoxide was also reported by same authors but the yields were lower (35–38%) [74]. The cedure was also applied to alcohol [75] or diphenol deriva- es acting as a nucleophile as shown for hydroquinone in Scheme 25: Use of H-phosphonothioate in the AT reaction (adapted from [78]). Scheme 25: Use of H-phosphonothioate in the AT reaction (adapted from [78]). Scheme 23: Use of phosphinous acid in AT reactions (adapted from [72]). engaged as a phosphorus-based precursor in the AT reaction [73]. The reaction proceeds in good yield (80%) with CCl4 as a solvent, an amine as a nucleophile and triethylamine as a base. The use of secondary phosphineselenoxide was also reported by the same authors but the yields were lower (35–38%) [74]. The procedure was also applied to alcohol [75] or diphenol deriva- tives acting as a nucleophile as shown for hydroquinone in Scheme 24 [76]. O,O-dialkyl thiophosphite was also engaged in AT reactions to produce dinucleotide designed as an anti-HIV prodrug [77]. Scheme 25: Use of H-phosphonothioate in the AT reaction (adapted from [78]). reported by Beletskaya et al. [79]. This activation was applied to AT reactions which used α-aminophosphonate as a nucleo- phile in the presence of CCl4 and triethylamine. The authors reported that no reaction occured at rt, whereas phosphor- amidate was obtained in low yield (15–20%) when classical heating (24 h, 110 °C) was applied. However, phosphorami- dates were isolated in 63–93% yields with MW heating. The reaction time was significantly shorter with MW since a full conversion was reached after only 30 to 40 minutes of heating. H-phosphonothioates are intermediates considered for the syn- thesis of nucleotide analogues. This functional group can be used as a nucleophilic species in a coupling reaction with a pri- mary alcohol. This coupling reaction is usually achieved by I2 acting as an oxidant, but Stawinski et al. also tested other halide sources including CCl4 and CBr4 [78]. Despite iodine is the reagent of choice in this reaction, CBr4 is also efficient to produce the coupling product (Scheme 25), whereas CCl4 was inefficient. The recent works of Hayes et al. reported on the synthesis of phosphoramidates starting from dialkyl phosphite and primary amine without a halogen source [80]. Instead of using CCl4 or CBr4, the authors employed a catalytic amount of CuI as depicted in Scheme 26. 3. Scope and synthetic conditions The second protocol (Scheme 23-ii) is almost similar to the first one except that a hydrochloride salt (e.g., ethylamine hydrochloride) was used as a substrate without prior solubiliza- tion in water. Finally, for the third protocol (Scheme 23-iii), the phosphinous acid is added to a mixture of the other substrates placed in a biphasic solution (water/CH2Cl2). The yields were between 77 to 97%. 1. It is noteworthy that the usage of a primary amine as an educt in this reaction did not lead to the insertion of CO2 , and the product resulted from the reaction of the amine on the chlorospirophosphorane. This result may be explained by the higher nucleophilicity of the primary amine in this reaction media. The phosphorus species engaged in AT reactions mainly include dialkyl phosphites. It must be noted that the alkyl-chain length can be changed and extended to lipid chains as exempli- fied in Scheme 17 (oleyl chains). Diaryl phosphite can also be used for AT reactions as illustrated in a recent example published by Gaan et al. (Scheme 21) [70]. O-Alkyl phosphonite can also be engaged in AT reactions as recently illustrated by Montchamp et al. in a study dedicated to the hydrophosphinylation of terminal alkenes (Scheme 22) [71]. Shi et al. have also reported the reactivity of O-alkyl arylphos- phonite in AT reactions with azide as the nucleophilic species [67]. Thiophosphorus-based precursors can also be engaged in AT reactions. Recently, secondary phosphinethiooxide has been Scheme 21: AT reaction with diaryl phosphite (adapted from [70]). Scheme 21: AT reaction with diaryl phosphite (adapted from [70]). Scheme 22: AT reaction with O-alkyl phosphonite (adapted from [71]). Scheme 21: AT reaction with diaryl phosphite (adapted from [70]). Scheme 21: AT reaction with diaryl phosphite (adapted from [70]). Scheme 22: AT reaction with O-alkyl phosphonite (adapted from [71]). Scheme 22: AT reaction with O-alkyl phosphonite (adapted from [71]). 1178 Beilstein J. Org. Chem. 2014, 10, 1166–1196. Scheme 23: Use of phosphinous acid in AT reactions (adapted from [72]). Scheme 23: Use of phosphinous acid in AT reactions (adapted from [72]). Scheme 23: Use of phosphinous acid in AT reactions (adapted from [72]). cheme 23: Use of phosphinous acid in AT reactions (adapted from [72]). gaged as a phosphorus-based precursor in the AT reaction ]. The reaction proceeds in good yield (80%) with CCl4 as a vent, an amine as a nucleophile and triethylamine as a base. 3. Scope and synthetic conditions These new synthetic conditions, that required O2 for the reoxidation of copper, can be viewed as an AT reactions usually proceed at 0–5 °C but lower temperatures (−10 °C [41]) and higher temperatures (50°C [76]) were also reported. The use of microwave (MW) activation was also Scheme 24: AT reaction with secondary phosphinethiooxide (adapted from [76]). Scheme 24: AT reaction with secondary phosphinethiooxide (adapted from [76]). 1179 Beilstein J. Org. Chem. 2014, 10, 1166–1196. related deoxygenated derivatives represent an important family of natural or synthetic compounds exhibiting, for example, anti- tumor or antiallergic activities. The reduction of a phenol group implies its activation as a good leaving group. Phosphate is one of the possible activating groups. Its use for the desoxygenation of phenol was first reported by Kenner et al. [81] who imple- mented the reaction with liquid ammonia as a solvent in the presence of metal as a reducing agent (e.g., Na) (Scheme 27-i). A similar methodology was used to produce 2-substituted bornane in a three-step sequence as shown in Scheme 27-ii [82]. Firstly, a Friedel–Crafts reaction produced the para-substituted phenol in 42% yield. Then, the phenol group was transformed in diethyl aryl phosphate with an AT reaction in 99% yield. Finally, the reduction of the phosphate was achieved in 74% yield. Similar synthetic schemes (AT reaction and reduction with Li/NH3) were also reported in another publication [83]. This procedure was recently improved by Lusch et al. (Scheme 27-iii) by using a lithium di-tert-butylbiphenylide radical anion [84]. Accordingly, a wide panel of phenol deriva- tives was first transformed in diethyl aryl phosphate with the AT reaction (yields from 76 to 96%), and then these phos- phates were reduced to the corresponding aromatic hydrocar- bons with moderate to good yields (10 to 83%). extension of the AT reaction which facilitates the avoidance of a halide source. Even though the yield is usually lower compared to classical AT conditions, this result opens new perspectives for the development of green processes. Scheme 26: AT-like reaction with CuI as catalyst and without halide source (adapted from [80]). Scheme 26: AT-like reaction with CuI as catalyst and without halide source (adapted from [80]). 4.1 Synthetic utilities of AT reactions In this section we will discuss the use of AT reactions to produce aryl phosphates or phosphoramidates which are impor- tant intermediates for a variety of subsequent transformations or can be used as organocatalysts. Aryl dialkyl phosphates, prepared by the AT reaction from phenol derivatives, were employed to reduce phenol functional groups. It is noteworthy that phenol compounds and their The phosphoramidate group can also be used to enhance the re- activity of an amine. This activation role of phosphoramidates Scheme 27: Reduction of phenols after activation as phosphate derivatives (adapted from [81] i ; [82], ii; and [83], iii). Scheme 27: Reduction of phenols after activation as phosphate derivatives (adapted from [81] i ; [82], ii; and [83], iii). Scheme 27: Reduction of phenols after activation as phosphate derivatives (adapted from [81] i ; [82], ii; and [83], iii). 1180 Beilstein J. Org. Chem. 2014, 10, 1166–1196. shown in Scheme 29. This example shows that the AT reaction was achieved with CCl4 as a halogenating agent. Then, the stan- nylation reaction produced the arylstannane in 75 to 90% yield [88]. was illustrated for the synthesis of medium and large-sized cyclic diaza-compounds by Zhao et al. [85]. This sequence started with the AT reaction (Scheme 28) to produce ω-difunc- tionalized phosphoramidatecarboxylic acid. In the last step, the phosphoramidate group activates the amine which is cyclized by a copper-catalyzed N-arylation. In the last step, the phos- phoramidate is cleaved by an acidic treatment. This sequence illustrates that the AT reaction is compatible with the presence of free carboxylic acid functional group. The Suzuki–Miyaura cross-coupling reaction is another type of reaction with the phosphate group acting as a leaving group. As exemplified in Scheme 30, the aryl dialkyl phosphate was produced in good yield by an AT reaction [89]. Then, this phos- phate group reacted with aryl Grignard, as a Kumada–Tamao- Corriu cross-coupling, in the presence of a nickel catalyst [90]. Aryl dialkyl phosphate, easily prepared starting from phenol in an AT reaction, can also be transformed into arylstannane derivatives by a SRN1 reaction by the photostimulation of trialkyl [86] or triarylstannyl [87] ion in liquid ammonia as Aryl dialkyl phosphate, which was readily obtained by the AT reaction from phenol as shown in Scheme 31 [91], can be Scheme 28: Synthesis of medium and large-sized nitrogen-containing heterocycles (adapted from [85]). 4.1 Synthetic utilities of AT reactions Scheme 28: Synthesis of medium and large-sized nitrogen-containing heterocycles (adapted from [85]). Scheme 29: Synthesis of arylstannane from aryl phosphate prepared by an AT reaction (adapted from [86]). Scheme 29: Synthesis of arylstannane from aryl phosphate prepared by an AT reaction (adapted from [86]). Scheme 30: Synthesis and use of aryl dialkyl phosphate for the synthesis of biaryl derivatives (adapted from [89]). Scheme 30: Synthesis and use of aryl dialkyl phosphate for the synthesis of biaryl derivatives (adapted from [89]). 1181 Beilstein J. Org. Chem. 2014, 10, 1166–1196. Scheme 31: Synthesis of aryl dialkyl phosphate by an AT reaction from phenol and subsequent rearrangement yielding arylphosphonate (adapted from [91]). Scheme 31: Synthesis of aryl dialkyl phosphate by an AT reaction from phenol and subsequent rearrangement yielding arylphosphonate (adapted from [91]) Scheme 31: Synthesis of aryl dialkyl phosphate by an AT reaction from phenol and subsequent rearrangement yielding arylphosphonate (adapted from [91]). Beside the use of phosphorus species as a substrate in reactions as those reported above, phosphoramidates, prepared by AT reactions, can also act as an organocatalyst. Indeed, with the strongly polarized P–O bond on one hand, and the P–N or P–NH bond on the other hand phosphoramides are good Lewis bases [96-98]. Hexamethylphosphoric triamide (HMPA) (or analogues) was the first phosphoramide derivative that was extensively studied as an organocatalyst [98,99]. However, HMPA was classified as a human carcinogen [100]. One of the first examples of the use of chiral phosphoramide ligands (Scheme 32-i) in organocatalysis was described by Denmark et al. who studied the enantioselective crossed aldol reaction of employed for the production of aryl phosphonate by applying a phospho-Fries rearrangement (a rearrangement initially reported by Melvin et al. [92]). This rearrangement proceeds by an ortho-metallation that can be characterized at a low temperature [93]. Then, the o-lithium species rearranges to produce 2-hydroxyarylphosphonate [94]. The recent publication of Yang et al. [95] reports the amination of either triaryl phosphate or dialkyl aryl phosphate catalyzed by nickel organometallic complexes. It is an additional illustra- tion of the use of aryl phosphate, which can be readily obtained by AT reactions. Scheme 32: Selected chiral phosphoramidates used as organocatalyst; i) chiral phosphoramidate used in the pioneer works of Denmark et al. [101]; ii, iii) synthesis of organocatalysts by using AT reaction (adapted from [107]). 4.2 Flame retardants aldehydes with trichlorosilyl enol ethers. They obtained the aldol products in high yields with moderate to good enantio- selectivities [101]. The chiral phosphoramidates used in this study and tested in many other enantioselective reactions (aldol reaction [102], Michael addition [103], Diels–Alder reaction [104], Friedel–Crafts alkylation [105]) illustrate the use of phosphoramidates as organocatalysts. These phosphoramidates were not synthesized by an AT reaction. Instead, the reaction of an amine with an electrophilic phosphorus species (P–Cl based compounds) afforded the phosphoramidates. Only a few exam- ples (shown in Scheme 32-ii,iii) of chiral phosphoramidates were synthesized with the AT reaction likely due to the commercial accessibility of some chlorophosphine or chlorophosphate that render the AT reaction pathway less attractive. However, as reported in Scheme 32-ii, some chiral phosphoramidates and thiophosphoramidates can be readily synthesized with the AT reaction in high yield (85–88%) from dimethyl phosphite or O,O-dimethyl thiophosphite [106]. When the chiral amine was functionalized with a thiol group (Scheme 32-iii), the expected phosphoramidate was jointly isolated with 5 to 10% of thiophosphoramidate resulting from a cyclization reaction. The chiral phosphoramidates (Scheme 32-ii and iii) were tested as a chiral catalyst for the nucleophilic addition of diethylzinc [107] on benzaldehyde or for the asymmetric borane reduction of a prochiral ketone. The phosphoramidate (Scheme 32-iii) was the most efficient catalyst for these two reactions (ee: 95–98%, conversion 87–98%). Since the early age of the development of synthetic fibers, the production of flame retardants became an industrial and acad- emic challenge aiming to identify efficient compounds for this purpose but also to elucidate the mechanisms involved in the burning process of polymers. This goal was also extended, for evident safety reasons, to natural fibres (e.g., cotton). Halogen- based flame retardants were extensively employed (e.g., OctaBDE) but for safety reasons that pointed out carcinogenic risks and/or to the production of toxic fumes when burning (e.g., HBr) several of these halogenated flame retardants were withdrawn from the market (e.g., octaBDE) [109]. Phosphorus- based compounds [110] are another class of flame retardants. This type of compounds contributes to the extinction of the flame by, at least, two distinct mechanisms. First, some phos- phorus compounds (e.g., DOPO, Scheme 34) act on the gas phase by the production of non-flammable compounds (e.g., water) or by the production of reactive species that act as hydroxyl radical scavengers. 4.1 Synthetic utilities of AT reactions Scheme 32: Selected chiral phosphoramidates used as organocatalyst; i) chiral phosphoramidate used in the pioneer works of Denmark et al. [101]; ii, iii) synthesis of organocatalysts by using AT reaction (adapted from [107]). Scheme 32: Selected chiral phosphoramidates used as organocatalyst; i) chiral phosphoramidate used in the pioneer works of Denmark et al. [101]; ii, iii) synthesis of organocatalysts by using AT reaction (adapted from [107]). 1182 Beilstein J. Org. Chem. 2014, 10, 1166–1196. 4.2 Flame retardants Alternatively, phosphorus-based flame retardants may act on the solid phase by forming a thermal barrier between the solid phase and the gaseous phase. This charring process results from the formation of polyphos- phoric acid derivatives. Interestingly, the association of phos- phorus and nitrogen-based flame retardants proved to be effi- cient probably due to synergic effects. Melamine polyphos- phate (MPP, Scheme 34), illustrates this possibility: melamine decomposes endothermically and produces NH3 when burning (a gas-phase active agent), while polyphosphate, which is simultaneously produced, favors the charring process. The synergic effects of phosphorus and nitrogen-based flame retar- dants led scientists to investigate the synthesis of organic com- pounds characterized by the presence of these two elements. Accordingly, phosphoramidates constitute an interesting family of potential flame retardants and the AT reaction is an efficient tool for the production of this type of compounds. In relation with asymmetric synthesis, the determination of the enantiomeric excess (ee) is usually achieved by different methodologies including chiral HPLC or CPG. In a recent study, Montchamp et al. used the AT reaction for the determin- ation of the ee of P-chiral H-phosphinates by the formation of diastereoisomers (Scheme 33) [108]. The ee determined by this method, which can be achieved directly in the NMR tube before recording 31P NMR spectra, was consistent with those deter- mined by other methods (e.g., chiral HPLC). Phosphoramidate derivatives were investigated as flame retar- dants for many years [111,112]. However, some recent studies, Scheme 33: Determination of ee of H-phosphinate by the application of the AT reaction with a chiral amine (adapted from [108]). 1183 Beilstein J. Org. Chem. 2014, 10, 1166–1196. Scheme 34: Chemical structure of selected flame retardants synthesized by AT reactions; (BDE: polybrominated diphenyl ether; DOPO: 9,10- dihydro-9-oxa-10-phosphaphenanthrene-10-oxide; DEPA: diethyl phosphoramidate; PAHEDE: phosphoramidic acid-N-(2-hydroxyethyl) diethyl ester; EHP: diethyl 3-hydroxypropylphosphoramidate; MHP: dimethyl 3-hydroxypropylphosphoramidate; PAEDBTEE: phosphoramidic acid-1,2- ethanediylbis-tetraethyl ester; PAPDBTEE: phosphoramidic acid-1,4-piperazinediyl tetraethyl ester; DMAPR: dimethyl allylphosphoramidate; DPAPR: diphenyl allylphosphoramidate; DMAPR: dimethyl diallylphosphoramidate). AT reactions; (BDE: polybrominated diphenyl ether; DOPO: 9,10- Scheme 34: Chemical structure of selected flame retardants synthesized by AT reactions; (BDE: polybrominated diphenyl ether; DOPO: 9,10- dihydro-9-oxa-10-phosphaphenanthrene-10-oxide; DEPA: diethyl phosphoramidate; PAHEDE: phosphoramidic acid-N-(2-hydroxyethyl) diethyl ester; EHP: diethyl 3-hydroxypropylphosphoramidate; MHP: dimethyl 3-hydroxypropylphosphoramidate; PAEDBTEE: phosphoramidic acid-1,2- ethanediylbis-tetraethyl ester; PAPDBTEE: phosphoramidic acid-1,4-piperazinediyl tetraethyl ester; DMAPR: dimethyl allylphosphoramidate; DPAPR: diphenyl allylphosphoramidate; DMAPR: dimethyl diallylphosphoramidate). groups (MHP) was more efficient [115]. 4.2 Flame retardants The mechanism of de- gradation states that the MHP produced covalent bonds with cotton cellulose while the diethyl analogue (EHP) did not produce any such bonds. In this study, MHP was produced by an AT reaction as reported for PAHEDE [113]. For the syn- thesis of MHP, the aminoalcohol engaged in the AT reaction exhibited complete chemoselectivity since no trace amount of phosphate was reported. Some bis(phosphoramidates) were also considered as flame retardants. PAEDBTAEE and PAPBDTEE (Scheme 34) were compatible with cotton acetate and enhance the formation of char [116]. These two bis(phosphoramidates) were produced at a 0.1 mol scale (31 g of pure PAEDBTEE) with the AT reaction carried out in THF with a stoichiometric amount of CCl4 (0.2 mol) and diethylphosphite (0.2 mol). Yields greater than 95% were reported for these crystalline solids. which will be discussed below, have reinvestigated the use of this type of compounds. We focus on the synthetic procedures of phosphoramidates. These derivatives can be divided in two broad categories depending on the presence of polymerizable groups. When polymerizable groups are present, the fire-resis- tant molecule can be included in polymers by copolymerization or can act as a crosslinking agent. This type of compounds is also employed for the surface modification of fibers or poly- mers (e.g., plasma technique). Without a polymerizable group, phosphoramidates can be used as an additive in polymers. Gaan et al. [113] have studied the thermal decomposition of cotton cellulose treated with different phosphoramidates. More specifi- cally, they have shown that a secondary phosphoramidate (e.g., PAHEDE) was more efficient than a primary phosphoramidate (DEPA; Scheme 34). Additional results reported by Rupper et al. [114] indicated that PAHEDE produced phosphoric acid moieties at the surface of cellulose after burning. Moreover, cellulose interacts with phosphoramides to produce C–O–P bonds. In these studies, secondary phosphoramidate (e.g., PAHEDE) was synthesized by reacting diethyl phosphite with ethanolamine in CCl4 and with one equivalent of triethylamine. After filtration and concentration, the phosphoramidate was isolated in quantitative yield after distillation. It is noteworthy that the synthesis is readily achieved and can be applied to large-scale productions. 4.2 Flame retardants More recently, the comparison of the flame-retardant properties of MHP and EHP (Scheme 34), led to the conclusion that phosphoramidate with two methoxy Other studies reported the modification of DOPO (a flame retar- dant in epoxy resins, Scheme 35-i) with the aim to react the P–H bond (phosphinate) to incorporate either nitrogen or oxygenated groups. Attempts to produce P–N bonds by using the AT reaction with diethanolamine (DEolA) failed because no chemoselectivity was observed between the secondary amine and the primary alcohol [117], whereas in other studies involving dialkyl phosphite a chemoselectivity was observed [107]. It can therefore be concluded that the phosphinate modi- fies the reactivity of the phosphorus group leading to this 1184 Beilstein J. Org. Chem. 2014, 10, 1166–1196. Scheme 35: Transformation of DOPO (i) and synthesis of polyphosphonate (ii) by the AT reaction (adapted from [117] and [118]). Scheme 35: Transformation of DOPO (i) and synthesis of polyphosphonate (ii) by the AT reaction (adapted from [117] and [118]). and in vivo transfection assays. For the synthesis of cationic lipids, simple, efficient, modular procedures must be developed because the current applications require multifunctional carrying systems. Indeed, many nano-carriers are designed to respond to a specific stimulus (pH, red/ox, light) to trigger the drug release. To achieve the synthesis of polyfunctional amphiphilic compounds, the synthetic scheme must therefore be efficient, flexible and versatile. The AT reaction, which was used as a key step for the synthesis of neutral or cationic lipids, matches several of these properties. As a first example, cationic lipophosphoramidates can be produced in a three-step sequence that includes an AT reaction (Scheme 36). First, dioleyl phos- phite can be readily prepared by a transesterification-like reac- tion between diphenyl phosphite and oleylalcohol. This reac- tion tolerates a large panel of lipid alcohol. These phosphite intermediates can be produced on a large scale (more than 50 g) and can be stored for a long period of time without any degrad- ation in contrast to dialkyl chlorophosphate (another possible absence of selectivity. This surprising reactivity of DOPO with alcohol functional groups inspired the authors to produce cyclic phosphonates with the AT reaction. Accordingly, polymer P1, obtained by polycondensation was functionalized by an AT reaction to produce polymer P2 in 65% yield (Scheme 35-ii) [118]. 4.2 Flame retardants All these recent studies, which report flame-retardant prop- erties of phosphorus-based compounds illustrate that the AT reaction is easy to implement and, interestingly, high yields are usually obtained. Accordingly, phosphoramidates are cheap to manufacture and we assume that this reaction will be employed to design novel flame retardants. 4.3. Phosphorus-based amphiphiles and biological applications Cationic lipids are a promising class of compounds with the capacity to carry nucleic acids (DNA, RNA) for both in vitro 1185 Beilstein J. Org. Chem. 2014, 10, 1166–1196. Scheme 36: Synthesis of lipophosphite (bisoleyl phosphite) and cationic lipophosphoramidate with an AT reaction (adapted from [119]). Scheme 36: Synthesis of lipophosphite (bisoleyl phosphite) and cationic lipophosphoramidate with an AT reaction (adapted from [119]). groups from the nucleic acids. The replacement of trimethylam- monium with either trimethylphosphonium or trimethylarso- nium offers better transfection efficacies probably owing to a better compromise between the stability and the instability of lipoplexes [120]. The synthesis of trimethylarsonium-based phosphoramidate was also achieved by using the AT reaction which proved to be an efficient tool to link the lipid part with the polar head region of the cationic lipid as exemplified in Scheme 37. With the same synthetic scheme, guanidinum-based lipophosphoramidates [121] obtained from natural amino acids, methylimidazolium [122], spermine-based amphiphile [123], dicationic lipophosphoramides [119] and arsonium or phospho- nium [120] cationic lipids were synthesized. intermediate to produce amphiphilic compounds), which are water sensitive. In a second step, these phosphites can be engaged in the AT reaction as illustrated in Scheme 36. Bis- oleyl phosphite was reacted with dimethylaminopropylamine to produce the expected phosphoramidate in good yield (80%) [119]. The reaction is carried out by adding DIPEA to a mix- ture of dioleyl phosphite, the nucleophilic amine and CBrCl3 in anhydrous dichloromethane. This addition was conducted at 5 °C followed by stirring the solution at 5 °C for 1 h. The concentration and extraction with a low polar solvent (hexane, diethyl ether), produced the expected phosphoramidate in almost quantitative yield. Purification was then achieved by column chromatography. In the last step, the cationic lipid was produced by reacting the tertiary amine functional group with iodomethane. Again, a simple washing (e.g., Et2O/water) produced the expected cationic phosphoramidate with good purity and in good yield (>80%). The lipid domain of lipophosphoramidates also influences the transfection process by acting on the physic-chemical prop- erties of the supramolecular aggregates. Indeed, it was observed that bisphytanyl derivative BSV18 was particularly efficient for in vivo experiments. Presumably, this efficacy is based on the formation of an inverted hexagonal phase [124]. 4.3. Phosphorus-based amphiphiles and biological applications Once more, the AT reaction is a versatile reaction that allows for the produc- tion of a large panel of cationic lipids with unsaturated (e.g., KLN47 [125,126]), polyunsaturated (e.g., BSV4 [127]) or substituted alkyl chains (BSV18 [124]) as shown in Scheme 38. In this sequence (Scheme 36) the AT reaction occupies a central place and with some adaptations a series of cationic lipids were synthesized, each of which characterized by different polar heads. These cationic lipids interact with nucleic acids mainly by electrostatic interactions, while the hydrophobic domains help to produce supramolecular aggregates (lipoplexes). Ideally, these aggregates must be as strong as possible to protect and to compact nucleic acids when these lipoplexes are localized in the systemic circulation (in vivo experiments) or in the supernatant media (in vitro experiments). However, after cell internaliza- tion, presumably by endocytosis processes, these lipoplexes must be as fragile as possible in order to escape lysosomal de- gradation. Consequently, the stability of the lipoplexes must be carefully tuned. The nature of the cationic polar head is one of the molecular features of the cationic lipids which directly influence the stability of lipoplexes by acting on the strength of the ionic interactions with the anion charge of the phosphate In association with cationic lipids, helper lipids are frequently added to liposomal solutions with the aim to enhance transfec- tion efficacies. DOPE (1,2-dioleyl-sn-glycero-3-phospho- ethanolamine) is a natural phospholipid that was frequently employed as a helper lipid, since it favors the formation of a hexagonal phase at a pH of 6 [128,129]. In addition to natural helper lipids synthetic co-lipids were also developed. Neutral lipophosphoramidates were synthesized by AT reactions (Scheme 39) [130]. These helper lipids were obtained in a one- step reaction with 29–30% yield from dioleyl phosphite. The 1186 Beilstein J. Org. Chem. 2014, 10, 1166–1196. Scheme 37: Use of AT reactions to produce cationic lipids characterized by a trimethylphosphonium, trimethylarsonium, guanidinium and methylimi- dazolium polar head. Scheme 37: Use of AT reactions to produce cationic lipids characterized by a trimethylphosphonium, trimethylarsonium, guanidinium and methylimi- dazolium polar head. Scheme 37: Use of AT reactions to produce cationic lipids characterized by a trimethylphosphonium, trimethylarsonium, guanidinium and methylimi- dazolium polar head. Scheme 38: Cationic lipid synthesized by the AT reaction illustrating the variation of the structure of the lipid domain. The arrows indicate the bond formed by the AT reaction. 4.3. Phosphorus-based amphiphiles and biological applications Scheme 38: Cationic lipid synthesized by the AT reaction illustrating the variation of the structure of the lipid domain. The arrows indicate the bond formed by the AT reaction. Scheme 38: Cationic lipid synthesized by the AT reaction illustrating the variation of the structure of the lipid domain. The arrows indicate the bond formed by the AT reaction. 1187 Beilstein J. Org. Chem. 2014, 10, 1166–1196. Scheme 39: Helper lipids for nucleic acid delivery synthesized with the AT reaction (adapted from [130]). Scheme 39: Helper lipids for nucleic acid delivery synthesized with the AT reaction (adapted from [130]). lipids were destabilized in the presence of a reducing agent presumably caused by the breakdown of the S–S bond. lipids were destabilized in the presence of a reducing agent presumably caused by the breakdown of the S–S bond. low yield was explained by the low solubility of the amine, which imposed the use of methanol as a solvent. Consequently, methanol acts as a second nucleophilic agent competing with the amine (histidine methyl ester or histamine). These yields can be improved (up to 50%) by reducing the volume of methanol used as a solvent in this reaction [131]. Interestingly, the liposomal solutions which incorporated the histamine-based helper lipid were also efficiently used to prepare lipopolyplexes (association of cationic lipid, cationic polymers and nucleic acid) employed for pDNA [132], RNA [133] and siRNA delivery [134]. low yield was explained by the low solubility of the amine, which imposed the use of methanol as a solvent. Consequently, methanol acts as a second nucleophilic agent competing with the amine (histidine methyl ester or histamine). These yields can be improved (up to 50%) by reducing the volume of methanol used as a solvent in this reaction [131]. Interestingly, the liposomal solutions which incorporated the histamine-based helper lipid were also efficiently used to prepare lipopolyplexes (association of cationic lipid, cationic polymers and nucleic acid) employed for pDNA [132], RNA [133] and siRNA delivery [134]. The introduction of a lipid domain on a molecule exhibiting specific properties (e.g., fluorescent or targeting group) is another goal needed to design tools for vectorization purposes. The use of click reactions for the production of polyfunctional amphiphiles (e.g., Huisgen cycloaddition) is very attractive. The combination of the AT reaction and click reaction (CuAAC) was reported to produce fluorescent lipids. 4.3. Phosphorus-based amphiphiles and biological applications The ‘clickable’ lipids (N3 or alkyne-functionalized phosphoramidates) were obtained by an AT reaction (Scheme 41-i). These intermediates were isolated with moderate to good yields (67–92%) at a 1 g scale after a purification step on silica gel [136]. These inter- mediates were then engaged in copper-catalyzed Huisgen cyclo- addition to produce efficiently a series of fluorescent lipids like those shown in Scheme 41-ii. The question of the destabilization of lipoplexes after cell inter- nalization can also be addressed by designing red/ox-sensitive cationic lipids. Accordingly, the incorporation of a disulfide bond, which can be cleaved by reducing agents naturally present in cytosol (glutathione), can induce a destabilization of the supramolecular aggregates. The AT reaction was also used for the synthesis of such red/ox-sensitive amphiphiles as illus- trated in Scheme 40 [135]. The synthesis of lipophosphites incorporating two disulfide moieties was a key step. These phosphites were then engaged in the AT reaction to produce ammonium (BSV76) or phosphonium (BSV42 and BSV69) red/ ox-sensitive cationic lipids in one or two steps. It was subse- quently shown that lipoplexes prepared from these cationic Recently, Le Gall et al. [137] have reported that some lipophos- phoramidates synthesized by AT reactions exhibited a remark- able bactericidal effect even on clinically relevant strains (S. aureus N315). Interestingly, the bactericidal effect was inde- pendent of the resistance profile of bacteria (e.g., MRSA). In a structure–activity study it has been shown that the presence of a trimethylarsonium polar head combined with a lipid domain 1188 Beilstein J. Org. Chem. 2014, 10, 1166–1196. Scheme 40: AT reaction used to produce red/ox-sensitive cationic lipids (adapted from [135]). Scheme 40: AT reaction used to produce red/ox-sensitive cationic lipids (adapted from [135]). Scheme 40: AT reaction used to produce red/ox-sensitive cationic lipids (adapted from [135]). Scheme 41: Alkyne and azide-functionalized phosphoramidate synthesized by AT reactions,(i); illustration of some fluorescent lipids synthesised from these intermediates, (ii) (adapted from [136]). Scheme 41: Alkyne and azide-functionalized phosphoramidate synthesized by AT reactions,(i); illustration of some fluorescent lipids synthesised from these intermediates, (ii) (adapted from [136]). Scheme 41: Alkyne and azide-functionalized phosphoramidate synthesized by AT reactions,(i); illustration of some fluorescent lipids synthesised from these intermediates, (ii) (adapted from [136]). 1189 Beilstein J. Org. Chem. 2014, 10, 1166–1196. have a toxic effect on bacteria (bactericidal action) and a trans- fection capability for eukaryotic cells [137]. were two structural features deeply influencing the bactericidal efficacies. 4.3. Phosphorus-based amphiphiles and biological applications The most efficient bactericidal agents were BSV77 and BSV4 (Scheme 42). Moreover, it was shown that lipoplexes, formed by the association of pDNA with BSV4, kept its bactericidal action. Additional experiments demon- strated that BSV4-based lipoplexes were able to simultaneously β-Cyclodextrin (β-CD) is another molecular platform that was chemically modified with a lipid moiety introduced by an AT reaction [138]. As exemplified in Scheme 43, Djedaïni-Pilard et Scheme 42: Cationic lipids exhibiting bactericidal action – arrows indicate the bond formed by the AT reaction (adapted from [137]). Scheme 42: Cationic lipids exhibiting bactericidal action – arrows indicate the bond formed by the AT reaction (adapted from [137]). Scheme 42: Cationic lipids exhibiting bactericidal action – arrows indicate the bond formed by the AT reaction (adapted from [137]). Scheme 43: β-Cyclodextrin-based lipophosphoramidates (adapted from [138]). Scheme 43: β-Cyclodextrin-based lipophosphoramidates (adapted from [138]). Scheme 43: β-Cyclodextrin-based lipophosphoramidates (adapted from [138]). 1190 Beilstein J. Org. Chem. 2014, 10, 1166–1196. merization of 4-methyl-2-oxo-2-hydro-1,3,2-dioxaphopsholane with triisopropylaluminium (Scheme 44) [139]. Then, the AT reaction was carried out in a mixture of DMF/CCl4 with doubly protected spermidine (protection with trifluoroacetamide groups) as a nucleophile to produce a polyphosphoramidate. The deprotection of the primary amine with NH3 yielded the polyphosphoramidate branched with polyamine, which was subsequently used as a gene carrier. The functionalization of the polyphosphite with the AT reaction was compatible with a large panel of primary and secondary amines [140]. The post-func- tionalization of this polyphosphoramidate with monosaccharide or disaccharide groups has also been reported as a better trans- fection agent, especially for hepatocytes [141]. al. reported the use of the AT reaction to introduce a lipophos- phoramidate fragment on a permethylated β-cyclodextrin possessing one primary amine (Scheme 43-i). After purifica- tion on silica gel, the expected lipophosphoramidate was isolated with 35% yield. The use of a spacer placed between the lipophosphoramide and the β-CD moiety (Scheme 43-ii) produced another permethylated β-CD with a better yield (66%). It is noteworthy that for this last reaction, an excess of CBrCl3 and DIPEA was used, which might explain the better yield. Finally, the same reaction achieved on non-methylated β-CD produced the lipophosphoramidate with very low yield (4%). It is probable that the alcohol functions and the residual water molecules compete as nucleophilic species in the AT reaction. Chitosan is another type of polymer functionalized with an AT reaction as shown in Scheme 45 [142]. 4.3. Phosphorus-based amphiphiles and biological applications The primary alcohol functions of chitosan were first protected with a trityl group. Then, the protected chitosan was engaged in an AT reaction that The AT reaction was also employed for the functionalization of polymeric materials that were subsequently used as a gene carrier. A polyphosphite was synthesised by ring-opening poly- Scheme 44: Polyphosphate functionalized by an AT reaction (adapted from [139]). Scheme 44: Polyphosphate functionalized by an AT reaction (adapted from [139]). Scheme 44: Polyphosphate functionalized by an AT reaction (adapted from [139]). Scheme 44: Polyphosphate functionalized by an AT reaction (adapted from [139]). Scheme 45: Synthesis of zwitterionic phosphocholine-bound chitosan (adapted from [142]). Scheme 45: Synthesis of zwitterionic phosphocholine-bound chitosan (adapted from [142]). Scheme 45: Synthesis of zwitterionic phosphocholine-bound chitosan (adapted from [142]). 1191 Beilstein J. Org. Chem. 2014, 10, 1166–1196. or CBCl3 instead of using them as a solvent. AT reactions can be used by chemists as a tool to activate phenol or amine func- tional groups, which may subsequently be engaged in reactions such as cyclization, reduction, cross coupling, the production of organometallic species or for the synthesis of arylphosphonates. Beside these synthetic applications, phosphoramidates or phos- phates produced by AT reactions can be used for a variety of applications including organocatalysis, improvement of the fire resistance of polymers, prodrugs, and vectorization purposes. Among the recent published works, we would like to point out two promising domains, in which the AT reaction seems excep- tionally attractive and worthwhile to be studied in depth. First, the current developments of vectorization systems aiming at being applied in the realm of personalized medicine require the synthesis of amphiphilic derivatives with several functionalities, e.g., fluorescent moieties, targeting groups, and PEG fragments, to produce stealthy nanoparticles. The accessibility of phos- phite with two lipid chains renders the AT reaction very attrac- tive for the incorporation of hydrophobic domains and thus provides a synthetic path to functionalized amphiphilic com- pounds. The second domain concerns the activation of small molecules like CO2 as recently illustrated by Y. F. Zhao et al. In these works, hydrospirophosphorane reacts with CO2 and a sec- ondary amine to produce a phosphorylated carbamate deriva- tive. Currently, the hydrospirophosphorane is used stoichiomet- rically. The development of a catalytic system characterized by hydrospirophosphorane or analogues acting as organocatalyst represents an alternative to organometallic catalysis in the field of CO2-based chemistry. 4.3. Phosphorus-based amphiphiles and biological applications used a mixture of solvent (isopropanol/DMA), triethylamine as a base and CCl4 as a halogenating agent. The deprotection of the trityl group under basic conditions (NH3, H2O) led to the loss of one choline moiety. The AT reaction was also used to produce prodrugs as exempli- fied by the works of Zhao et al (Scheme 46), who reported the synthesis of phosphoramidates that included two lipid chains and one AZT moiety [143]. All phosphoramidates produced by this synthetic scheme exhibited high anti-HIV activities. The AT reaction can also be used to produce nucleoside phosphor- amidate monoester [144] or thiophosphoramidates-based dinu- cleotides [77]. Other prodrugs were prepared by functionaliza- tion of polymers via a phosphoramidate tether. Yang et al. have reported the synthesis of chitosan functionalized with d4T (stavudine), a nucleoside reverse transcriptase inhibitor [145,146]. Conclusion The discovery of the reaction of dialkyl phosphite with amine in the presence of a base and CCl4 by Atherton, Openshaw and Todd in 1945 opened the way to a series of studies, which were initially focused on the investigation of the mechanism of this reaction. The most likely mechanism produced chloro- or bromophosphate as intermediate reactive species. This inter- mediate reacts in situ with a nucleophile in the presence of a base, which is involved to trap hydrochoride or hydrobromide, the byproducts of the AT reaction. The synthetic conditions were improved by employing stoichiometric quantities of CCl4 Scheme 46: Synthesis of AZT-based prodrug via an AT reaction (adapted from [143]). Scheme 46: Synthesis of AZT-based prodrug via an AT reaction (adapted from [143]). Scheme 46: Synthesis of AZT-based prodrug via an AT reaction (adapted from [143]). 1192 Beilstein J. Org. Chem. 2014, 10, 1166–1196. Acknowledgements 26. Hulst, R.; Zijlstra, R. W. J.; Feringa, B. L.; De Vries, N. K.; ten Hoeve, W.; Wynberg, H. Tetrahedron Lett. 1993, 34, 1339–1342. doi:10.1016/S0040-4039(00)91790-8 Acknowledgements We thank CNRS, the University of Brest, Conseil régional de Bretagne (Grant for SSLC), Association Française contre les Myopathies (AFM, Evry, France), and FEDER (Fond Européen de Développement Régional) for funding. 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The license is subject to the Beilstein Journal of Organic Chemistry terms and conditions: (http://www.beilstein-journals.org/bjoc) The definitive version of this article is the electronic one which can be found at: doi:10.3762/bjoc.10.117 License and Terms This is an Open Access article under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The license is subject to the Beilstein Journal of Organic Chemistry terms and conditions: (http://www.beilstein-journals.org/bjoc) The definitive version of this article is the electronic one which can be found at: doi:10.3762/bjoc.10.117 Beilstein J. Org. Chem. 2014, 10, 1166–1196. License and Terms doi:10.3762/bjoc.10.117 1196
https://openalex.org/W4252792974	https://journals.plos.org/plosbiology/article/file?id=10.1371/journal.pbio.1001848&type=printable	English	null	Correction: Impairment of TrkB-PSD-95 Signaling in Angelman Syndrome	PLoS biology	2,014	cc-by	275	Correction 1. Cao C, Rioult-Pedotti MS, Migani P, Yu CJ, Tiwari R, et al. (2013) Impairment of TrkB-PSD-95 Signaling in Angelman Syndrome. PLoS Biol 11(2): e1001478. doi:10.1371/journal.pbio.1001478 The PLOS Biology Staff The PLOS Biology Staff JM wishes to declare the following competing interest, which should have been indicated in the research article. JM owned shares in Angelus Therapeutics. However, for clarification, no materials or support were received from this company, and no agreements were in place concerning the execution or publication of this work. In addition, a US patent application entitled ‘‘Long term potentiation with cyclic-GluR6 analogs’’ (US Patent Appli- cation #20120149646) has been filed by Brown University, with JM listed as co-inventor. Reference Citation: The PLOS Biology Staff (2014) Correction: Impairment of TrkB-PSD-95 Signaling in Angelman Syndrome. PLoS Biol 12(4): e1001848. doi:10.1371/journal. pbio.1001848 Published April 8, 2014 PLOS Biology \| www.plosbiology.org Citation: The PLOS Biology Staff (2014) Correction: Impairment of TrkB-PSD-95 Signaling in Angelman Syndrome. PLoS Biol 12(4): e1001848. doi:10.1371/journal. pbio.1001848 Published April 8, 2014 Copyright: 2014 The PLOS Biology Staff. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Published April 8, 2014 Copyright: 2014 The PLOS Biology Staff. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. PLOS Biology \| www.plosbiology.org 1 April 2014 \| Volume 12 \| Issue 4 \| e1001848 April 2014 \| Volume 12 \| Issue 4 \| e1001848
https://openalex.org/W4287331760	https://zenodo.org/records/4682717/files/HIS-V55-I1-C1-NOV-2020%20(PP-01-06).pdf	Bengali	null	উনিশ ও বিশ শতকে ভারতে হিন্দু সাম্প্রদায়িকতাবাদ প্রসারের রূপরেখা	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	1,915	NAME OF THE AUTHOR ORIGINAL RESEARCH ARTICLE ADVANCE RESEARCH JOURNAL OF MULTIDISCIPLINARY DISCOVERIES I Vol.55 I Issue-1 I Chapter-1 E-ISSN : 2456-1045 উনিশওনিশশতকেভারকতনিন্সাম্ানরেতািা্ পসাকরররূকরো ORIGINAL RESEARCH ARTICLE NAME OF THE AUTHOR ISSN : 2456-1045 (Online) ICV Impact Value: 72.30 GIF- Impact Factor: 5.188 IPI Impact Factor: 3.54 Publishing Copyright @ International Journal Foundation Article Code: HIS-V55-I1-C1-NOV-2020 Category : HISTORY Volume : 55.0 ( NOVEMBER-2020 EDITION ) Issue: 1(One) Chapter : 1 (One) Page : 01-06 Journal URL: www.journalresearchijf.com Paper Received: 12.11.2020 Paper Accepted: 15.01.2020 Date of Publication: 10-02-2021 Doi No.:10.5281/zenodo.4682717 Chaina Debnath* Department of History Berhampore Girls College Nadia, West Bengal ABSTRACT আধুনিকভারতবর্ষরইনতহাসচচষাাএকটজটিস্ষকাতর' নব্াহরিাসাম্ানাকতাবা্তভারতবর্ষ সাম্ানাক উন ন া ্া া ন ADVANCE RESEARCH JOURNAL OF MULTIDISCIPLINARY DISCOVERIES I Vol.55 I Issue-1 I Chapter-1 E-ISSN : 2456-1045 ADVANCE RESEARCH JOURNAL OF MULTIDISCIPLINARY DISCOVERIES I Vol.55 I Issue-1 I Chapter ADVANCE RESEARCH JOURNAL OF MULTIDISCIPLINARY DISCOVERIES I Vol.55 I Issue-1 I Chapter- E-ISSN : 2456-1045 পচার চািারিা হা মুসিমাির্র নবুরদ বরটর্র নবুরদ িাত নকন যুনক ন্রা নবচার কররি নবররানধতাকররতহরতান্ট্সরকারররনবুরদত কারর সবরচরা দবন্ দোা হত্া করা হরতা দসিা ন্নবর গনিরত, কারজ এই আরনািি সাম্ানাক রপরিাত দোা-হত্ানবররাধাআরনািিবনহরা যাা১৮৯৬ সারিতনকনসাম্ানাকতাভাাবহ রপ নিরা নিরর আরস নবং্ ্তরকর ন্তাা ্্রকআইিসভাা্রক্াকন্রমরধ্ন্রাতনবং্ ্তরকর দোা়ার ভারতাা রাজিানতরত চরমপনার উদবএবংআযষসমারজরসরসচরমপনাদিতরবররনর ঘনি্ সমরকষ উ্ররা্র বরনদ দপরত োরকত পাঞারবর িািা িাজপত রাা এবং নতিক ন্বাজা উৎসব োরপনত উৎসব সংঘটত করর জাতাাতাবা্রক নহনুরুর আবররর আবদ রারখিত ১৯০৬ এ বসভস-নবররাধা সর্্া আরনািরির সািি্, মুসিমাির্র সতা রাজনিনতক সংোঠি োর়তুিরতঅিুপানরতকররত১৯০৯মরি- নমর্া সংংারমুসনিমর্রজি্আসিসংরকরকররনহনু সাম্ানাকমরিাভাবদকউরংন্রানছিতভাররতর পধািরাজনিনতক্িনহরসরবকংর্রসরকাযষকিাপ বহদোাড়ানহনরকঅসন্কররনছিতকাররকংর্স দকাি সাম্ানাক ্ি নছি িাত কারজই মুসনিম সাম্ানাকতার পসার, ইংররজর্র মুসনিম দতা্র িানতএবংকংর্নস িানত ওকাযষকিাপসমরকষ এই দ্রার নহনুর অিাসা- এই নতিট কারর নহনুসাম্ানাকতারউদরবরদক্প্তকররত সাম্ানরেতািা্পসাকরনিন্মিাসভারভূনমো সাম্ানরেতািা্পসাকরনিন্মিাসভারভূনমো উনিশশতকেভারকতসাম্ানরেতািা্-এর সূচিা ১৮৭০এর্্রকভাররতরসাম্ানাকতাবিরত নকছুইনছিিা।কারর১৮৫৭-রনবর্ারহনহনু-মুসিমািরা এরকঅপরররসহকানরররপন্ট্্াসরিরনবররানধতা করররছএবংনিরজর্রঅনধকারপুিঃপনত্াাব্েষহরিও তার্রমরধ্কখরিাসারেষরসংঘাতদচারখপর়নি।নকন এইনচর্ররপব্রিযাা১৮৮০্্রক।মারেনকছুন্রির জি্এই্ুইসম্ারারসাম্ানাকসমানতওসদাব পরাধািতারিাোপা্দেরকমুনকরতিতুিোনতসঞার কররিওপারসনরকসরনহওঅনবশারসরবাতাবরররততনর করর। সাম্ানাক বরি দয সময দিতারা ব্িাম কনররারছতারাদযপরত্রকভারতনবভাজিদচরানছরিি তানকনিা।নিজ সম্ারারমািুর্রউনািকরন, জানতনহরসরবোর়তুিরত, কসংংারদেরকমুককরর আধুনিককররতুিরতদচরানছরিিনহনুর্রপভাবদেরক মুককরর।তসা্আহম্১৮৮৪সারিবরিনছরিি, " Remember that Hindus and Muslims are religious terms. Other wise Hindus Muslims and Christians who live in this country are by virtue of this fact one quem (nation or community).২) নকনএইনচর্ররপব্রিযাা ১৮৮০্্রকই।কাররতসা্আহরম্খািজাতাা কংর্রসরনবররানধতাকররনছরিিসাম্ানাক্রন্রকার দেরকই। Chaina Debnath* Department of History Berhampore Girls College Nadia, West Bengal Open access, Peer-reviewed and Indexed journal (www.journalresearchijf.com) Page \| 1 আধুনিকভারতবর্ষরইনতহাসচচষাাএকটজটিস্ষকাতর' নব্াহরিাসাম্ানাকতাবা্তভারতবর্ষ সাম্ানাক সমস্ামূিতউপনিরবন্ক্াসরিরসারেযুককররদ্খাহাতপাচািভাররতদযসবজিরোা্ারকআকমরকারারভূনমকাা দ্খান্রানছি, ভাররতরইনতহারসতারানবিািহরাযাাতনকন ইসিামএবংএর্র্আনবভষারবরপরদেরকইতার্র নিজসসাতা্বজাাদররখনছিতশুদেরকইভারতবর্ষনহনুওমুসিমাি- এই্ুটসম্াা ্ুইনবপরাতন্নবরর নবভকহরাপা্াপান্বসবাসকরররছতঅতারতরএইধাররারনভন্রতপাচািযুোরক নহনুএবংমধ্যুোরকমুসিমািযুো নহরসরবনচন্তকরাহাতঅধ্াপকC.A. Bayly সাম্ানাকসমস্ার(উিনবং্্তরক) োনতপকরনতআরিািাপসরস এইনসদারাউপিাতহিদয, " There is little basis for the assumption made by many writers that Hindu - Muslim conflict did not occur until the creation of local representative bodies and the emergency of 'Modern ' politics in the post - Mutiny era". খুবসহজকোা, সাম্ানাকতাবা্হিএমিএকনবশাস, দয এক্িমািু্একটনবর্্ধরমষনবশাসকররিতার্রসামানজক, রাজনিনতকএবংঅেষনিনতকসােষসবএকইহাত সাম্ানাকতাবা্হরিাদসইনবশাস, অিুযাাাভাররতনহনু, মুসিমাি, ন্রািওন্খরানবনভনওসতাসম্াাযারা সাধািভারবএবংসতাভারবনবি্যএবংসংহতত(১) CITATION OF THE ARTICLE Debnath C. (2020) উনি্ওনব্্তরকভাররতনহনুসাম্ানাকতাবা্পসাররর রপররখা; Advance Research Journal of Multidisciplinary Discoveries; 55(1) pp. 01-06 * Corresponding Author আধুনিকভারতবর্ষরইনতহাসচচষাাএকটজটিস্ষকাতর' নব্াহরিাসাম্ানাকতাবা্তভারতবর্ষ সাম্ানাক সমস্ামূিতউপনিরবন্ক্াসরিরসারেযুককররদ্খাহাতপাচািভাররতদযসবজিরোা্ারকআকমরকারারভূনমকাা দ্খান্রানছি, ভাররতরইনতহারসতারানবিািহরাযাাতনকন ইসিামএবংএর্র্আনবভষারবরপরদেরকইতার্র নিজসসাতা্বজাাদররখনছিতশুদেরকইভারতবর্ষনহনুওমুসিমাি- এই্ুটসম্াা ্ুইনবপরাতন্নবরর নবভকহরাপা্াপান্বসবাসকরররছতঅতারতরএইধাররারনভন্রতপাচািযুোরক নহনুএবংমধ্যুোরকমুসিমািযুো নহরসরবনচন্তকরাহাতঅধ্াপকC.A. Bayly সাম্ানাকসমস্ার(উিনবং্্তরক) োনতপকরনতআরিািাপসরস এইনসদারাউপিাতহিদয, " There is little basis for the assumption made by many writers that Hindu - Muslim conflict did not occur until the creation of local representative bodies and the emergency of 'Modern ' politics in the post - Mutiny era". খুবসহজকোা, সাম্ানাকতাবা্হিএমিএকনবশাস, দয এক্িমািু্একটনবর্্ধরমষনবশাসকররিতার্রসামানজক, রাজনিনতকএবংঅেষনিনতকসােষসবএকইহাত সাম্ানাকতাবা্হরিাদসইনবশাস, অিুযাাাভাররতনহনু, মুসিমাি, ন্রািওন্খরানবনভনওসতাসম্াাযারা সাধািভারবএবংসতাভারবনবি্যএবংসংহতত(১) আধুনিকভারতবর্ষরইনতহাসচচষাাএকটজটিস্ষকাতর' নব্াহরিাসাম্ানাকতাবা্তভারতবর্ষ সাম্ানাক সমস্ামূিতউপনিরবন্ক্াসরিরসারেযুককররদ্খাহাতপাচািভাররতদযসবজিরোা্ারকআকমরকারারভূনমকাা দ্খান্রানছি, ভাররতরইনতহারসতারানবিািহরাযাাতনকন ইসিামএবংএর্র্আনবভষারবরপরদেরকইতার্র নিজসসাতা্বজাাদররখনছিতশুদেরকইভারতবর্ষনহনুওমুসিমাি- এই্ুটসম্াা ্ুইনবপরাতন্নবরর নবভকহরাপা্াপান্বসবাসকরররছতঅতারতরএইধাররারনভন্রতপাচািযুোরক নহনুএবংমধ্যুোরকমুসিমািযুো নহরসরবনচন্তকরাহাতঅধ্াপকC.A. Bayly সাম্ানাকসমস্ার(উিনবং্্তরক) োনতপকরনতআরিািাপসরস এইনসদারাউপিাতহিদয, " There is little basis for the assumption made by many writers that Hindu - Muslim conflict did not occur until the creation of local representative bodies and the emergency of 'Modern ' politics in the post - Mutiny era". খুবসহজকোা, সাম্ানাকতাবা্হিএমিএকনবশাস, দয এক্িমািু্একটনবর্্ধরমষনবশাসকররিতার্রসামানজক, রাজনিনতকএবংঅেষনিনতকসােষসবএকইহাত সাম্ানাকতাবা্হরিাদসইনবশাস, অিুযাাাভাররতনহনু, মুসিমাি, ন্রািওন্খরানবনভনওসতাসম্াাযারা সাধািভারবএবংসতাভারবনবি্যএবংসংহতত(১) ADVANCE RESEARCH JOURNAL OF MULTIDISCIPLINARY DISCOVERIES I Vol.55 I Issue-1 I Chapter-1 সাম্ানরেতািা্পসাকরনশন্ত মধ্নিি শ্রেরভূনমো পাঞাবনহনুমহাসভাপনতন্তহা১৯০৯সারি। এরপধািস্স্নছরিিইউ, এিমুখানজষওরাাবাহা্ুর িািচাড্।িািচাড্কংর্সরকনহনুর্রসআররানপত্ুভষাো্ এবংনিছকনহনুসারেষরজি্্ূবষিতরউৎসবরিবরষিা কররি।নতনিআররামরিকররিমুসিমাির্রদতা্র করারজি্নহনুসােষনবসজষিন্র্কংর্স।িািচাড্র তারভা্ররবরিনছরিি'আনমপেমনহনু, তারপর ভারতাাএকোএকজিনহনুরকশধুনবশাসকররিই চিরবিাএরকতারদ্হ-মরির, তারজাবরির, তার আচররএরঅপনরহাযষঅং্কররওতুিরতহরব'(৩) এই একই সমরা জন নিরানছি নহনু সাম্ানাকতাবা্ত ১৮৭০ এর ্্ক দেরকই নহনু জনম্াররা, মহাজি', মধ্নব্ ন্ককর্র একাং্ মুসিমাি নবররাধা মরিাভাব দক জানোরা তুিরত অ্রাভূনমকা ্হর কররনছিতএই দ্রা ভাররতর ইনতহাসসমরকষ উপনিরবর্র্রন্ভনসসমনদমরি নিরা মুসিমাি ্াসকর্র 'অত্াচারা ''মুসিমাি 'নিপা়রি'র হাত দেরক নহনুর্র রকা কররত ন্ট্রা 'মুনক্াতা' ভূনমকা পািি করররছি বরি পচারচািাাত ১৮৯০এর্্রকরদোা়ারন্রকসারার্্ জুর়চািারিাহাদোাহত্ানবররাধাপচার,এবংএই alresearchijf com) Page \| 2 ১৯০৯ন্রার্অর্াবরএ-পাঞারবরপার্ন্ক নহনুসর্িিঅিুন্তহা।এইঅিু্ারিবকব্রাখরত ADVANCE RESEARCH JOURNAL OF MULTIDISCIPLINARY DISCOVERIES I Vol.55 I Issue-1 I Chapter-1 ADVANCE RESEARCH JOURNAL OF MULTIDISCIPLINARY DISCOVERIES I Vol.55 I Issue-1 I Chapter-1 E-ISSN : 2456-1045 সাধািতাপুিুদারকররতনকনকংর্রসরমরতাএকট সবষভারতাাজাতাাতাবা্াসংোঠিোকাসর্ওনহনু মহাসভাউদরবরপরাাজিদকিদ্খান্ি-তানচাাকরার নব্া। নোরািািািাজপতরাাবরিনছরিি-"নহনুরানিরজরাই একট' জানত'কাররতার্রনিজসনবর্্ধররিরএকট সভ্তার পনতনিনধু করর। "(৪)নহনু নবন্নতাবা্া ্নকগনির উদরবর দপছরি অি্তম কারর নিরাপ্াহািতাদবাধ।ন্ট্্াসিভাররতরসানাু িারভরসরসসরসএর্র্একিতুিধররিরপ্াসনিকও অেষনিনতকব্বসাপচিিকররযারিরিপচনিত সামানজক, রাজনিনতকওঅেষনিনতকব্বসামূি্হািহরা পর়। এইরপ সংকট দেরক নিরজর্র নিরর দপরত আতঅরঅ্ররনিজনিজদোা্ারক সঙবদকরারমরধ্ ন্রাঅনধকাং্দকর্পাচািধরমষরমহািঐনতরহ্র তার্রদপররাযুনোরানছি।(৫) পেম নবশযুদ চিাকািাি নহনু মহাসভার ্রন্ভনসরতএকটাব়ধররিরপনরবতষিআরস।যুদ জনিতজুনরঅবসাাচাকনররসংখ্াবহগররবরনদপাা। আরোরমতনিজনিজঅঞরিদকািমরতন্িযাপিিা করর ভারতবাসা ভাো্ারঅ্রর ভারতবর্ষর নবনভন জাাোাাছন়রাপর়।তার্রসংকারষ্রন্ভনসপনরবতষি আরস।ভারতবর্ষর রাজিানতরতদযভারতবাসাআরও সনকাঅং্্হরকররতপাররএইসরচতিতাএখি িতুিভারবদজরোওরঠ।পাঞারবরনহনুমহাসভারআ্রি নবহারওযুকপর্্নহনুমহাসাভার্াখাপনতন্তহা। ১৯১৫সারিহনর্াররকমরমিাানহনুমহাসভাোঠতহা ম্িরমাহিমািব্এরদিতররু।এইসর্িরিসভাপনতর ভা্ররমহারাজামিানচনিনাএইআ্ঙাপকা্ কররিদযHindus might be reduce even to a minority for they made no coverts to increase their numbers , on the countrary they were faced with the problem of how best to worest their diminution. এইসংোঠরিরউর্্্নছি নহনুর্রসামানজকওরাজনিনতকসােষরকাকরা।এই সংোঠি১৯১৬ন্রার্কংর্সওমুসনিমিাোকতরষক সংঘটতিখরিĝ চুনকরনবররানধতাদযমিকররদতমিই প্াসনিকপর্ওআইিসভারস্স্পর্আররাদবন্ সংখ্কনহনুনিরাারোরপরচ্াচািাা।যন্ও ১৯১৫- ১৯২২ন্রা্পযষাএইসভারঅনযু কাোরজ-কিরমই সামাবদনছি।ব্তসাম্ানাকসংসানহরসরবমুসনিম িারোরপার্নহনুমহাসভানছিঅরিকাংর্ইমাি।কারর নছিখুবইস্।নহনুসভাব়জনম্ারবাসমাাভূসামা এবংপূবষতি্াসকর্রারমুখপা্র্রনিরাোঠতহওাাা সাধাররমািুর্রদেরক্ূররোরক।অি্ন্রকন্ট্ সরকারমুসিমািসম্ারারপনতযরতাটাকুিারহাত বান়রানছরিিনহনুর্রপনতততটাস্াহানি।তার পরাাজিহারতানছিিাআবারউভাসম্ারার সমািুপারতরসন্করাসমবনছিিা। যাইরহাক নহনু পুিুুাবি বার্র অি্তম পেম নবশযুদ চিাকািাি নহনু মহাসভার ্রন্ভনসরতএকটাব়ধররিরপনরবতষিআরস।যুদ জনিতজুনরঅবসাাচাকনররসংখ্াবহগররবরনদপাা। আরোরমতনিজনিজঅঞরিদকািমরতন্িযাপিিা করর ভারতবাসা ভাো্ারঅ্রর ভারতবর্ষর নবনভন জাাোাাছন়রাপর়।তার্রসংকারষ্রন্ভনসপনরবতষি আরস।ভারতবর্ষর রাজিানতরতদযভারতবাসাআরও সনকাঅং্্হরকররতপাররএইসরচতিতাএখি িতুিভারবদজরোওরঠ।পাঞারবরনহনুমহাসভারআ্রি নবহারওযুকপর্্নহনুমহাসাভার্াখাপনতন্তহা। ১৯১৫সারিহনর্াররকমরমিাানহনুমহাসভাোঠতহা ম্িরমাহিমািব্এরদিতররু।এইসর্িরিসভাপনতর ভা্ররমহারাজামিানচনিনাএইআ্ঙাপকা্ কররিদযHindus might be reduce even to a minority for they made no coverts to increase their numbers , on the countrary they were faced with the problem of how best to worest their diminution. এইসংোঠরিরউর্্্নছি নহনুর্রসামানজকওরাজনিনতকসােষরকাকরা।এই সংোঠি১৯১৬ন্রার্কংর্সওমুসনিমিাোকতরষক সংঘটতিখরিĝ চুনকরনবররানধতাদযমিকররদতমিই প্াসনিকপর্ওআইিসভারস্স্পর্আররাদবন্ সংখ্কনহনুনিরাারোরপরচ্াচািাা।যন্ও ১৯১৫- ১৯২২ন্রা্পযষাএইসভারঅনযু কাোরজ-কিরমই সামাবদনছি।ব্তসাম্ানাকসংসানহরসরবমুসনিম িারোরপার্নহনুমহাসভানছিঅরিকাংর্ইমাি।কারর নছিখুবইস্।নহনুসভাব়জনম্ারবাসমাাভূসামা এবংপূবষতি্াসকর্রারমুখপা্র্রনিরাোঠতহওাাা সাধাররমািুর্রদেরক্ূররোরক।অি্ন্রকন্ট্ সরকারমুসিমািসম্ারারপনতযরতাটাকুিারহাত বান়রানছরিিনহনুর্রপনতততটাস্াহানি।তার পরাাজিহারতানছিিাআবারউভাসম্ারার সমািুপারতরসন্করাসমবনছিিা। পাচািনহনুঐনতহ্পুিঃপনত্া করার্ানব, নহনুজিতারকসংোঠতকরারপরচ্াপেমদেরকইএর একটাসাম্ানাকরপন্রানছি।১৮৬৭ন্রার্নহনু দমিারপবতষি, মহারাষন্বাজা,োরপনতউৎসবপচিি এবং১৮৯৯ন্রার্নভ. নিসাভারকারররদিতররু অনভিব ভারত দসাসাইট োঠি একন্ত করার জি্পুিুরািবা্ার্রএরকমসকিপরচ্াইএমিনছি দসখারিনহনুছা়াঅি্ধমষমরতর( নবর্্তমুসিমাির্র) অং্্হরসমবনছিিা।দসযুরোরঅি্তমপাচাি পুিুরািবা্া সংোঠিটনছিআযষসমাজ।পেমন্রক নহনুমহাসভারউর্্্নছিদযদকািউপারানহনুধরমষর নবশদতারকাকরা। বতষমািভারতাারাজিানতরতঅি্তমসংকরটর কাররনবন্নতাবা্া্নকগনিরতৎপরতা।নকনএট আধুনিকরাজিানতরতদকািিতুিসংরযাজিিা।ভারত ব্ষ সাধািতা িারভর অরিক আরো দেরকই এই নবন্নতাবা্া ্নকগনি নকাা্াি নছি এবং জাতাাতাবা্াআরনািরিরমূিধারাটরকএরাবরাবর আঘাতকরররছ-কনত্যকরররছতারসাধািতাপরবষর নবন্নতাবা্া্নকগনিরপসরসআরিাচিাকররতদোরি পেরমআসারমরমুসনিমিারোরকোসরররআরস।আমরা ভুরিযাইদযপা্াপান্আরওকরাকটসাম্ানাক্নক তখিমাোচা়ান্রাউরঠনছিএবংএর্রআরপা্হাি মরিাভাবতৎকািািরাজিানতরকযরে্জটিকরর তুরিনছি। নহনু মহাসভা এমিই একট রাজনিনতক সংোঠি।কংর্সওনহনুমহাসভাররাজনিনতকিরক্ মূিতদকািপােষক্নছিিা।উভাচাইরতাভাররতর alresearchijf.com) Page \| 3 যাইরহাক নহনু পুিুুাবি বার্র অি্তম গুুপূরষ পযষারার সূচিা হা ১৯২২-২৩ সারি। যাইরহাক নহনু পুিুুাবি বার্র অি্তম গুুপূরষ পযষারার সূচিা হা ১৯২২-২৩ সারি। Open access, Peer-reviewed and Indexed journal (www.journalresearchijf.com) Page \| 3 সংোঠি।কংর্সওনহনুমহাসভাররাজনিনতকিরক্ মূিতদকািপােষক্নছিিা।উভাচাইরতাভাররতর যাইরহাক নহনু পুিুুাবি বার্র অি্তম গুুপূরষ পযষারার সূচিা হা ১৯২২-২৩ সারি। ADVANCE RESEARCH JOURNAL OF MULTIDISCIPLINARY DISCOVERIES I Vol.55 I Issue-1 I Chapter- E-ISSN : 2456-1045 আযষসমানজরা'শনদ'ও'সংোঠি' পকরনতরওপরগুু আররাপ করর। সুনমত সরকার যোেষই বরিরছি মুসিমাির্রতবনিোওতিনজমনছিআযষসমাজর্র শনদওসংোঠরিরজবাব।১৯২৩সারিরআোরমারস ম্িরমাহিমািব্সভাপনতরুবারারসারতনহনুমহাসভার অনধরব্িবরস।এইঅনধরব্রিশনদওসংোঠিদক সানবষকনহনুকমষসূনচরঅাভুষককরাহা।এইভারবনহনু মহাসভারসারেআযষসমারজরদযাোসু্সানপতহাদ্র্র নবনভনঅংর্নহনুর্রসংঘবদকরারজি্ নহনু মহাসভার ্াখা ছন়রা প়রত োরক। সাম্ানাক অসরাা্পাঞাবন্লা, উ্রপর্্, নবহারপভরনতরারজ্ তা্আকারধাররকররনছি।এমিনকমা্াজবাংিাা দযখারিনহনুমহাসভারদকারিাঅনযুইনছিিাদসখারি নহনুসংোঠিগনি্নক্ািাহরাওরঠ।নহনুমহাসভার অ্মঅনধরব্রিরসভাপনতুকররতনোরািািারাজপে রাাপকার্্বরিিদয-'কংর্রসরঅনহংসঅসহরযাো আরনািিনহনুসংোঠিগনিরক্ুবষিদতাকরররছইএমিনক সাধািতাআরনািিরকও নপনছরান্র্'(৬) তরবনব্ ্তরককংর্রসরঅরিকনহনুদিতাইনহনুমহাসভাদক দপররাজুনোরানছরিি।কংর্রসরএমআরজাাকর, নস ওাাইনচাামনরএবংআররাঅরিরকইকংর্রসরস্স্ হরাওনহনুমহাসভাসারেঘনি্ভারবযুকনছরিি। জহরিািদকআরকপকররবরিনছরিি" সংরকরপবিরত দোরিভাররতকখরিাএতখারাপঅবসাহানি।অসহরযাো আরনািরিরদযপনতনকাা১৯২২-২৩ এশুহা, ত ধাররনকননিন্তভারবইজিোরররসরসসংন্্সময কাজকমষি্কররন্র্...।জিসাধাররদকবিএকটাই ন্কাপার্-তাহিসাম্ানাকঘররা" সাম্ানরেতািা্পসাকরআর. এস. এসএর ভূনমো সাম্ানরেতািা্পসাকরআর. এস. এসএর ভূনমো ১৯২৫সারিিাোপুরররাষাাসাংরসবকসংরঘর পনত্াভাররতনহনুসাম্ানাকতারনবকার্উরলখরযাো্ ভূনমকা্হরকরর।দকনবদহিরোওাারএরদিতররু আরএসএসঅি্তমপধািসাম্ানাক্নকররপ। আতপকা্ করর। নহনু সাম্ানাকতার দপকাপট আরিাচিাকররতনোরাসুনমতসরকার,তনিকা সরকার তপিবসুপমূখ' Khaki Shorts and Saffron Flags ' - পুনযকাা বরিরছি আরএসএস আ্াসা নহনু সাম্ানাকতারমূিউৎসওসরপ।আরএসএস- ও কংর্সদকতার্রপধাি্তনহসারবদ্খত।এইকটর সাম্ানাক সংোঠরির পধাি এম এস ওাািকার আরএসএসত্সূ্পাতকররনছরিিতার' We' িামক পুনযকাা।১৯৩৯-এনতনিদঘা্রাকররিসংখ্ািঘুর্র ্ানবদমরিদিওাাহরি'নহনুজাতাাজাবিধংসহওাার নবপ্ দ্খা দ্রব('৯ নহনুর্র রকার পা্াপান্ দোাাািকরআকমরকররনছরিিমুসিমাির্রওকংর্স দিতার্র।দোনিমরিকররিকংর্রসরসরন্হরারছজাতাা দচতিারকধংসকরারহানতাারররপ।জাতাাতাবা্ার্র আকমরকররনতনিবরিনছরিিআমার্রসবরচরাগুতর ্তর্র( মুসিমাির্র) বুরকজন়রাধররআমার্র অনযুইনবপনকররছি।নতনিভাররতঅি্াি্ধম্া সম্াাওমুসিমাির্রপরাম্ষন্রানছরিিনহনুসারির ওনহনুর্রনহনুসংংরনতওভা্া্হরকররতহরবনহনু ধমষরক্দাকররতওএইধরমষরমাহাতদকহ্রািািি কররতহরব,নহনুজানতওসংংরনতরমনহমাকাতষিকরা ছা়াআরদকািভাবিাদকপ্াদ্ওাাযারবিা, তার এইভা্ররপনরষারহরাযাাভারতবর্ষরনবর্ন্বরি দকউোকরবিাযারাোকরবতারাশধুমা্নহনুজানতর হকরমর্াসহরাোকরতহরব, দকারিা্ানবকররতপাররব িা,দকারিাসুরযাো-সুনবধাচাইরতপাররবিা, নবর্্ অনধকারদতাপ্ইওরঠিা, এইভারবনতনিমুসিমাি O P i d d I d d j l ( j ১৯৩৭সারিনহনুমহাসভার্ানাুঅনপষতহা নবিাাক ্ারমা্র সাভারকর এর হরয।সাভারকর মুসিমািআনধপরত্রনবপ্সমরকষনহনুর্রবারবার সতকষ কররআসনছরিি।১৯৩৭-এ'নতনিবরিনছরিি নহনুর্রওনহনুসারিরঅি্াি্অমুসিমািঅংর্র কপারিিাঞিাওমুসিমািআনধপরত্রনতিকএডরকন্রত চাাএবংনহনুর্রচাাতার্রনিজদ্র্কাত্ারস পনররত কররত '।(৭)১৯৩৮ এ সভাপনতর ভা্রর কংর্রসর সমারিাচিা করর বরিি 'কংর্স কম্রা পনতপর্নহনুসারেষরপনতনবশাসঘাতকতাকররএবং মুসিমািিারোরকারছএকপারাখা়াহরাোরক '.(৮)নবং্্তরকভাররতরনবনভনঅঞরিরমহাসভাোর় ওরঠ।সংোঠরিরমুসনিমনবররাধাএবংকংর্সনবররাধা কাযষকিাপমুসনিমর্রমরধ্সাম্ানাকতারকদযমি বান়রাতুরিনছিদতমিইতর্রন্কদেরককংর্স ধমষনিররপকরাজনিনতকসংোঠিহওাাসর্ওকমাোত সাম্ানাকপচারএরকারছিনতসাকারবাধ্হা। কংর্রসরঅি্তমপধািদিতামনতিািদিরহুপু্ Page \| 4 Page \| 4 ADVANCE RESEARCH JOURNAL OF MULTIDISCIPLINARY DISCOVERIES I Vol.55 I Issue-1 I Chapter-1 E-ISSN : 2456-1045 নহনুধরমষপুিুুাবরিরসপরকবকব্রারখি।দযমি, রারজনিািনম্, নোনর্চনদঘা্, অমরতিািবসুপমুখ। উিনবং্্তরকবাংিা, নহনন, উ্ুষএবংঅি্াি্ভা্াা বহসানহত্, কাব্্নপকান্তহা।যারবকব্নছি অং্তসাম্ানাক।এইসমাবাংিাসানহরত্রএকজি অি্তমপধািসানহনত্কনছরিিবনঙমচনচরটাপাধ্াা।' রাজনসংহ' উপি্াসরচিাকররনচরন্রিরজি্নতনি মুসিমাির্রনব্ িজররপর়ি।নহনুরবারুোাো োাইরতনোরাএবংসাধািতারবাধওজাতাাতাবার্রআ্্ষ পচারকররতনোরামুসিমাি্াসকর্রদহাপনতপন করারপবরতাসাম্ানাকমরিাভাবইনিদযাোাা।দয দকািসম্াাতারআ্্ষঐনতহ্নিরাোবষকররতপারর। নকনদসইোবষরবাধযখিঅি্সম্াারকআঘাতকরর, তখিতারপনররনতভারিাহরতপাররিা।রবানিাে অরিকআরোইতাই্ুঃখকররনিরখনছরিি- "একটান্ি নছিযখিনহনুআপিনহনুুিইাাদোĝরবকনররতউ্্ত হইি।তখিমুসিমািযন্নহনুরদোĝরবমানিাািইাা নিরজরাচুপচাপকনরাাোকরতাতরবনহনুখুবখুন্হইত সরনহিাই।নকনদযকারররনহনুরনহনুরুঅ্হইাা উঠিদসইকারররইমুসিমারিরমুসিমানিমাোতুনিাা উঠি।এখিদসমুসিমািররপইপবিহইরতচাা।নহনু সনহতনমন্াানোাাপবিহইরতচাািা।" (১২) সম্াাদক্ূররসনররাদ্া।তারপনতটবকরব্ মুসিমাির্রপনততারনবর্্ঘররারই বনহঃপকা্ ঘরটনছি।তার্রএইসাম্ানাকপররাচিাা'নহনুধমষ নবপন', 'নহনুধমষনবশাসনবপন' ও'নহনুসংংরনতনবপন' বরিদ্াররোািকররতদ্খাযাা(১০)।পাানতি্্ক আরোআরএসএসএরমুখপা্Organiser নিরখনছরিা সমযমুসিমািরামরকতার্রনবিাাকবরিদমরিনিি, সমযসাম্ানাকসমস্ানমরটযারব। উনিশশতকেিাংলারসাম্ানরেতািা্পসরস উিনবং্্তা্ারদ্্ারধষধমষওসমাজজাবরিনহনু জাতাাতাবার্রওপুিুুাবি পভাববাংিারতবরনদ দপরতোরক।ন্ট্সরকারইংররনজন্কারপবতষি কররিবাঙানিসম্াাসািরন্হরকররি।িরি আধুনিকন্কাান্নকতন্ট্অিু্হপু্বাঙানি মধ্নবর্রদোা়াপ্িহা।ইংররনজন্কাান্নকত দ্রারমরধ্জাতাাতারবাধোর়ওঠারসারেসারেনিজস ধমষ, সানহত্সমরকষসরচতিতাকম্বরনদপাা।বাংিাা নহনুজাতাাতাবা্ওনহনুধরমষপুিুুাবিবার্রপধাি মুখপা্নছরিিরাজিারাারবসু।১৮৭২ন্রার্১৫ই দসর্টররাজিারাারবসু'নহনুধরমষরদ্্ু' নব্রাদয বকরতা দ্ি তা দেরকই Hindu Revivalist আরনািরিরসূ্পাতহা।ন্নকতমুসনিমদিতরবরনযখি একটসতাপরেমুসিমাির্রসংোঠতকরারপরচ্াা রততখিন্নকতনহনুসম্ারারমরিনকধররির পনতনকাাসরন্কররতারাজিারাারবসুরবকব্হরা ওরঠ। ১৯৮১ ন্রার্ রাজিারাার বসু দিরখি মুসিমািরাযখিি্া্িািমহারমিািঅ্ারসানসরা্রির সংঘবদহর্ি, তখিনহনুর্রওমহানহনুসনমনতোঠি করাউনচত।নতনিআররাবরিি" মুসিমািওভারতবাসা অি্াি্জানতরসরসআমরারাজনিনতকওঅি্াি্ নব্রাযত্ূরপানরদযাোন্ব, নকনকর্কদযমি পনরনমতজনমক্ষরকরর, সমযদ্্ক্ষরকররিা, দসইরপনহনুসমাজইআমার্রকারজরদক্হইরব "।(১১)বসু্ামোরঞওমহানহনু সনমনতর্াখা-প্াখা োঠরিরপরাাজিাাতাতুরিধররি।এর্ারাসামানজক তব্ম্োকাসরুওনহনুর্রমরধ্একাতদবাধোর় উঠরব।এইসমরাআরওঅরিককরাকজিনহনুদিতা নিসাভারর-নহনুরাষ্্ষি, একরিক্ি অি্াদপনসরিনোািনসরচস, দবাটাই, ১৯৪, পর্া, ২১ [8] নবপাি চন আধনিক ভারত ও [10] নবপািচন, মর্ুিামুখানজষ, আন্ত্মুখানজষ, দকএম পানিকর, সুরচতামহাজি-ভাররতরসাধািতাসং্াম (১৯৫৭-১৯৪৭),দকনপবাোনচএনদকামানি, ১৯৯৪ [11] কাজাআব্ুিও্ু্-বাংিারজাোরর, নবশভারতা, কিকাতা,১৩৬০বসা্, পর্া-১২৭ [12] রবানরচিাবিা, জন্তবান্ষকা সংংরর, ্রাা্্খন, পন্মবসসরকার১৯৯০,পর্া১৮২- ৮৩ [13] নবপািচন-আধুনিকভারতওসাম্ানাকতা বা্, দকনপবাকনচ,এনদকামানি, -২পর্া **** ADVANCE RESEARCH JOURNAL OF MULTIDISCIPLINARY DISCOV জানতবরিনকছুনছিিা, োকরতপাররিা, ভারতনচরকাি নিছকইএকট'ধম্াসম্ারারসমূরহরসংঘ'নছি, আরছ এবংোকরব।" (১৩) তে্সূ্ [1]নবপাি চন -আধুনিক ভারত ও সাম্ানাকতাবা্,দকনপবাোচা,১৯৮৯পর্ািটার- 1 [2] সমরকমারমনলক-আধুনিকভাররতররপাার, রাজ দেরক সরাজ, ১৮৫৭-১৯৪৭,ওরার দবসি পাবনি্াসষকিকাতা, ২০০৭,পর্ািং৪১৪ [3]িািচাড্- দসিি অ্াবরিরো্ি ইি পনিটি, িারহার,1938 [4] নসদােষ গহ রাা -আধুনিক ভারতবর্ষর ইনতহাস,১৮৫৮-১৯৬৪, পর্নসভ পাবনি্াসষ, কিকাতা, পর্ািাটার২০৬ [5]Walter k.Anderson and Sridhar D.Dumle-The Brotherhood in saffron : The Rastriya Swayamsebak s Sanga and Hindu Revivalist , page no.1 [6]B.B Misra -The Indian political parties : An historical analysis of political behaviour upto 1947, Oxford University Press, 1976 , p-164 [7] নভ. নিসাভারর-নহনুরাষ্্ষি, একরিক্ি অি্াদপনসরিনোািনসরচস, দবাটাই, ১৯৪, পর্া, ২১ [8] নবপাি চন- আধুনিক ভারত ও সাম্ানাকতাবা্,দকনপ, বাোনচএনদকামানি, কিকাতা১৯৮৯,পর্া১১৫ [9]M.S Gwalkar -We or power Nationhood design, Nagpur 1939, p-58 E-ISSN : 2456-1045 ) \| কংর্রসর বান্ষকঅনধরব্রিবনঙমচনরনচত বরনমাতরমোািটব্বহারনিরাওমুসিমািওকংর্রসর মরধ্মতনবররাধিক্করাযাা।োািটপনতমাউপাসিা করানকনআিনমঠকাব্্ননতনিরচিাকররনছরিি মুসনিমনবররাধা একট কানহিার দপকাপরট। এই সাম্ানাকঅসরকহানতাারকররইভাররতরপধাি্ুই রাজনিনতকসংোঠিজাতাাকংর্সওমুসনিমিাোএর রাজনিনতক্রক্াকন্ভারতনবভাজরিরদক্প্ত করর।পেরমকংর্সদিতরুনব্াটরকগুুিান্রিও অরিকপররহরিওসাধািভাররতএরগুুবুেরত দপররনছি। ভারতরক নবভাজি করর সরন্ হরানছি সাম্ানাকতার নভন্রত্ুটসতারাষভারতএবং পানকযাি।দোĝরবআরঐনতহ্রদর্াররন্রতদকাি সম্াাদবন্িাভহিআবারকারকনতহরিাএই নবতরকষিানোরাবিাভারিাএইসাম্ানাকদর্াররন্ ভারতবর্ষঐরক্রবনিদকন্নেিদেরকন্নেিতার কররতুরিনছি।নবপািচনঠকইবরিরছি- "ভারতাা en access, Peer-reviewed and Indexed journal (www.journalresearchijf.com) Page \| 5 ADVANCE RESEARCH JOURNAL OF MULTIDISCIPLINARY DISCOVERIES I Vol.55 I Issue-1 I Chapter-1 E-ISSN : 2456-1045 জানতবরিনকছুনছিিা, োকরতপাররিা, ভারতনচরকাি নিছকইএকট'ধম্াসম্ারারসমূরহরসংঘ'নছি, আরছ এবংোকরব।" (১৩) তে্সূ্ [1]নবপাি চন -আধুনিক ভারত ও সাম্ানাকতাবা্,দকনপবাোচা,১৯৮৯পর্ািটার- 1 [2] সমরকমারমনলক-আধুনিকভাররতররপাার, রাজ দেরক সরাজ, ১৮৫৭-১৯৪৭,ওরার দবসি পাবনি্াসষকিকাতা, ২০০৭,পর্ািং৪১৪ [3]িািচাড্- দসিি অ্াবরিরো্ি ইি পনিটি, িারহার,1938 [4] নসদােষ গহ রাা -আধুনিক ভারতবর্ষর ইনতহাস,১৮৫৮-১৯৬৪, পর্নসভ পাবনি্াসষ, কিকাতা, পর্ািাটার২০৬ [5]Walter k.Anderson and Sridhar D.Dumle-The Brotherhood in saffron : The Rastriya Swayamsebak s Sanga and Hindu Revivalist , page no.1 [6]B.B Misra -The Indian political parties : An historical analysis of political behaviour upto 1947, Oxford University Press, 1976 , p-164 [7] নভ. নিসাভারর-নহনুরাষ্্ষি, একরিক্ি অি্াদপনসরিনোািনসরচস, দবাটাই, ১৯৪, পর্া, ২১ [8] নবপাি চন- আধুনিক ভারত ও [10] নবপািচন, মর্ুিামুখানজষ, আন্ত্মুখানজষ, দকএম পানিকর, সুরচতামহাজি-ভাররতরসাধািতাসং্াম (১৯৫৭-১৯৪৭),দকনপবাোনচএনদকামানি, ১৯৯৪ [11] কাজাআব্ুিও্ু্-বাংিারজাোরর, নবশভারতা, কিকাতা,১৩৬০বসা্, পর্া-১২৭ [12] রবানরচিাবিা, জন্তবান্ষকা সংংরর, ্রাা্্খন, পন্মবসসরকার১৯৯০,পর্া১৮২- ৮৩ [13] নবপািচন-আধুনিকভারতওসাম্ানাকতা বা্, দকনপবাকনচ,এনদকামানি, -২পর্া ** ADVANCE RESEARCH JOURNAL OF MULTIDISCIPLINARY DISCOVERIES I Vol.55 I Issue-1 I Chapter-1 E-ISSN : 2456-1045 জানতবরিনকছুনছিিা, োকরতপাররিা, ভারতনচরকাি নিছকইএকট'ধম্াসম্ারারসমূরহরসংঘ'নছি, আরছ এবংোকরব।" (১৩) তে্সূ্ [1]নবপাি চন -আধুনিক ভারত ও সাম্ানাকতাবা্,দকনপবাোচা,১৯৮৯পর্ািটার- 1 [2] সমরকমারমনলক-আধুনিকভাররতররপাার, রাজ দেরক সরাজ, ১৮৫৭-১৯৪৭,ওরার দবসি পাবনি্াসষকিকাতা, ২০০৭,পর্ািং৪১৪ [3]িািচাড্- দসিি অ্াবরিরো্ি ইি পনিটি, িারহার,1938 [4] নসদােষ গহ রাা -আধুনিক ভারতবর্ষর ইনতহাস,১৮৫৮-১৯৬৪, পর্নসভ পাবনি্াসষ, কিকাতা, পর্ািাটার২০৬ [5]Walter k.Anderson and Sridhar D.Dumle-The Brotherhood in saffron : The Rastriya Swayamsebak s Sanga and Hindu Revivalist , page no.1 [6]B.B Misra -The Indian political parties : An historical analysis of political behaviour upto 1947, Oxford University Press, 1976 , p-164 [7] নভ. ADVANCE RESEARCH JOURNAL OF MULTIDISCIPLINARY DISCOVERIES I Vol.55 I Issue-1 I Chapter-1 জানতবরিনকছুনছিিা, োকরতপাররিা, ভারতনচরকাি নিছকইএকট'ধম্াসম্ারারসমূরহরসংঘ'নছি, আরছ এবংোকরব।" (১৩) তে্সূ্ [1]নবপাি চন -আধুনিক ভারত ও সাম্ানাকতাবা্,দকনপবাোচা,১৯৮৯পর্ািটার- 1 [2] সমরকমারমনলক-আধুনিকভাররতররপাার, রাজ দেরক সরাজ, ১৮৫৭-১৯৪৭,ওরার দবসি পাবনি্াসষকিকাতা, ২০০৭,পর্ািং৪১৪ [3]িািচাড্- দসিি অ্াবরিরো্ি ইি পনিটি, িারহার,1938 [4] নসদােষ গহ রাা -আধুনিক ভারতবর্ষর ইনতহাস,১৮৫৮-১৯৬৪, পর্নসভ পাবনি্াসষ, কিকাতা, পর্ািাটার২০৬ [5]Walter k.Anderson and Sridhar D.Dumle-The Brotherhood in saffron : The Rastriya Swayamsebak s Sanga and Hindu Revivalist , page no.1 [6]B.B Misra -The Indian political parties : An historical analysis of political behaviour upto 1947, Oxford University Press, 1976 , p-164 [7] নভ. নিসাভারর-নহনুরাষ্্ষি, একরিক্ি অি্াদপনসরিনোািনসরচস, দবাটাই, ১৯৪, পর্া, ২১ [8] নবপাি চন- আধুনিক ভারত ও সাম্ানাকতাবা্,দকনপ, বাোনচএনদকামানি, কিকাতা১৯৮৯,পর্া১১৫ [9]M.S Gwalkar -We or power Nationhood design, Nagpur 1939, p-58 [10] নবপািচন, মর্ুিামুখানজষ, আন্ত্মুখানজষ, দকএম পানিকর, সুরচতামহাজি-ভাররতরসাধািতাসং্াম (১৯৫৭-১৯৪৭),দকনপবাোনচএনদকামানি, ১৯৯৪ [11] কাজাআব্ুিও্ু্-বাংিারজাোরর, নবশভারতা, কিকাতা,১৩৬০বসা্, পর্া-১২৭ [12] রবানরচিাবিা, জন্তবান্ষকা সংংরর, ্রাা্্খন, পন্মবসসরকার১৯৯০,পর্া১৮২- ৮৩ [13] নবপািচন-আধুনিকভারতওসাম্ানাকতা বা্, দকনপবাকনচ,এনদকামানি, -২পর্া **** জানতবরিনকছুনছিিা, োকরতপাররিা, ভারতনচরকাি নিছকইএকট'ধম্াসম্ারারসমূরহরসংঘ'নছি, আরছ এবংোকরব।" (১৩) [10] নবপািচন, মর্ুিামুখানজষ, আন্ত্মুখানজষ, দকএম পানিকর, সুরচতামহাজি-ভাররতরসাধািতাসং্াম (১৯৫৭-১৯৪৭),দকনপবাোনচএনদকামানি, ১৯৯৪ [10] নবপািচন, মর্ুিামুখানজষ, আন্ত্মুখানজষ, দকএম পানিকর, সুরচতামহাজি-ভাররতরসাধািতাসং্াম (১৯৫৭-১৯৪৭),দকনপবাোনচএনদকামানি, ১৯৯৪ তে্সূ্ [11] কাজাআব্ুিও্ু্-বাংিারজাোরর, নবশভারতা, কিকাতা,১৩৬০বসা্, পর্া-১২৭ [12] রবানরচিাবিা, জন্তবান্ষকা সংংরর, ্রাা্্খন, পন্মবসসরকার১৯৯০,পর্া১৮২- ৮৩ [8] নবপাি চন- আধুনিক ভারত ও সাম্ানাকতাবা্,দকনপ, বাোনচএনদকামানি, কিকাতা১৯৮৯,পর্া১১৫ [9]M.S Gwalkar -We or power Nationhood design, Nagpur 1939, p-58 Open access, Peer-reviewed and Indexed journal (www.journalresearchijf.com) Page \| 6
https://openalex.org/W4312746020	https://re.public.polimi.it/bitstream/11311/1204260/1/GPPS-TC-2021_paper_349.pdf	English	null	Automatic extraction of Chemical Reactor Networks from CFD data via advanced clustering algorithms	Proceedings	2,022	cc-by	6,847	Automatic extraction of Chemical Reactor Networks from CFD data via advanced clustering algorithms Alberto Cuoci Politecnico of Milan Alberto.cuoci@polimi.it Milan, Italy Matteo Savarese Université Libre de Bruxelles Matteo.savarese@ulb.be Bruxelles, Belgium Matteo Savarese Université Libre de Bruxelles Matteo.savarese@ulb.be Bruxelles, Belgium Ward De Paepe Université de Mons Ward.depaepe@umons.ac.be Mons, Belgium Alessandro Parente Université Libre de Bruxelles Alessandro.parente@ulb.be Bruxelles, Belgium Ward De Paepe Université de Mons Ward.depaepe@umons.ac.be Mons, Belgium Alessandro Parente Université Libre de Bruxelles Alessandro.parente@ulb.be Bruxelles, Belgium ABSTRACT The design of cleaner and more sustainable combustion technologies represents nowadays a key task. Reliable numerical models, able to cope with a large variety of configurations, combustion processes and fueling mixtures are needed, especially for future applications in combustion monitoring and control, for thermal and environmental performances, which are of critical importance. In this work, alternative, low-computational cost modelling tools for pollutants and thermal efficiency predictions, represented by Chemical Reactor Networks (CRN), are designed from Computational Fluid Dynamic (CFD) simulations assessing a novel methodology, by exploring new possibilities offered by Machine Learning (ML) algorithms. In particular, unsupervised learning approaches are employed, in order to extract the key features of the system flow-field, adopting advanced clustering algorithms, such as Local Principal Component Analysis (LPCA) and K-Means, thus providing an efficient and automatic identification of similar thermo-chemical state compartments in the computational domain. The identified zones are modelled in a post-processing phase as a network of interconnected chemical reactors, and detailed kinetic mechanisms are employed for low concentration pollutants predictions. The case study, a quasi-industrial, flameless-capable combustion furnace, fed with methane-hydrogen mixtures in different compositions at a nominal power of 15 kW, has been investigated numerically by performing 2D CFD simulations with reduced chemistry and subsequently CRN simulations has been carried out with detailed kinetics, adopting the aforementioned approach. Results are validated upon experimental data, in order to provide a novel methodology for CRN design applications, which can be suited for future GTs applications. Proceedings of Global Power and Propulsion Society ISSN-Nr: 2504-4400 GPPS Xi’an21 18th – 20th October, 2021 www.gpps.global Proceedings of Global Power and Propulsion Society ISSN-Nr: 2504-4400 GPPS Xi’an21 18th – 20th October, 2021 www.gpps.global Proceedings of Global Power and Propulsion Society ISSN-Nr: 2504-4400 GPPS Xi’an21 18th – 20th October, 2021 www.gpps.global Proceedings of Global Power and Propulsion Society ISSN-Nr: 2504-4400 GPPS Xi’an21 18th – 20th October, 2021 www.gpps.global Proceedings of Global Power and Propulsion Society ISSN-Nr: 2504-4400 GPPS Xi’an21 18th – 20th October, 2021 www.gpps.global Automatic extraction of Chemical Reactor Networks from CFD data via advanced clustering algorithms This work is licensed under Attribution 4.0 International (CC BY 4.0) See: https://creativecommons.org/licenses/by/4.0/legalcode INTRODUCTION In the context of the energy transition, increasing attention needs to be paid to develop sustainable combustion technologies. A valid alternative for an efficient and clean energy conversion system is represented by micro Gas Turbines combined with non conventional combustion regimes, such as Moderate and Intense Low-oxygen Dilution (MILD) combustion (Cavaliere and de Joannon, 2004), thus providing high thermal efficiency, large fuel flexibility and low pollutants emissions (NOx, CO and Soot). MILD combustion, also referred to as Flameless (Wünning and Wünning, 1997) or Colorless Distributed Combustion (Arghode and Gupta, 2010) is achieved by means of preheating reactants above their self-ignition temperature and through a strong entrainment of inert combustion products within the reaction region, thus lowering the oxygen concentration at which combustion takes place, resulting in a suppression of temperature peaks, which in turn leads to the abatement of thermal pollutants, such as NOx, SOx and soot (Christo and Dally, 2005). The development of a Colorless Distributed Combustion (CDC) chamber for Gas Turbines application has been shown by (Arghode and Gupta, 2011), while (Zornek et al., 2015) have demonstrated experimentally the applicability of the flameless regime to a Turbec T100 to burn low- calorific, biomass derived fuels with a consistent reduction in NOx and CO emissions. This work is licensed under Attribution 4.0 International (CC BY 4.0) See: https://creativecommons.org/licenses/by/4.0/legalcode In order to assist the design of such advanced technologies, reliable numerical models are required, especially for predicting low concentration pollutants, a crucial aspect in assessing environmental performances of combustion systems. Computational Fluid Dynamics (CFD) has been extensively used for modelling turbulent reacting flows, but predicting pollutants emissions still remains an open research challenge, due to computational cost limitations. In this framework, detailed kinetic mechanisms should be employed, in order to account for all the possible formation pathways of minor chemical species, thus leading to unfeasible computational costs associated with the high dimensionality of the problem, despite the constant increase in computational resources (Lu and Law, 2009). For this reason, alternative tools to model complex reactive systems, such as equivalent Chemical Reactor Networks (CRN), have increased their popularity in the combustion community, since they are capable of handling highly detailed chemical mechanisms, consisting of hundreds of species and thousands of reactions, while maintaining the computational requirements on an affordable level. INTRODUCTION Stemming from the theory proposed by Levenspiel (Levenspiel, 1997), complex reactive flows may be approximated by a series of interconnected, ideal chemical reactor models, namely Perfectly Stirred Reactors (PSR) and Plug Flow Reactors (PFR), reducing the problem to a 0-D or 1-D configuration, where equations for conservation of mass and species are solved only in few “blocks”, thus considerably lowering the number and the complexity of the equations to be solved, with respect to a CFD simulation with detailed kinetics. This modelling technique has been applied to industrial furnaces (Faravelli et al., 2001) and burners (de Toni et al., 2013), showing its versatility towards different configurations. It has also been successfully applied to premixed combustion in gas turbines (Lee et al., 2011; Park et al., 2013). In this framework, several works (Hao, 2014; Lee et al., 2011; Nguyen, 2019; Nguyen et al., 2017; Novosselov et al., 2006) focused on the manual design of small-sized CRN of gas turbine combustors from CFD simulations carried out with simplified kinetics, in order to reduce the associated computational cost. The system domain is then arbitrarily divided into different zones, according to temperature, composition and velocity similarities, by observing the main flow-field features provided by CFD simulations, obtaining CRN models able to predict emissions (NO and CO) and thermal performances over a certain range of operating conditions (i.e. equivalence ratio). However, manually designing an equivalent CRN model is a time- consuming and experience-required task, since an automatic and more systematic methodology to obtain a reduced and simple CRN model from CFD data is still missing, thus particularly relying on the user ability. Examples of automatically generated CRN from CFD data are available in literature. An integrated CFD-CRN procedure is firstly presented by Falcitelli (Falcitelli et al., 2002) and more recently improved by (Monaghan et al., 2012), (Cuoci et al., 2013) and (Stagni et al., 2014) with a fully coupled CFD-CRN approach for detailed pollutants formation analysis by post-processing CFD data with a kinetic post processor. Even though this approach has obtained remarkable results, its adoption aimed at obtaining also a spatial representation of minor chemical species, and the elevated number of reactors included (~103 – 104) only partially reduce the complexity of a CFD computational grid, and the CPU time associated with the solution of such a large network remains still high (~50 min) (Stagni et al., 2014) . INTRODUCTION What makes CRN modelling very appealing, is the possibility to use simple configurations to perform fast input-output kinetics calculations, in order to employ this tool for combustion monitoring and control applications. In this sense, a first example of a small-sized CRN of a jet stirred reactor to predict in real time lean blowout for gas turbines applications is presented by Kaluri (Kaluri et al., 2018) and improved by Gupta (Gupta et al., 2019) by building a CRN-based control system also able to prevent the blowout itself. However, even in this case the CRN has been manually designed by observing the general features of the flow-field from CFD simulations, and a procedure to automatically identify a simple network configuration from CFD data is still missing in literature, according to the authors’ knowledge, thus limiting the use of CRN modelling only to experienced designers. In this framework, Machine Learning (ML) algorithms may represent an effective way to understand and extract flow- field key features from CFD simulations data. ML is receveing increasing attention from the combustion community. Advanced statistical tools, such as Principal Component Analysis (PCA), have been used to identify lower-dimensional manifolds in turbulent reactive flows (Parente et al., 2009; Parente and Sutherland, 2013), moreover, PCA in its local formulation (Local PCA), based on a local reconstruction error minimization, has also been used as an unsupervised clustering approach to partition thermo-chemical data for adaptive chemistry applications, to speed up CFD calculations (D’Alessio et al., 2020b, 2020a). In this context, post-processing CFD data by unsupervised clustering algorithms, such as K-Means or Local PCA (LPCA), can represent an effective solution for the identification of similar thermo-chemical state zones in computational domains, by grouping computational cells that exhibit similar temperature and chemical composition. The application of clustering algorithms for the extraction of CRN from CFD data is investigated in this work, to establish a novel and automatic methodlogy for the design of equivalent CRN models of simplified configurations. The objective is to develop new solutions for an efficient design of reduced-order and physics-based combustion models, which can be used for fast input-output predictions of thermal and environmental performances, providing a useful modelling tool for future applications in combustion processes optimization, real-time monitoring and control. 2 Case study The furnace analysed in this paper is schematically represented in Figure 1. The stainless steel combustion chamber has a cubic internal section of 700mm on each side and it is well insulated with a layer of ceramic fiberboards of 200mm thick (2). The furnace has a global nominal power of 20 kW and it is equipped with a finned heat exchanger (1) to recover heat from the flue gases. Fuel and air are preheated through the recuperative heat exchanger and injected co-axially at the bottom-center of the chamber (3). The internal recirculation of flue gases, necessary to achieve MILD regime, is guaranteed by the high velocity jet. Moreover, 4 finned air-cooling tubes (4) are present, which allow for flexible variation of extracted heat from the system, thus simulating an external load. For experimental measurements, an air-cooled suction pyrometer equipped with a 1.5 mm diameter N-type thermocouple (Nicrosil/ Nisil) is used to measure the in-flame temperature profiles, while the temperature of the furnace Tf (5) and the temperature of the flue gases Tfg (6) are measured by two shielded N-type thermocouples with the position showed in Figure 1. Finally, exhaust gas composition is measured with electrochemical sensors with nominal accuracies for CO (±2 ppm), NO (±5 ppm), NO2 (±5 ppm), and O2 (±0.8% of reading) and with a non-dispersive infrared (NDIR) sensor for CO2 (±1% of reading + 0.3%). The experimental campaign showed in this paper is already available from previous works (Ferrarotti et al., 2018, Ferrarotti, 2020). Figure 1: Schematic representation of the MILD combustion furnace: (left) front section (right) view from top Figure 1: Schematic representation of the MILD combustion furnace: (left) front section (right) view from top METHODOLOGY In this work, a novel methodology for the automatic extraction of equivalent CRN models from CFD data is presented, representing a first step for future GTs applications. In particular, a numerical CFD campaign has been performed on a quasi-industrial MILD combustion furnace (Ferrarotti et al., 2017), operating at a nominal power of 15 kW and fueled with CH4 – H2 mixtures in variable proportion, with an equivalence ratio of 0.8. The CFD data have been post-processed by applying clustering algorithms, namely K-Means and Local PCA, to identify different zones in the domain by partitioning computational cells showing similar characteristic in terms of temperature and chemical composition. The identified compartments are then modeled as a network of Perfectly Stirred Reactors (PSR) by calculating their volume and the mass flowrates exchanged between clusters. The mass flowrates exchanged across the identified compartments are meant to represent the connections between the reactors, thus schematically approximating the complex flow-field of the original system. Results are then exported for detailed kinetic calculations using Chemkin Pro®, and the reactor network is solved. A sensitivity analysis with the number of clusters (or reactors) has been performed on a single case, e.g. a fixed fuel composition. The most accurate network has been tested on all the cases available, to observe the level of generalization of this approach, employing different kinetic mechanisms, to assess also the effect of the chosen kinetics. Finally, objective criteria to determine an a priori number of reactors, for each available case have been tested, relying on clustering evaluation indexes. Results are then validated upon experimental data available from previous works (Ferrarotti et al., 2018; Ferrarotti, 2020). Data pre-processing When dealing with multi-variate dataset, it is a good practice to standardize the data before applying any clustering algorithm. In particular, to the i-th variable of each observation the mean value of the i-th variable of all the observations is subtracted, in order to center the data (centering). Subsequently, each centered observation needs to be divided by a scaling value (scaling), according to the chosen criteria, in order to normalize raw variables, that usually may show very different values and scales between each other. The process of centering and scaling the i-th variable of the j-th observation can be indicated as shown in eq. 6: x2i,j= xi,j-x3i,j di (6) x2i,j= xi,j-x3i,j di (6) 𝑑# represents the scaling factor, which can be obtained following different criteria available in statistics (Parente and Sutherland, 2013). In this work, the autoscale criterium has been used, which means that 𝑑# is calculated as the standard deviation of the i-th variable considering all the observations. 𝑑# represents the scaling factor, which can be obtained following different criteria available in statistics (Parente and Sutherland, 2013). In this work, the autoscale criterium has been used, which means that 𝑑# is calculated as the standard deviation of the i-th variable considering all the observations. CFD Numerical model In order to provide the required data for subsequent CRN design, CFD simulations of the experimental cases investigated have been performed. In particular, Reynolds-Averaged Navier-Stokes (RANS) equations are solved on a 2- dimensional grid, since it has been observed that 2-dimensional domains are able to preserve spatial cell connectivity when clustering algorithms are applied. To model turbulence, the standard 𝑘−𝜖 model was employed. For chemistry, the Kee chemical mechanism, comprising 17 species and 34 chemical reactions, was chosen, representing a trade-off between chemistry details and 3 computational cost. Special attention needs to be paid to Turbulence-Chemistry Interactions (TCI) modelling, since it is known that in MILD regime, due to the diffuse and slowered combustion kinetics, induced by the oxygen dilution, chemical and turbulent mixing timescales become comparable (Lewandowski et al., 2020), thus making the hypothesis of infinitely fast kinetics not suitable for this regime. For this reason, the Partially Stirred Reactor (PaSR) (Zhiyi et al., 2017) model was employed, to correctly account for both chemical and mixing timescales. In the PaSR model, the computational cell is divided into a reactive and a non reactive part, and the final species concentration of the cell is determined by the mass exchanged from the two zones, driven by turbulence. In particular, the mass fraction of the reactive zone with respect to the computational cell is calculated as: κ = τC τmix+τC (1) κ = τC τmix+τC (1) where 𝜏! and 𝜏"#$ are the chemical and mixing timescales, respectively, that can be estimated in different ways. In this work, the approach used on the same case study, from (Ferrarotti et al., 2018), has been employed, with the static version for 𝜏"#$ evaluation, with the model constant 𝐶"#$ = 0.5 (Ferrarotti et al., 2019; Li et al., 2018). where 𝜏! and 𝜏"#$ are the chemical and mixing timescales, respectively, that can be estimated in different ways. In this work, the approach used on the same case study, from (Ferrarotti et al., 2018), has been employed, with the static version for 𝜏"#$ evaluation, with the model constant 𝐶"#$ = 0.5 (Ferrarotti et al., 2019; Li et al., 2018). CFD Numerical model The mean source term for RANS equation closure is given by a mass transfer between the reactive and the non reactive part of the cell, as follows: ωi+ = κ ρ ,Yi -Yi 0- τ (2) (2) where 𝜌 is the mixture density and 𝜏∗ represents the residence time in the reactive zone, which is modelled as an ideal reactor, evolving from 𝑌# & (nass fraction of i-th species in the non-reactive zone) to 𝑌# ∗ (mass fraction of the i-th species in the reactive zone), following a time-splitting approach: where 𝜌 is the mixture density and 𝜏∗ represents the residence time in the reactive zone, which is modelled as an ideal reactor, evolving from 𝑌# & (nass fraction of i-th species in the non-reactive zone) to 𝑌# ∗ (mass fraction of the i-th species in the reactive zone), following a time-splitting approach: dYi * dt = ωi ̇ ρ (3) Clustering CFD data Temperature and chemical composition data available from a single CFD simulation, representing the value of state variables in each computational cell, are exported, and we can indicate as 𝐱𝐣 the vector of length n)+ containing the data in the i-th cell, that may also be referred to as the i-th observation in the dataset. The data matrix X will be formed by stacking in rows the available observations, thus having n,-. rows (number of observations) and n)*+ columns. The data needs to be pre-processed, by performing centering and/or scaling according to statistics criteria, and, subsequently, a clustering algorithm is applied, by partitioning the observations into a user-defined number of clusters, following an unsupervised learning approach. Data clustering Once the data have been pre-processed, a clustering algorithm can be applied, following an unsupervised approach. K-Means is a popular algorithm (Lloyd, 1982) which aims to group the observations in k partitions by finding the best 𝜇/, or cluster centroids, according to the following minimization problem: min μj 6 6 78xj-μj87 2 (7) xj∈Cj k j=1 where 𝐶/ denotes the subdomain of data belonging to the j-th cluster, so that the sum of within-clusters Euclidean distances is minimized. The minimization is not solved as a pure optimization problem, but it is indirectly solved following an heuristic algorithm, described below: where 𝐶/ denotes the subdomain of data belonging to the j-th cluster, so that the sum of within-clusters Euclidean distances is minimized. The minimization is not solved as a pure optimization problem, but it is indirectly solved following an heuristic algorithm, described below: 1. The k-means cluster centroids are initialized and the Euclidean distance between each observation to each centroids is computed 1. The k-means cluster centroids are initialized and the Euclidean distance between each observation to each centroids is computed p 2. Each observation is labelled with the corresponding cluster index that minimize the Euclidean dista 2. Each observation is labelled with the corresponding cluster index that minimize the Euclidean distance 3. The means of all the observations in each cluster are computed, thus updating the cluster centroids. The distance i l l d i b h b i d h d d id l d (2) i d 2. Each observation is labelled with the corresponding cluster index that minimize the Euclidean distance 3. The means of all the observations in each cluster are computed, thus updating the cluster centroids. The distance is calculated again between the observations and the updated centroids values, and step (2) is repeated 4. The steps 2 and 3 are repeated until convergence is reached 3. The means of all the observations in each cluster are computed, thus updating the cluster centroids is calculated again between the observations and the updated centroids values, and step (2) is repea g 4. The steps 2 and 3 are repeated until convergence is reached 4. Data clustering The steps 2 and 3 are repeated until convergence is reached Local Principal Component Analysis (LPCA), on the other hand, is an unsupervised learning approach that also involves dimensionality reduction and it is based on the minimization of a local reconstruction error: ϵr=;xi p-xi; (8) where 𝑥# 1 is the reconstructed observation from the p-truncated dimensional space obtained by performing PCA (Kambhatla and Leen, 1997). The original, centered and scaled, data matrix 𝑿∈ℝ𝒏𝒗𝒂𝒓 is partitioned in k clusters, and in each cluster PCA is performed, thus finding k reduced basis of Local Principal Components (LPCs) 𝑨/ ∈ ℝ1 with p < nvar. At this point, for each observation it is possible to iteratively compute k local reconstruction errors, and label the observation with the cluster index for which the reconstruction error is minimum. The algorithm proceeds as follows: where 𝑥# 1 is the reconstructed observation from the p-truncated dimensional space obtained by performing PCA (Kambhatla and Leen, 1997). The original, centered and scaled, data matrix 𝑿∈ℝ𝒏𝒗𝒂𝒓 is partitioned in k clusters, and in each cluster PCA is performed, thus finding k reduced basis of Local Principal Components (LPCs) 𝑨/ ∈ ℝ1 with p < nvar. At this point, for each observation it is possible to iteratively compute k local reconstruction errors, and label the observation with the cluster index for which the reconstruction error is minimum. The algorithm proceeds as follows: 1. Initialization of the centroids 2. Partition each observation according to equation (8) 3. The cluster centroids are updated according to the new partition 4. PCA is performed in each cluster 4. PCA is performed in each cluster The steps from 2 to 4 are repeated until a convergence criteria is met: in this case until the global mean reconstruction error is below a fixed threshold and/or the cluster centroids no longer change. CRN modelling g Once the clustering has been performed, it is possible to visualize the different clusters as the identified zones in the computational domain that exhibit similar thermo-chemical characteristic. A typical output resulting from applying clustering to CFD data is represented in Figure 3. Figure 2: A typical visualization on the CFD domain of a clustering output Figure 2: A typical visualization on the CFD domain of a clustering output The volume of each cluster is calculated by summing the individual volumes of the cells belonging to that cluster. The mass flowrates across neighbors clusters are then retrieved from the CFD simulation. Results are exported in Chemkin Pro®, creating a network of Perfectly Stirred Reactors with the same volume obtained by the clustering and assigning the connections according to the computed mass flowrates. The choice of using only PSRs is justified by the small volume of the in-flame reactors, which is obtained by the clustering. To account for initial incomplete mixing of the reactants, the The volume of each cluster is calculated by summing the individual volumes of the cells belonging to that cluster. The mass flowrates across neighbors clusters are then retrieved from the CFD simulation. Results are exported in Chemkin Pro®, creating a network of Perfectly Stirred Reactors with the same volume obtained by the clustering and assigning the connections according to the computed mass flowrates. The choice of using only PSRs is justified by the small volume of the in-flame reactors, which is obtained by the clustering. To account for initial incomplete mixing of the reactants, the 5 cluster representing the injection zone is modelled as a PSR with an infinitesimal volume, thus working as a mixer, since the residence time is too low and ignition cannot take place. The boundary conditions are then specified, namely inlets, outlet and heat losses, which are estimated from the experiments and arbitrarily assigned to the largest post-flame reactor, while assuming the other reactors adiabatic. Once a detailed kinetic mechanism is specified, including all the minor pollutants, the network (mass and energy conservation equations) can be finally solved by the global solver of Chemkin Pro®, which ensure rapid convergence (~10 s). cluster representing the injection zone is modelled as a PSR with an infinitesimal volume, thus working as a mixer, since the residence time is too low and ignition cannot take place. Chemical Reactor Network results First, the effect of the clustering algorithm and the main clustering set-up parameter, namely the number of clusters chosen, needs to be assessed both in a qualitative and quantitative manner, by observing the physical shape of the resulting clustering output and by comparing the predictions of temperature and NO emissions of the extracted network with experimental data. Attention is focused on a single experimental case, namely the 75% H2 – 25% CH4 mixture, and a sensitivity analysis with the number of reactors and the clustering algorithms employed, namely K-Means and LPCA, is performed in order to assess their impact on the predictions. The GRI 3.0 (Smith et al., 2021) mechanism has been used at this stage. Then, the network which showed better agreements with experimental values, was used to simulate all the experimental cases, to assess the level of generalization of this CRN approach. The effect of the kinetic mechanism employed was evaluated at this stage, by performing simulations with three different chemical mechanisms: the GRI 3.0, GRI 2.11 (C.T. Bowman et al., 2021) and the POLIMI C1-C3 (RANZI et al., 2014). Finally, since an objective criteria to efficiently select a reasonable a priori number of cluster is needed, to have a more systematic CRN design process, a network was extracted on every case available, by choosing the number of clusters which optimizes clustering evaluation indexes available in literature (Davies and Bouldin, 1979). CFD results CFD results are shown in Figure 4. Results are in good agreement with experimental data, even though a mismatch is observed in the cases corresponding to Figure 4 (b) and 4 (e). Regarding the case in Figure 4 (b), an explaination can be related to the fact that a slight addiction of hydrogen can greatly affect the position of the reactive zone, and in that specific case can result in a transition from a pure MILD to a partially flame regime, thus being difficult to predict correctly. As for the case in Figure 4 (e), the mismatch is probably due to the model constant, which may overestimates the reactive zone. Figure 3: Comparison between the experimental and the CFD flame temperature profile Figure 3: Comparison between the experimental and the CFD flame temperature profile Chemical Reactor Network results CRN modelling The boundary conditions are then specified, namely inlets, outlet and heat losses, which are estimated from the experiments and arbitrarily assigned to the largest post-flame reactor, while assuming the other reactors adiabatic. Once a detailed kinetic mechanism is specified, including all the minor pollutants, the network (mass and energy conservation equations) can be finally solved by the global solver of Chemkin Pro®, which ensure rapid convergence (~10 s). Effect of clustering parameters The K-Means and the LPCA clustering algorithm were used to cluster the computational domain of the 75% H2 – 25% CH4 mixture, by using 5, 8 and 11 clusters, since we want to maintain a very low amout of units, to speed up the calculations. The GRI 3.0 chemical mechanism was employed and the network was solved with Chemkin ProÒ. Predictions of outlet temperature, maximum temperature and NO are compared with experimental values. Results are reported in Table 1. We can observe that increasing the number of reactors employed also results in increased accuracy, meaning that the flow-field is better represented by the augmented network structure, since more features of the flow-field itself are included. Regarding the algorithms, Local PCA shows better predictions with respect to K-Means, and it is able to match very closely experimental results in the 11 reactors case. This can be explained by observing the typical clustering output differences in Figure 3, where the Local PCA seems to better capture the flame extension, resulting in a correct prediction of the residence time spent by the mixture in the reactive zone, which is of key importance for pollutants emission characterization. The 6 maximum temperature appears a very sensitive parameter, which closely depends by the recirculation and the residence time in the ignition reactor, values that are changing when clustering parameters are modified. maximum temperature appears a very sensitive parameter, which closely depends by the recirculation and the residence time in the ignition reactor, values that are changing when clustering parameters are modified. Table 1: Results of different networks extracted on the 75% H2 case using K-Means and Local PCA with different number of reactors K-Means Variable Exp value 5 reactors 8 reactors 11 reactors Outlet temperature [K] 1255 ± 20 1281 1275 1275 Max temperature [K] 1837 ± 20 1753 1787 1944 Outlet NO [ppm] 35.6 ± 5 2.1 10.7 22.1 Local PCA Variable Exp value 5 reactors 8 reactors 11 reactors Outlet temperature [K] 1255 ± 20 1275 1275 1275 Max temperature [K] 1837 ± 20 1524 2041 1981 Outlet NO [ppm] 35.6 ± 5 3.2 51.7 36.3 sults of different networks extracted on the 75% H2 case using K-Means and Local PCA with different number of reactors CONCLUSIONS In this work, a novel methodology is presented for the extraction of Chemical Reactor Networks from CFD data using unsupervised clustering algorithms. The effect of clustering settings and parameters on the CRN output results is discussed, and simulations with CRN and detailed kinetics are performed. A possible a priori criterion for the identification of the optimal number of clusters is also illustrated. Predictions of temperature and low concentration pollutants (NO) obtained from CRN appears in good agreement with experimental values. Special attention needs to be paid to the computational time required to solve the networks, which is very small (~10 s), making the whole CRN design process and modelling very appealing for control and optimisation applications. Network generalization Figure 5: Results of CRN extracted for each single case by minimizing the Davies-Bouldin index Figure 5: Results of CRN extracted for each single case by minimizing the Davies-Bouldin index Figure 6 shows that results improved with respect to using a single network to predict the whole case set. The variation of the DB index with the number of clusters along the different cases is reported in Figure 7. Figure 6: DB index variation with the number of clusters, along the different cases Figure 6: DB index variation with the number of clusters, along the different cases We can see that no substantial variation of this index appears in varying the number of clusters in the range of fuel composition investigated, except from the case with pure H2. This means that increasing the number of cluters does not greatly affect the clustering structure and explains the little sensitivity to predictions for the case with 75% H2. However, the DB index can be very useful to reveal substantial changes in the clustering effectiveness, as with the case with 100% H2, since going from 9 to 10 clusters results in a great increase of the index value, meaning that the cells are not yet grouped in an afficient way, which can also be non-optimal for CRN applications. Network generalization g The most accurate network identified before (LPCA, 11 reactors) was applied to all the experimental cases available, to determine to which extent a CRN generated on a certain case can generalize a wide range of operating conditions. Different chemical mechanisms were employed at this stage, to assess also the effect of chemical kinetics on the obtained results. Indeed, considering the composition variability of the fuel mixtures, the properties of the chemical mechanisms can play a major role. Results in Figure 5, are showing that the CRN extracted on the 75% H2 mixture provides good predictions in closeby conditions but it fails to capture the combustion features of mixtures richer in CH4. This is due to changes in the flow-field occurring at different fuel compositions, which cannot be captured completely by one representative case, highlighting the case-specific nature of the CRN approach. Figure 4: Results from the CRN generated on the 75% H2 case on all the cases, by using different chemical mechanisms Figure 4: Results from the CRN generated on the 75% H2 case on all the cases, by using different chemical mechanisms Network extracted optimizing clustering evaluation indexes To assess the impact of quantitative metric on the a priori determination of the number of clusters, a single CRN was extracted for each experimental point, optimising, for each case, the Davies-Bouldin index (DB) (Davies and Bouldin, 1979). The DB index is calculated as the ratio of within-cluster and between-cluster distance in the data space: 𝐷𝐵 = 3 4 ∑ 𝑚𝑎𝑥 /5# F𝐷#,/G 4 #73 (9) where 𝐷#,/ is the within-to-between cluster distance ratio for the i-th and j-th clusters, or in other terms: where 𝐷#,/ is the within-to-between cluster distance ratio for the i-th and j-th clusters, or in other terms: Di,j= di++dj+ di,j (10) 7 In Eq 10, 𝑑8+ and 𝑑9+ are the average distances between each point in the i-th and j-th cluster and the centroid of i-th and j-th cluster, respectively, while 𝑑#,/ is the Euclidean distance between the i-th and j-th cluster centroids. ,/ For each experimental case, a parametric study varying the number of clusters using LPCA was performed. Then, the number of clusters minimising the DB index was chosen, and a network with that number of clusters was extracted and simulated,. Results are reported in Figure 6. The authors acknowledge the support of the Energy Transition Fund of Belgium. 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https://openalex.org/W2751775535	https://www.intechopen.com/citation-pdf-url/56524	English	null	The Role of the Informal Sector in a Rurbanised Environment	InTech eBooks	2,017	cc-by	11,925	Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) Interested in publishing with us? Contact book.department@intechopen.com Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com Open access books available Countries delivered to Contributors from top 500 universities International authors and editors Our authors are among the most cited scientists Downloads We are IntechOpen, the world’s leading publisher of Open Access books Built by scientists, for scientists 14% 191,000 210M TOP 1% 154 7,200 Chapter 3 Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.70169 http://dx.doi.org/10.5772/intechopen.70169 ( ) p Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, nd reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Economic activities performed by rural populations linked to informal trading and mar- kets have not received a broad attention in the literature. Thus, the question of the pres- ent investigation is the role of the informal sector in a rurbanised environment, and if there are differences in the waste management activities of the informal sector in cities and in an urbanised rural environment. To obtain information about the informal waste pickers in the rural areas, data were collected directly through a questionnaire from the following coun- tries (sorting in alphabetic order): Austria, the Czech Republic, Germany, Jordan, Mexico, Nepal, South Africa and Vietnam. The methodology used for the data collection consisted of a background analysis (with a literature review), complemented with the collection of empirical evidence, field interviews and partially local field analysis. The informal collection of waste is a phenomenon that results in principle from social differences within society and the population. Therefore, it is not surprising that the perception of the activities of infor- mal waste collectors in the scientific literature refers to developing and emerging countries, since social differences are more pronounced. These informal waste management systems in low- and middle-income countries exist usually in parallel with formal waste management systems, a fact that applies for urban as well as rural areas, and might be considered as a result of rurbanisation. The case studies show the development of the informal sector as an important part of the waste management activities, when a country evolves. With increasing economic development, the importance of the informal sector is shrinking step by step in relation with the improvement of the formal activities. Even this development goes faster in urban areas; the conclusion applies also to rural areas. Keywords: informal waste collection, informal recycling, waste collection in rural areas The Role of the Informal Sector in a Rurbanised Environment Petra Schneider, Le Hung Anh, Jan Sembera and Rodolfo Silva and Rodolfo Silva Additional information is available at the end of the chapter 1. Introduction The term ‘rurban’ refers to a region which has both urban and rural characteristics. Rurbanisation may be due to either urban expansion or rural migration, leading to urban– rural interactions, which result in an urbanised lifestyle in rural areas. This development manifests in rapid urbanisation of the rural population—lifestyles and mind sets—perceiv- ing cities as a source of income, stability and a possibility for better living conditions. The perception of rurbanisation goes back nearly a century. The term was firstly used by Sorokin & Zimmerman [1]. Also, Parson [2] highlighted the idea of rurbanisation, describing rurban communities as rural socio-geographic spaces where styles of life and the standard of liv- ing have changed so much that they resemble those in urban localities. This phenomenon also found in massive migration from rural to urban areas. Later research on rurbanisation by Chapuis & Brossard [3] described a population growth phenomenon observed in the rural environment due to the effect of changing the rural–urban migration patterns from the urban to the rural direction. This phenomenon was named ‘rural rebirth’, characterised by community policies [4], receptivity, land use [5], utilising neighbours [6], agricultural development [7], tourist sites, secondary residences and available homes [8], as well as end- less options [9]. Rural rebirth describes the migratory flow caused by the effects of rurbanisation on rural livelihood [9]. The rural rebirth phenomenon also reflects a special economic situation: the financial potential to afford to live a separated life in the countryside. The definition of rurban- isation exposes its effect on rural patterns: ‘rurbanisation is a process of altering rural forms with pre-selected urban patterns and lifestyles, which creates new genetically altered rurban forms’ [10, 11]. Nowadays, both types of migration are observed in parallel: rural–urban migration mainly in emerging countries resulting in the formation of megacities, and urban–rural migration mainly in industrial countries. Also, the medial impact forces the urbanisation of rural liveli- hood through advertising and sales strategies. Rurbanisation leads to a habit change in waste generation: while poor population from rural areas mostly produces organic and fast biode- gradable wastes, the more rurbanised population is consuming in a different way, causing a double consumption in comparison to traditional lifestyle and an increased waste generation of plastic, glass, metal and electronics [12]. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Solid Waste Management in Rural Areas 28 1. Introduction Recyclable materials are of interest for recyclable waste dealers, leading to the situation that rurbanisation causes activities of informal waste pickers also in the rural area. The term ‘informal’ does not give a clear definition in the literature yet. According to Chi et al. [13], informal activities are possible to be carried out ‘due to lack of legislation, structure or insti- tutionalisation in a way out of the different levels and mechanisms of the official governmental power’. Furthermore, they can be characterised as ‘not registered, and characterised as illegal’. Informal actions can therefore not be equated with such illegal acts, since the term ‘informal’ addition- ally involves legal grey zones. The term ‘informal’ thus also includes non-regulated acts and unclear defined rules [14]. The informal sector is characterised by labour-intensive, largely unregulated and unregistered, low-technology manufacturing or provision of waste collection services [15]. Informality is usually associated with undesirable developments such as tax eva- sion, unregulated enterprises and even environmental degradation [16]. Mainly in low- and The Role of the Informal Sector in a Rurbanised Environment http://dx.doi.org/10.5772/intechopen.70169 29 29 middle-income countries, the informal sector especially in the urban area reaches a significant proportion of the waste collection activity in solid waste management (SWM) as reported by Scheinberg et al. [17]: Belo Horizonte, Brazil—6.9%; Canete, Peru—11%; Delhi, India—27%; Dhaka, Bangladesh—18%; Managua, Nicaragua—15%; Moshi, Tanzania—18%; Quezon City, Philippines—31%. For rural areas, information on the percentage of informally collected waste is very rare. Even informal sector entrepreneurs in the past did not pay taxes, not have a trad- ing license and are not included in social welfare or government insurance schemes [18], since a few years there are strong activities in many developing countries to include the informal sector into the official waste management system [19, 20]. This leads to the situation that the informal sector generally achieves high recovery rates (up to 80%) because the ability to recy- cle is vital for the livelihood of people involved [19, 20]. The official waste management system in urban and urbanised areas could not be managed without waste pickers, scrap collectors, traders and recyclers. Although not officially recog- nised, they often perform a significant percentage of waste collection services, in many cases at no cost to local authorities, central governments or residents. By its nature, the activity of the informal sector is market-driven, leading to highly adaptable and flexible demand-driven informal waste collection forces. 1. Introduction Generally, the volume of waste generation in rural areas is smaller than in urban areas due to the different consumption habits of inhabitants caused by a generally smaller income. Depending on the country development level, the mean rural waste generation is reported between 0.1 (countries in Asia [12], the Middle East [21] and Latin America [12]) and 0.4 kg/cap/d (rural areas in Eastern Europe [22], the Middle East [21, 23], Asia [24] and Africa [12]). The waste generation in rural areas increases rapidly up to 0.9 kg/cap/d when a touristic infrastructure is installed, becoming comparable with urban waste generation rates in developing countries, as documented for instance from Cyprus [25] and Romania [26]. In a variety of countries, only a small share of rural population has access to waste collection services [27]. Usually, informal waste collection is carried out by poor and marginalised social groups who decide for waste picking for income generation and some even for everyday sur- vival [28]. Although urbanisation takes place in rural areas, still there are typical rural waste streams caused from rural industries like agriculture. Rural industries create waste that can be prob- lematic to manage, like silage wrap, chemical drums and chemicals. Anyhow, those materials are not of interest for potential informal collectors as they cannot be valorised by them. As in urban areas, the main focus of waste pickers of the informal sector is on recyclable materi- als, especially metals and plastics, sometimes also glass as well as paper and cardboard. The waste generation rates in rural areas of developing countries are quite comparable in the range of 0.3 (Shah et al., [2, 29], for rural areas in India) up to 0.8 kg/capday, as reported from several sources. In countries where rurbanisation goes faster, the waste generation rate is in the upper range, for instance 0.75 kg/capday (with a content of mineral recyclables of about 22%) in Iran [30]. Economic activities performed by rural populations linked to informal trading and markets have not received a broad attention in the literature [31]. Thus, the question of the present investiga- tion is the role of the informal sector in a rurbanised environment. Are there differences in the waste management activities of the informal sector in cities and urbanised rural areas? Solid Waste Management in Rural Areas 30 2. Research methods As is visible from the map, the urbanisation percentage in Austria, the Czech Republic, Germany, Jordan and Mexico was high (up to 80%), while South Africa’s level reached approximately 50%, Vietnam 30% and Nepal 10%. The information was collected from the Czech Republic, Mexico, Nepal, Vietnam, South Africa through the indicated information sources and methods: The information was collected from the Czech Republic, Mexico, Nepal, Vietnam, South Africa through the indicated information sources and methods: Austria: local data collection from primary and secondary sources, as well as information collection at a Resource Management Workshop in Austria in April 2017, Austria: local data collection from primary and secondary sources, as well as information collection at a Resource Management Workshop in Austria in April 2017, zech Republic: local data collection from primary and secondary sources, Czech Republic: local data collection from primary and secondary sources, Germany: local data collection from primary and secondary sources, as well as interviews with representatives of local waste management authorities, Germany: local data collection from primary and secondary sources, as well as interviews with representatives of local waste management authorities, Jordan: local data collection from primary and secondary sources, as well as information collection in March 2017, Jordan: local data collection from primary and secondary sources, as well as information collection in March 2017, Mexico: local data collection from primary and secondary sources, Mexico: local data collection from primary and secondary sources, Nepal: local data collection from primary and secondary sources, information received from the Solid Waste Management and Resource Mobilization Centre (SWMRMC) in Nepal. The SWMRMC made an investigation in each municipality [33], categorised them into urban and rural wards as smallest administrative unit. The rural wards are characterised through lesser population density than urban areas and without commercial activities, where the represen- tative households in each municipality were selected randomly by employing the right-hand- rule technique (Asian Development Bank [48, 49]). South Africa: local data collection from primary and secondary sources, as well as field research and interviews with the waste pickers from the informal sector in February 2017, Vietnam: local data collection from primary and secondary sources, as well as information collection at the National Farmers Union in January 2017. 2. Research methods By nature, the collected data had inhomogeneous composition, due to two reasons: firstly, the data availability strongly varied in the countries, and even concerned the type of data; secondly, not all types of data could be collected from all countries. Anyhow, for the recent scope of the investigation, the data were sufficient, as the aim of the chapter is to give an overview on the variety of settings for the informal sector. 2. Research methods The methodology for data collection consisted of a background analysis (with a literature review), complemented with the collection of empirical evidence, field interviews and par- tially local field analysis. For data collection, the interviews included the following questions: • Which are the rural waste generation rates, especially in comparison with those of urban areas? • Which are the rural waste generation rates, especially in comparison with those of urban areas? • What is the waste composition in rural areas? • What is the percentage collected by informal waste pickers? • General organisation of the rural collection systems, and especially the informal sector (informal waste pickers on the streets/landfills)? • General organisation of the rural collection systems, and especially the informal sector (informal waste pickers on the streets/landfills)? • What kind of waste do informal waste pickers collect? • Are they an official part in the official waste management system? Generally, the status of the informal sector is hardly documented in the literature, and most of the available data on the informal sector were collected for urban areas. Some information for rural areas is from Latin America (Colombia, Brazil), and from Africa, which was collected for this study. The reason for the poor documentation is supposed to be the informal status of the waste pickers and their ‘hiding’ from the statistics. To obtain information about the informal waste pickers in the rural areas, the information was collected directly by a question- naire from the following countries (sorting in alphabetic order): Austria, the Czech Republic, Germany, Jordan, Mexico, Nepal, South Africa and Vietnam. Figure 1 shows the location of the investigated countries in the UNICEF map of the urbanised population percentage by Figure 1. Urbanised population percentage by country in 2006. Map source: UNICEF, The State of the World’s Children 2008 (p. 134) [32]. Figure 1. Urbanised population percentage by country in 2006. Map source: UNICEF, The State of the World’s Children 2008 (p. 134) [32]. The Role of the Informal Sector in a Rurbanised Environment http://dx.doi.org/10.5772/intechopen.70169 31 31 country in 2006 [32]. As is visible from the map, the urbanisation percentage in Austria, the Czech Republic, Germany, Jordan and Mexico was high (up to 80%), while South Africa’s level reached approximately 50%, Vietnam 30% and Nepal 10%. country in 2006 [32]. 3.1. Situation in high-income countries: Germany, Austria, and the Czech Republic According to OECD information, Germany has 80.6 million inhabitants with an average household income of 34,700 US$. The average waste generation rate varies between 0.65 and 1.37 kg/cap/d in rural areas, in comparison to urban areas with waste generation rates between 1.37 and 2.2 kg/cap/d, having a total average of 1.68 kg/cap/d [34]. The waste col- lection system is a selective system, which separates recyclables (glass, paper, plastic (PET) bottles, other plastics, metals and biodegradable waste) from residual waste. The waste man- agement system is operated by municipal or communal operators, and only exceptionally by private operators, a situation which applies for rural and urban areas. The activities that can be considered as a type of informal sector activity are some private poor people who collect bottles and cans from the streets in order to transfer them to the bottle deposit refund system, which exists in an automated way in each supermarket or rural discounter. For instance, in Germany, the refund for one PET bottle or one metal can is 0.25 € (0.27 US$), while the refund for a glass bottle is 0.08 € (0.09 US$). The deposit refund was calculated according to the environmental risk (PET bottles) or the material value (metal cans), and is equal in rural and in urban areas. In Germany, a deposit-refund- ing system for bottles of alcoholic beverages does not yet exist, but it is under governmen- tal preparation (status as of February 2017). For that reason, the waste bottles most often found in the environment are bottles of alcoholic beverages which are not of interest to informal collectors. Formal collectors provide glass containers, where consumers put those types of bottles and packaging glass for material valorisation. The existing system fulfils the scope of a clean environment, and private bottle pickers are the exception. Furthermore, all municipal landfills were closed by law in 2005, and landfills for the disposal of untreated municipal waste have not existed anymore since then. All generated waste has to undergo a pre-treatment, before recycling or re-using as priority options, and only hazardous waste is disposed of. The described situation is the reason that informal waste collectors on landfills do not exist at all in Germany. 3. Investigation results The results are a summary of the collected data for each country, which gives information on the collection scheme, as well as the involvement and the activity of the informal sector in the respective countries. Generally, it was observed that the informal sector existed in urban and rural areas; even the quantity of waste collected was smaller in the rural areas. Furthermore, settlements of the informal sector can be found in the areas of communal dumpsites and landfills, collecting already recyclable materials before the waste goes to the dumpsite and Solid Waste Management in Rural Areas 32 landfill. Furthermore, the extent of the activity of the informal sector depends on the type and structure of the collection system in the country. Usually, when the collection system is not a selective system separating the recyclables, there is a larger activity of the informal sec- tor. This is usually the case in low- and middle-income countries. In high-income countries like Germany in Central Europe, a real informal sector does not exist. 3.1. Situation in high-income countries: Germany, Austria, and the Czech Republic Other waste is not collected by private waste pickers, as all waste streams are collected in selective collection schemes through formal collection systems of the municipalities, which is valid for urban as well as for rural areas, centralised in civic amenity areas. In zones which are close (up to 50 km) to the East European border (with Poland or the Czech Republic), there are informal East European waste collectors (especially from Romania, Hungary, Poland and the Czech Republic) [35] waiting outside the civic amenity centres to collect usable waste directly from the customers who are bringing waste to the centres. Usually, they are collecting household appliances, textiles, toys and other items for children, sports equipment, electrical appliances such as TV sets, washing machines or refrigerators, tires, scrap metals and other The Role of the Informal Sector in a Rurbanised Environment http://dx.doi.org/10.5772/intechopen.70169 33 bulky waste, for instance from furniture. The transfer of this kind of waste is free of charge, and even it is not really allowed, it is tolerated, and in this way informal by nature. A comparable situation does exist in Austria. The country has 8.5 million inhabitants with an average household income of 45,500 US$. The average waste generation rate being 1.58 kg/cap/d is slightly lower than in Germany, having generally comparable dimensions to Germany for rural and urban areas. A visit to Austria in April 2017 indicated a certain per- centage of waste bottles in the urban area spread around public collection bins while there was nearly no waste in rural areas in the environment. The result of the interview indicated that there is a comparable deposit refund system like in Germany, but obviously it appeared not to be efficient everywhere, maybe because the deposit refund was too small. In Austria, bottles are partly pledged. For simple reusable beer bottles, 0.09 € (0.10 US$) are refunded and 0.36 € (0.40 US$) for special types of beer bottles. For reusable PET bottles as used by some mineral water and lemonade manufacturers, a 0.29 € (0.33 US$) deposit is charged, as well as for 1-l mineral water glass bottles. Anyhow, relevant informal activities are prac- ticed for the same materials as in Germany, which can be considered as a particularity of a high-income country. Also an informal waste transfer from Austria to Eastern Europe coun- tries by informal waste collectors does exist. According to Obersteiner et al. 3.1. Situation in high-income countries: Germany, Austria, and the Czech Republic [35], 69% of the informal waste collectors in Austria originate from Hungary and 19% from Austria. The rest comes from Bulgaria, the Czech Republic, Slovenia, Slovakia and Romania. Istvan et al. [36] reported that informal waste collectors from Hungary even travel for waste collection to the Netherlands. According to Obersteiner et al. [37], a verification at the Hungarian border showed that the collected items were 47.21% by volume of furniture, 18.77% by volume of electrical appliances and 13.19 Vol% of metals. Also, the Czech Republic is considered a high-income country; even waste collectors from the Czech Republic come to the neighbouring countries like Germany and Austria, as the income there is even higher. The Czech Republic has 10.5 million inhabitants and an average house- hold income of 17,542 US$. According to the income, which is proportionally lower than in Germany or Austria, the average waste generation rate is also lower: 0.8 kg/cap/d, and also significantly lower than the EU average of 1.3 kg/cap/d. No refund is applied for aluminium cans or plastic bottles in the Czech Republic, only some kinds of glass bottles are refunded for 3 Kč (approximately 0.11 US$). That is why the ‘secondary’ collection of this type of waste is negligible there. The Waste Law of the Czech Republic orders the municipalities and communes to arrange waste collection places so that some parts of the waste (esp. glass, paper, plastic, metals and biowaste) should be collected separately. All rural areas are administrated by their central municipalities, meaning that law and waste management in rural and in urban areas are the same. The informal waste collectors are active in the Czech Republic, even being gypsies like in Romania and Hungary. The waste proportion collected by them is finally included into the waste that is recycled by the recycling companies and in that way included into the statis- tics. The informal sector usually collects metals that can be simply sold. They sometimes also steal some metal parts of working systems (electrical wires, railway security systems, monu- ments, sewer covers, etc.) and sell them as metal waste. They are not foreseen to be a part Solid Waste Management in Rural Areas 34 of the official system even there are some laws and procedures to prevent them. 3.1. Situation in high-income countries: Germany, Austria, and the Czech Republic Generally, the informal waste pickers are much more active in the poor areas of the country (Northern Bohemia or Northern Moravia) than in the rich regions. An investigation carried out by Tydlitatova et al. [38] in several rural communes in the Czech Republic on the impact of the implementation of the system pay as you throw (PAYT) showed that the villages, which applied Local Tax system, produced 47% more of mixed municipal waste. The villages that applied Local Tax generated an average of 0.52 t of mixed municipal waste per 5 years, and more than the villages that applied the fee by Act on Waste [38]. The results according to Tydlitatova et al. [38] are given in Table 1. The example from the Czech Republic shows that not only the average household income has an impact on the waste generation rate but also the system of payment of waste fees. Higher fees have a regulating impact and cause lower waste generation rates. Municipality Population (2011) Applied waste law (2011) Fee per person or dustbin Fee per person or dustbin Distance to landfill, km Horažďovice 5578 Local Tax CZK 600/person US$ 24/person 43 Horoměřice 3335 Local Tax CZK 480/person US 19/person 6 Jílové u Prahy 4222 Local Tax CZK 500/person US$ 20/person 1 Mnichovice 3069 Fee by Act on Waste CZK 1750/120 l US$ 70/120 l 35 Psáry 3331 Fee by Act on Waste CZK 2145/120 l US$ 86/120 l 53 Říčany 13,499 Contractual form by Act on Waste CZK 2520/120 l US$ 101/120 l 36 Statenice 1261 Local Tax CZK 600/person US$ 24/person 6 Table 1. Waste management system in several rural municipalities [38]. Table 1. Waste management system in several rural municipalities [38]. Table 1. Waste management system in several rural municipalities [38]. In the following discussion, the focus is on low- and middle-income countries which all face the issue of informal waste pickers, in the urban as well as in the rural areas. The respective countries are considered in alphabetic order. In the following discussion, the focus is on low- and middle-income countries which all face the issue of informal waste pickers, in the urban as well as in the rural areas. The respective countries are considered in alphabetic order. 3.2. Jordan (lower middle-income country) Jordan is a lower middle-income country in the Middle East, with an original number of inhabitants of 6.5 million in 2013 (data of OECD), which increased through migrants from Iraq and Syria recently by at least 2 million; 21.2% of the inhabitants live in the rural areas [39]. The annual average household income is approximately 5160 US$, with a large varia- tion. The average waste generation is 0.9 kg/cap/d in urban areas and 0.6 kg/cap/d in rural areas. Jordan is quite densely populated, and the existing informal waste collection sector has undergone an even higher competition after a large number of migrants entered the coun- try to search for possibilities to ensure their income for living, as reported in interviews in March 2017. This kind of situation was recently also observed in Turkey, where the existing The Role of the Informal Sector in a Rurbanised Environment http://dx.doi.org/10.5772/intechopen.70169 35 The Role of the Informal Sector in a Rurbanised Environment http://dx.doi.org/10.5772/intechopen.70169 35 Figure 2. Flow chart for solid waste streams and scavengers role in Jordan ([39], adapted). Figure 2. Flow chart for solid waste streams and scavengers role in Jordan ([39], adapted). well-organised informal sector got quite under economic pressure caused by a stronger com- petition. Resource recovery and recycling are practised in a limited way, even those of urban areas are clean and free from street waste. In the rural areas, there is a higher percentage of waste beside the roads, and it is obvious that there is cleaned or collected much more seldom. well-organised informal sector got quite under economic pressure caused by a stronger com- petition. Resource recovery and recycling are practised in a limited way, even those of urban areas are clean and free from street waste. In the rural areas, there is a higher percentage of waste beside the roads, and it is obvious that there is cleaned or collected much more seldom. A well-documented study on the informal sector in the rural and rurbanised environment of Jordan was provided by Aljaradin et al. [39], which analysed the informal recycling activities carried out by a scavenger in the Tafila region of Jordan. The general situation is given in Figure 2, and it is a typical situation for a variety of low- and lower-middle-income countries. 3.2. Jordan (lower middle-income country) There is no legislation which forbids scavengers to pick and recycle waste but the Ministry of Social Development always tracks them for children working as waste pickers [39]. The infor- mal recycling in Jordan was estimated to be around 10% from the total municipal solid waste (MSW) generated. As shown in Figure 2, their activities are carried out before the solid waste reaches the final disposal sites for the separation of recyclable materials, but the majority of informal collection is done at the disposal sites. The informal waste collectors are welcomed as they reduce the cost of formal waste management systems. The materials most often collected are aluminium, plastic, paper, cardboard, glass, copper and iron [39]. The average quantity collected by 100 scavengers per day is reported with 150-kg soft drink cans, 5-kg aluminium stripes, 2-kg copper wires and 90-kg scrap metals. The average waste composition contains biodegradable waste (52%), plastics (17%), paper/ cardboard (14%), glass (3%), metals (1%) and others (17%) (Karak et al. [40]). The composition of the scavenger crowd in the Tafila region is 99% men and 1% woman [39], with 80% being less than 25 years old. The majority of the informal waste pickers in Tafila (78%) obtain a monthly income of >250 € (268 US$), the others <250 €. As Aljaradin et al. [39] reported, scav- engers usually have no concept of the essential role of their work in the waste management activities, and their social status is very low. 3.3. Mexico (upper middle-income country) Mexico is a country in Latin America with 122 million inhabitants. The annual average household income is 12,800 US$. The average waste generation in rural communities is 0.68–1.09 kg/cap/d [41]. In other studies, carried out in rural communities in Mexico the interval found is between Solid Waste Management in Rural Areas 36 0.28 and 0.58 kg/cap/d [41, 42], indicating urbanised behaviour. It can be assumed that the dif- ferences result from the consideration of agricultural wastes. A study carried out in eight com- munities from Michoacan, Mexico [42], points a composition of 44% of food scraps, 8% of yard trimmings, 2% of cardboard, 2.8% of paper and 0.6% of textiles [41]. In comparison, the per-capita MSW generation in the urban area ranged from 327 to 361.35 kg/inhabitant/year from 1995 to 2012 [43]. As in other developing countries, also in Mexico, the informal sector exists, which is con- cerned with the recovery of waste, but an investigation to quantify the contribution regard- ing the recovery of recyclables [44] would be necessary. According to Taboada-González et al. [41], in some rural communities of Mexico, waste collection is provided by the munici- pality through the Department of Waste Management (DWM) at no charge. The waste is collected once a week at the curbside where residents place their garbage bins. Afterwards, waste is disposed of in each community’s dumpsite. The percentage of coverage of waste col- lection services in the rural area is 60%, making it clear that the DWM does not totally collect the waste generated by the communities, being inefficient in most of the cases. The rest of the waste is usually mismanaged and burned outdoors or discarded at ravines, uncultivated land and canals. Also, an unquantified fraction is collected by informal collection services that offer their services in exchange of a gratuity. Also in Mexico a deposit refund system exists. Figure 3. Informal refuse collection in Netzahualcoyotl, Mexico. Photo by Medina [45]. Figure 3. Informal refuse collection in Netzahualcoyotl, Mexico. Photo by Medina [45]. In Mexico, scavenging and informal refuse collection (IRCs) is very common (Figure 3) [45]. In many cases, rag pickers recover some valuable materials (aluminium, tin can and ferrous waste) and the rest is dispersed to be burned outdoors. Waste picking is done near the source, that is, after collection has taken place at the generating sources and previous to being transported to the dump or landfill. Table 2. Recyclables prices in Mexico [46]. Table 2. Recyclables prices in Mexico [46]. 3.3. Mexico (upper middle-income country) The most common way of selling the collected material is directly to the companies that attend the site daily [43]. Materials such as aluminium, tin cans and fer- rous waste are collected by waste pickers in rural communities [41]. Waste pickers working in The Role of the Informal Sector in a Rurbanised Environment http://dx.doi.org/10.5772/intechopen.70169 37 the landfill collect mainly plastic (PET), also aluminium cans, plastic (HDPE) and metals. The material is selected for collection in terms of the market for each product [44]. According to Medina [43], in some towns, informal refuse collectors pick up garbage and charge each home a fee between US$ 0.10 and 0.50 (Figure 4). In many cases when they operate in a place far from the municipal disposal sites, they take the collected waste to privately operated transfer sta- tions and pay a fee of US$ 1–4 for unloading wastes there, depending on the amount. Hence, in addition to collection fees, they recover recyclables from the wastes, which, considering the fees they pay, results in an average income of US$ 9–15 a day, which is between three and five times the minimum wage. Being so, in many cases IRC is a highly paid activity. Figure 4. Informal refuse collection in Tultepec, Mexico. Photo by Medina [45]. Figure 4. Informal refuse collection in Tultepec, Mexico. Photo by Medina [45]. MXN Peso/kg US$/kg Paper 3.00 0.156 Newspaper 1.50 0.078 Glass 0.60 0.031 Plastic PET 3.00 0.156 Cardboard 2.00 0.104 Aluminium 17.00 0.884 Food tins 17.00 0.884 Metal 2.00 0.104 Magazines 3.00 0.156 Solid Waste Management in Rural Areas 38 In many towns not just in Mexico but in Latin America, the informal sector has been used as a semi-official tool to bring services to low-income areas, which offers a more open system, responding to basic needs and demands [44]. Table 2 shows the recyclable prices in Mexico [46]. In many towns not just in Mexico but in Latin America, the informal sector has been used as a semi-official tool to bring services to low-income areas, which offers a more open system, responding to basic needs and demands [44]. Table 2 shows the recyclable prices in Mexico [46]. Generally, there is a direct relationship between producers and consumers in the informal sector, requiring low capital, which allows for more rapid growth. 3.4. South Africa (upper middle-income country) The country located in the Southern Africa has 70 million inhabitants with an annual aver- age income of 5845 US$. The average waste generation is 1.7 kg/cap/d in urban regions and 0.35 kg/cap/d in rural regions [47]. Results of a topic-related research of the Council for Scientific and Industrial Research South Africa on the informal waste sector indicated that between 60,000 and 90,000 waste pickers earn a livelihood from the recovery of recyclables from municipal waste in South Africa. This intensive informal sector, which especially also works in the rural areas, provides a valuable, and low-cost recycling solution. While the informal sector in the urban areas is going to be formalised step by step, the informal sector in rural areas is mainly living from the activity of private recycling companies. Generally, the situation in the urban area is easier and more economic for an informal waste picker than in the rural area. In the urban area, for instance, in Bloemfontein, the informal sector was somehow formalised through green T-shirts, which must be bought, and represent the official allowance to collect recyclables. In this way formalised, the waste picker can act as glass recycler and earn up to 12,000 ZAR/y (approximately 923 US$/y). In the rural areas, the informal collection is a very difficult job. Usually, the informal sector col- lects the recyclables at landfills, means on landfills with an informal allowance to enter them, or in front of the landfill at the entrance, or on the rural road, which is connecting the landfill. There, the informal recyclers even stop cars, which are on the way to the landfill. Besides those activities, also conventional collection activities in the villages do exist, even they are not the majority of the activities leading to income for the informal waste recyclers. Generating income with informal activities in South Africa is a quite unpredictable activity. As the waste collectors reported in interviews in February 2017, they do not know when the private waste recycling company sends the trucks to collect the waste of the informal sector, which usually happens twice a year, sometimes only once a year, but the date is not announced. This leads to the situation that the informal waste collectors need to establish waste storage sites (usually outside landfills), where the recyclable waste fractions are already pre-sorted and packed to be ready for the collection by the recycler in each moment. 3.3. Mexico (upper middle-income country) However, this peculiar nature of the informal sector makes monitoring and regulation more difficult, for which it has resulted in the inefficiencies previously mentioned. Hence, as stated by Medina [45], incorpo- rating informal collection services into the municipal waste systems and formal programmes could bring some control over their operations and stop illegal dumping. 3.4. South Africa (upper middle-income country) Such constellation leads to informal settlements for the purpose of waste collection and manual pre-sorting. The payment is small: 2 ZAR per kilogram metal (0.15 US$), 1 ZAR per kilogram plastics (0.08 US$) and 1 ZAR per kilogram glass (0.08 US$). If the collection activity goes properly, and the private recy- cling company sends the collection truck, an informal waste recycler can earn approximately The Role of the Informal Sector in a Rurbanised Environment http://dx.doi.org/10.5772/intechopen.70169 39 39 6000 ZAR/y (approximately 461 US$/year). This annual income of an informal waste collector in the rural areas compares to half of an average monthly income of a worker in an urban area. There are nearly no women doing this kind of job in the rural recycling settlements. 6000 ZAR/y (approximately 461 US$/year). This annual income of an informal waste collector in the rural areas compares to half of an average monthly income of a worker in an urban area. There are nearly no women doing this kind of job in the rural recycling settlements. Figures 5–7 show impressions of an informal waste recycling settlement in the rural areas of eMalahleni, taken in February 2017. Figure 5. Informal waste-recycling settlement in the rural areas of South Africa, close to eMalahleni: PET collection (photos taken by the authors on 26 February 2017). Figure 5. Informal waste-recycling settlement in the rural areas of South Africa, close to eMalahleni: PET collection (photos taken by the authors on 26 February 2017). Figure 6. Informal waste-recycling settlement in the rural areas of South Africa, close to eMalahleni: glass collection (photos taken by the authors on 26 February 2017). Figure 6. Informal waste-recycling settlement in the rural areas of South Africa, close to eMalahleni: glass collection (photos taken by the authors on 26 February 2017). Solid Waste Management in Rural Areas 0 Figure 7. Informal waste-recycling settlement in the rural areas of South Africa, close to eMalahleni: living conditions at the informal recycling village (photos taken by the authors on 26 February 2017). Figure 7. Informal waste-recycling settlement in the rural areas of South Africa, close to eMalahleni: living conditions at the informal recycling village (photos taken by the authors on 26 February 2017). 3.5. Nepal (low-income country) Nepal is a country in Eastern Asia which consists of mountains, hills and a lowland region which is called Terai. The country has 28 million inhabitants and an annual average house- hold income of 701 US$. For the study of the Solid Waste Management and Resource Mobilization Centre [33], a total sample size of 3330 households from 60 municipalities in the rural areas selected from all ecological zones was considered, having 55 house- holds that gave an average per-capita household waste generation of 0.12 kg/cap/d [33]. The data base for Nepal shows that the household waste generation rates in new munici- palities varied depending upon the economic status. The average waste generation cor- relates with the monthly available household income. Households with a monthly budget of NRs ≥40,000 (about 389 US$) generate 0.88 kg/day, in comparison to 0.4 kg/day for households with a monthly budget of less than NRs ≤5000 (about 49 US$) [48]. The results of the study indicated a per-capita household waste generation from a minimum of 0.07 kg/cap/day (Bheriganga Municipality) to a maximum of 0.22 kg/cap/day (Bhojpur Municipality) [48]. The characteristics of MSW collected from any area depend on various factors such as con- sumer patterns, food habits, cultural traditions of inhabitants, lifestyles, climate, economic status, and so on. Composition of urban waste is changing with increasing use of packaging material and plastics. The average household waste composition investigated in 60 munici- palities in terms of the eight determining waste components (organics, plastics, paper and paper products, glass, metal, rubber and leather, textiles and others like inert and dust) is presented in Figure 8 [33]. The Role of the Informal Sector in a Rurbanised Environment http://dx.doi.org/10.5772/intechopen.70169 41 Figure 8. Average composition of household waste of 60 rural municipalities, with average values by % wet weight [33]. http://dx.doi.org/10.5772/intechopen.70169 Figure 8. Average composition of household waste of 60 rural municipalities, with average values by % wet weight [33]. The average composition of household waste comprises as highest fractions organic matter (68%), followed by plastics (10%), paper and paper products with 8% as well as other types of waste with 8.6% [33]. The rest, being below 4% wet weight, was glass, metal, rubber and leather, and textile components [33]. 3.5. Nepal (low-income country) Of total surveyed households from 60 municipalities, 51% responded that they are practicing segregation of waste at sources, which is higher than that of survey findings from 58 munici- palities conducted in 2012 [33]. The higher segregation at sources in new municipalities is because of rural nature of these municipalities where almost all households were found to segregate kitchen waste for their own purpose, for example, feeding cattle and using for tradi- tional type composting, and so on. Moreover, only 33% surveyed households have compost- ing practices of segregated waste while 37% do not have such composting practices in their households, which means that the segregated waste at source is mixed again during collection and transportation due to the lack of separate collection and treatment methods [48]. In many of the new municipalities, a solid waste collection system does not exist, and if the system exists, it is not satisfactory due to unscientific composting, or open burning, or throw- ing the waste in the open space around [33]. Only 2% of surveyed households sell segregated non-biodegradable fraction to informal sectors. 52% respondents told that they do burn of segregated non-biodegradable waste like plastics and papers, while remaining either throw into road drains or do both [49]. Collection, city cleaning and sweeping do not happen on a daily basis [33], and only main market and roads are served daily. Other areas are served intermittently, from twice a week to twice a month [33]. Many areas in the rural environ- ment are neglected due to inefficiency and inadequacy of service [49]. Although a concept of material recovery from MSW with legal provisions of sorting waste at sources has been Solid Waste Management in Rural Areas 42 already introduced in Nepal through the new solid waste management act (SWMA) promul- gated in July 2011, no formal municipal waste recovery and recycling programme exist in the municipalities [48]. Because of municipal budget constraints, municipalities try to create a sound budget without increasing cost-efficiency option, but arrive at the point that MSW has become environmental, financial and social burden to each municipality [33]. The conclusion of the investigation was that only 2% of surveyed households sell segregated reusable and recyclable fraction to informal sectors, being considered as very minimum resource recovery activities in the surveyed municipalities [33]. Figure 9 shows informal workers collecting recyclable materials, Belabari Municipality, Nepal. Figure 9. 3.5. Nepal (low-income country) Informal workers collecting recyclable materials, Belabari Municipality, Nepal (photo source: Solid Waste Management Technical Support Center [33], with friendly permission of SWMRMC Nepal). Figure 9. Informal workers collecting recyclable materials, Belabari Municipality, Nepal (photo source: Solid Waste Management Technical Support Center [33], with friendly permission of SWMRMC Nepal). 3.6. Vietnam (low-income country) Vietnam is a country in South East Asia, having a population heading towards 90 million inhabitants. The annual average household income was 1912 US$ in 2014. The economy of Vietnam is agriculture with paddy rice as major crop cultivated on 4.5 million ha land. In addition to the main product, rice grain, by-products such as rice straw and husk (renewable resource) are also produced, estimated to be around 38 million tonnes of straw and 6 to 7 million tonnes of husk per year for whole Vietnam [50]. The country is very densely populated. People live in all types of organisational forms, cover- ing sizes from the rural community to megacities. The agricultural sector in the rural areas plays a fundamental role in Vietnam, as it provides the nutrition for the growing population. The average waste generation in the rural area is 0.4–0.5 kg/cap/d (excluding agricultural wastes). The organic content of the generated waste is very high, up to 90%. Plastic bags form The Role of the Informal Sector in a Rurbanised Environment http://dx.doi.org/10.5772/intechopen.70169 43 43 a percentage up to 15%. A serious problem is the percentage of hazardous waste, especially bags, bottles and cans which contained pesticides. In private households, the waste is often burned. Only a small percentage is composted. The waste collection is organised in different ways in the provinces, usually comprising two levels: (a) Waste collection from the households through companies, NGOs (e.g. Farmers Union, Women Union, Veterans) or private waste pickers (informal sector). The waste collec- tion fee is stipulated by themselves, as well as the determination of the collected fees (payment for employees or investments). Informal waste pickers exist everywhere in the rural areas, and they mainly collect plastics, paper and metal. Only a small percent- age of the collected waste is recycled, the majority is put on landfills. In areas, which are far from the official collection and recycling infrastructure, the waste is deposited into illegal dumpsites. (b) Waste collection at the landfills of the province. The central waste management company URENCO allows private waste pickers to collect recyclables from the deposited waste and pays for this service. Vietnam produces many biodegradable wastes in the rural areas, which are mainly not recy- cled in the current state. The biomass contributes to the rural waste generation, and even it is recyclable, it is not yet properly valorised. 3.6. Vietnam (low-income country) For the informal sector, it is not an interesting waste stream. Currently, in the urban and rural areas in Vietnam, a bottle deposit refund system for beer and soft drink bottles does exist. The bottles refund for one box with 20 beer or 24 soft drink bottles is 20.000–40.000 VND (0.88–1.76 US$). The recyclable materials are bought by collectors from households with the following prices: Cardboard: 3000–4000 VND/kg (0.1–0.18 US$) Cardboard: 3000–4000 VND/kg (0.1–0.18 US$) Paper: 4000–5000 VND/kg (0.18–0.22 US$) White cleaned covers from nylon and plastics: 12,000 VND/kg (0.53 US$) Coloured cleaned covers from nylon and plastics: 10,000 VND/kg (0.44 US$) Dirty covers from nylon and plastics: 2000 VND/kg (0.09 US$) PET bottles: 4500 VND/kg (0.20 US$) Iron scrap: 4800 -10,000 VND/kg (0.21–0.44 US$) Aluminium scrap: 20,000 VND/kg (0.88 US$) Copper scrap: 60,000–90,000 VND/kg (2.65–3.97 US$). Paper: 4000–5000 VND/kg (0.18–0.22 US$) Figures 10 and 11 show informal workers collecting recyclable materials in the Mekong region in Vietnam. Solid Waste Management in Rural Areas 44 44 Figure 10. Informal workers collecting recyclable materials, Mekong region, Vietnam (photos taken by the authors on 14th august 2017). Figure 10. Informal workers collecting recyclable materials, Mekong region, Vietnam (photos taken by the authors on 14th august 2017) Figure 10. Informal workers collecting recyclable materials, Mekong region, Vietnam (photos taken by the authors on 14th august 2017). Figure 11. Informal workers collecting paper and cardboard, Mekong region, Vietnam (photos taken by the authors on 14th august 2017). Figure 11. Informal workers collecting paper and cardboard, Mekong region, Vietnam (photos taken by the authors on 14th august 2017). The Role of the Informal Sector in a Rurbanised Environment http://dx.doi.org/10.5772/intechopen.70169 45 4. Discussion The informal collection of waste is a phenomenon that results from social differences within society and the population. Therefore, it is not surprising that the perception of the activi- ties of informal waste collectors in the scientific literature refers to developing countries and emerging countries, since social differences are more pronounced. These informal waste man- agement systems in low- and middle-income countries usually exist in parallel with formal waste management systems, and this applies for urban as well as rural areas and might be considered as a result of rurbanisation. The case studies show the development of the infor- mal sector as an important part of the waste management activities, when a country starts to develop. With increasing economic development, the importance of the informal sector is shrinking step by step in relation with the improvement of the formal activities. Even this development goes faster in urban areas; the conclusion applies also to rural areas. Although organic waste is the main waste stream in rural areas, there is a relevant proportion of infor- mal activities on recyclables like metals, plastics, papers and glass. One of the main focuses of the formal waste management activities in the urban areas is to find solutions for the inclusion of the informal sector into the formal activities, and in this way, it is formalisation. In order to support the consideration of formalisation options of the informal sector, a further literature search was carried out. The aim was to reconcile the approaches used in other countries and to consider as far as possible a wide range of ideas. Thus, in principle, the following approaches exist in the literature: • Incentive systems for the disposal of certain wastes. • Umbrella organisation for informal collectors outside the waste regime. • Incorporating informal waste collectors into a commercial waste management company. • Incorporating informal waste collectors into a commercial waste management company. • Establishment of an umbrella organisation within the waste regime. • Second-hand goods trading through municipal waste management. • Consideration in national ReUse concepts. 4. Discussion In Brazil, the waste collector was recognised as a profession. There are three types of organisations: (1) unorganised and anonymous waste collectors, which are not affiliated with any organisation, (2) organised waste collectors organised in cooperatives and associations, and, as a rule, at least 10 years of professional experience, as well as contract-bound waste collectors, mainly working in scrap metal, in metal works and also in the municipal sector. In Brazil, there are 229,568 waste collectors, of which 67% are men, 25% are between 50 and 64 years old and 7% are over 65 years old. Only 14% of them have a school-leaving certificate. Approximately 4.5% work in a formal contract, which include 11,781 people. The contractually employed persons have a median income of about three to four times higher. (D) Official recognition of the waste collector is a profession. In Brazil, the waste collector was recognised as a profession. There are three types of organisations: (1) unorganised and anonymous waste collectors, which are not affiliated with any organisation, (2) organised waste collectors organised in cooperatives and associations, and, as a rule, at least 10 years of professional experience, as well as contract-bound waste collectors, mainly working in scrap metal, in metal works and also in the municipal sector. In Brazil, there are 229,568 waste collectors, of which 67% are men, 25% are between 50 and 64 years old and 7% are over 65 years old. Only 14% of them have a school-leaving certificate. Approximately 4.5% work in a formal contract, which include 11,781 people. The contractually employed persons have a median income of about three to four times higher. (E) Another possibility to improve the problematic situation of informal waste management would be to take account of informal collectors with the introduction of a national re-use system. The informal collectors could fulfil the need for new capacity in waste collection centres, such as storage areas and labour, or take over the transports between the munici- pal collection points and the socio-economic enterprises, or assist the employees of social economy enterprises with their repair knowledge. (F) Integration of the informal sector into the development of re-use and repair networks in co-operation with socio-economic integration companies. In this formalisation, the informal collectors function according to Scherhaufer et al. [51] as transporters for socio- economic enterprises. 4. Discussion The following basic options for the integration of the informal sector were determined in the lit- erature research in the form of case studies (in accordance with the investigations of Hold, [14]): The following basic options for the integration of the informal sector were determined in the lit- erature research in the form of case studies (in accordance with the investigations of Hold, [14]): (A) Involvement of informal waste collectors with the municipality by means of a contracted subcontract to an unionised unit, for example, for certain geographically defined admin- istrative units and/or for the lower-income areas. An example is Maputo (Mozambique): The informal waste collectors supply the recyclable waste to a centre for the sorting and pre-treatment of valuable substances. The recycling centre sells high-quality, recycled plastics to the local recycling industry and is independent of external financial support. (B) Involvement of informal waste collectors by specialising in a specific type of waste. In Delhi and Bangalore (India), numerous informal workers recycle electrical and electronic (B) Involvement of informal waste collectors by specialising in a specific type of waste. In Delhi and Bangalore (India), numerous informal workers recycle electrical and electronic Solid Waste Management in Rural Areas 46 waste. The informal recyclers received training in the risk of their work and appropriate recycling techniques. Finally, several small recyclers of electronic waste joined together and were able to register themselves officially. In the treatment of hazardous substances, the new company cooperates with an experienced, formal recycler. The newly founded and registered company has established itself on the market and offered its employees an improved job situation. (C) Specified waste refunds for certain recyclable waste types and quantities. In Bogota (Colombia), informal waste collectors receive 28.78 US$/tonne of waste collected at officially authorised collection points in the city; 13,754 informal waste collectors are found in Bogota, 58% of them are women; 1200 tonnes of waste are collected daily by them. The average income per collector is 3.41 US$/d. (C) Specified waste refunds for certain recyclable waste types and quantities. In Bogota (Colombia), informal waste collectors receive 28.78 US$/tonne of waste collected at officially authorised collection points in the city; 13,754 informal waste collectors are found in Bogota, 58% of them are women; 1200 tonnes of waste are collected daily by them. The average income per collector is 3.41 US$/d. (D) Official recognition of the waste collector is a profession. 5. Conclusions Scope of the current investigation was to collect data from literature and through field stud- ies in order to obtain information on the informal sector activities in the rural areas, work- ing in a rurbanised environment. A general conclusion from the questionnaires and field visits is that the informal sector exists also in rural areas; even the generation of recyclable waste is smaller than in urban areas. Therefore, the income of rural informal waste pickers is lower than that of urban waste pickers. As the informal sector in the rural area is usually concentrated near the landfills, they use recyclable materials going to the landfill in several ways to make their living. Usually they collect metals, glass, PET bottles and sometimes also papers. Potential differences in the waste management activities of the informal sec- tor in cities and in an urbanised rural environment can be stated at this investigation stage that the urban sector shall be usually formalised at a certain development stage, while this is usually not yet the case in the rural area. Further, like in other commercial sectors, the income in rural areas is usually less than in urban areas. The percentage of women in this sector is negligibly low. Most of the middle- and low-income countries deal with an informal waste sector. And usually, each respective country faces a number of unique socio-economic and political circumstances that may influence the integration of the informal sector into a formal sec- ondary resources economy. Anyhow, one question in this regard is: What model of social inclusion of waste pickers would be most appropriate in the respective country, means integration and/or formalisation? A discussion in a recent workshop at the Chamber of Commerce, held in Istanbul in October 2016 was on the subject of the inclusion of the informal sector into the official waste management system. The informal sector also par- ticipated. Surprisingly, not all of the members of the informal sector agreed to be included in the formal waste management organisation. Most of them told the freedom of their working conditions as reason. Having in view the process of rurbanisation and the economic development of the low- and middle-income countries, informal waste pickers are at present an important part of the system. It is to be expected that with increasing economic infrastructural development, their relevance will be decreasing on the long term, but not necessarily. 4. Discussion A separate collection of reusable items is carried out in the waste collection centres in question, which are then transported to the ReUse plants by the infor- mal collectors. The delivered items are then sorted according to their functions in ReUse operation. They are divided into items with or without the need for repairs [14]. The mentioned general options could be applied also in the rural areas; even the probability of the feasibility of some particular options is higher (in accordance with the investigations of Hold, [14]), as there are: • Option (A) could be applied also on communal basis in rural areas. • Option (B) appears not as an applicable option as the waste volumes in rural areas are small- er, and with this the proportion of usable recyclables is smaller. The example from South Africa shows a remarkable volume of recyclables needs time to be collected in the rural areas. The Role of the Informal Sector in a Rurbanised Environment http://dx.doi.org/10.5772/intechopen.70169 47 47 • Option (C) is an interesting option, which could allow for a constant cash flow or income. • Option (C) is an interesting option, which could allow for a constant cash flow or income. • Option (C) is an interesting option, which could allow for a constant cash flow or income. • Option (D) is a challenge. It would be useful to recognise waste recycling as profession, especially for the informal sector. This option could be combined with the special clothing of the waste recyclers in order to recognise them faster as officially working people. • Option (D) is a challenge. It would be useful to recognise waste recycling as profession, especially for the informal sector. This option could be combined with the special clothing of the waste recyclers in order to recognise them faster as officially working people. • Option (E): this option would work also in rural areas, but it requires as precondition that the country is already in a certain developed stage. • Option (E): this option would work also in rural areas, but it requires as precondition that the country is already in a certain developed stage. • Option (F): this option applies to a higher-developed society which already has a devel- oped waste management infrastructure. • Option (F): this option applies to a higher-developed society which already has a devel- oped waste management infrastructure. Acknowledgements Collection of data on the informal sector is not feasible without the support of many people, starting from the waste pickers through recycling companies to waste management authori- ties. We are grateful to all of them. Thanks to Pieter, Abraham, Vusi, Skumbulu, Sam, Given and Mandla from an informal collection location in South Africa; thanks to Prof. Dr. Christian Wolkersdorfer who gave technical support to conduct the interviews with the informal waste collectors in South Africa. Thanks also to Dr. Sumitra Amatya from the Solid Waste Management and Resource Mobilization Centre in Nepal, to Mrs. Birgit Dietz from the Vogtlandkreis Waste Authority in Germany; thanks to Ing. Danuše Hráská from The Czech Environmental Inspectorate. We hope that with this contribution we were able to put the situation of the informal sector in the awareness focus of the public and the special situation which exists in rural areas. 5. Conclusions In Vietnam, for instance, the informal sector is included in the waste management system as official power Solid Waste Management in Rural Areas 48 already to collect recyclables from landfill (e.g. Nam Son in Hanoi). On the other hand, it might be possible that the informal sector covers especially rural regions, which are less developed in infrastructure and/or which are far from the next recycling centres and not economically manageable with formal waste management activities. In such case, the infor- mal sector could be able to manage those regions with its technical means, for instance horse carriage (like in several East European countries), or smaller motorised vehicles. Anyhow, the main waste stream in rural areas which will not be managed by the informal sector is the organic waste. Author details Petra Schneider1, Le Hung Anh2, Jan Sembera3 and Rodolfo Silva4 Address all correspondence to: petra.schneider@hs-magdeburg.de 1 Department of Water, Environment, Civil Engineering, and Safety, University of Applied Sciences Magdeburg-Stendal, Magdeburg, Germany 2 Industrial University of Ho Chi Minh City, Ho Chi Minh City, Vietnam 3 Faculty for Mechatronics, Informatics and Interdisciplinary Studies, Technical University of Liberec, Liberec, Czech Republic 4 FGlez Consulting & Associates, Mexico City, Mexico Petra Schneider1, Le Hung Anh2, Jan Sembera3 and Rodolfo Silva4 Address all correspondence to: petra.schneider@hs-magdeburg.de p p g g 1 Department of Water, Environment, Civil Engineering, and Safety, University of Applied Sciences Magdeburg-Stendal, Magdeburg, Germany 1 Department of Water, Environment, Civil Engineering, and Safety, University of Applied Sciences Magdeburg-Stendal, Magdeburg, Germany 2 Industrial University of Ho Chi Minh City, Ho Chi Minh City, Vietnam 3 Faculty for Mechatronics, Informatics and Interdisciplinary Studies, Technical University of Liberec, Liberec, Czech Republic 3 Faculty for Mechatronics, Informatics and Interdisciplinary Studies, Technical University of Liberec, Liberec, Czech Republic 3 Faculty for Mechatronics, Informatics and Interdisciplinary Studies, Technical University of Liberec, Liberec, Czech Republic 4 FGlez Consulting & Associates, Mexico City, Mexico 4 FGlez Consulting & Associates, Mexico City, Mexico 4 FGlez Consulting & Associates, Mexico City, Mexico [1] Sorokin P, Zimmerman CC. 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https://openalex.org/W2605519552	http://pure-oai.bham.ac.uk/ws/files/55868087/Bowen_et_al_Moods_Clinical_Depression_Frontiers_Psychiatry.pdf	English	null	Moods in Clinical Depression Are More Unstable than Severe Normal Sadness	Frontiers in psychiatry	2,017	cc-by	6,118	Moods in Clinical Depression Are More Unstable than Severe Normal Sadness Bowen, Rudy; Peters, Evyn; Marwaha, Steven; Baetz, Marilyn; Balbuena, Lloyd DOI: 10.3389/fpsyt.2017.00056 License: Creative Commons: Attribution (CC BY) Citation for published version (Harvard): Bowen, R, Peters, E, Marwaha, S, Baetz, M & Balbuena, L 2017, 'Moods in Clinical Depression Are More Unstable than Severe Normal Sadness', Frontiers in Psychiatry, vol. 8, 56. https://doi.org/10.3389/fpsyt.2017.00056 Citation for published version (Harvard): Bowen, R, Peters, E, Marwaha, S, Baetz, M & Balbuena, L 2017, 'Moods in Clinical Depression Are More Unstable than Severe Normal Sadness', Frontiers in Psychiatry, vol. 8, 56. https://doi.org/10.3389/fpsyt.2017.00056 Link to publication on Research at Birmingham portal Download date: 24. 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Moods in Clinical Depression Are More Unstable than Severe Normal Sadness Objective: Current descriptions in psychiatry and psychology suggest that depressed mood in clinical depression is similar to mild sadness experienced in everyday life, but more intense and persistent. We evaluated this concept using measures of average mood and mood instability (MI). Method: We prospectively measured low and high moods using separate visual analog scales twice a day for seven consecutive days in 137 participants from four published studies. Participants were divided into a non-depressed group with a Beck Depression Inventory score of ≤10 (n = 59) and a depressed group with a Beck Depression Inventory score of ≥18 (n = 78). MI was determined by the mean square successive difference statistic. Edited by: Edited by: Renerio Fraguas, University of São Paulo, Brazil Reviewed by: Casimiro Cabrera Abreu, Queen’s University and Providence Care, Canada Angela Marie Lachowski, Ryerson University, Canada Serafim Carvalho, Hospital de Magalhães Lemos, Portugal Results: Mean low and high moods were not correlated in the non-depressed group but were strongly positively correlated in the depressed group. This difference between correlations was significant. Low MI and high MI were weakly positively correlated in the non-depressed group and strongly positively correlated in the depressed group. This difference in correlations was also significant. Conclusion: The results show that low and high moods, and low and high MI, are highly correlated in people with depression compared with those who are not depressed. Current psychiatric practice does not assess or treat MI or brief high mood episodes in patients with depression. New models of mood that also focus on MI will need to be developed to address the pattern of mood disturbance in people with depression. Correspondence: Rudy Bowen r.bowen@usask.ca Correspondence: Rudy Bowen r.bowen@usask.ca Specialty section: This article was submitted to Mood and Anxiety Disorders, a section of the journal Frontiers in Psychiatry Received: 13 December 2016 Accepted: 29 March 2017 Published: 12 April 2017 Take down policy down policy the University of Birmingham exercises care and attention in making items available there are rare occasions when an it ded in error or has been deemed to be commercially or otherwise sensitive. this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access ely and investigate. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we the work immediately and investigate. Download date: 24. Oct. 2024 Original Research published: 12 April 2017 doi: 10.3389/fpsyt.2017.00056 Keywords: depression, distress, low mood, high mood, mood instability INTRODUCTION The purpose of this paper is to distinguish between sadness and depression. Current descriptions suggest that depression is caused mainly by exposure to stress and the ups and downs of life (1, 2). If depression were simply extreme sadness, then patients would have more control over symptoms, and it would be less stigmatizing as a consequence (3, 4). Freud provided a clinical foundation for this idea by concluding that mourning and melancholia were comparable because the symptoms are similar, they are both precipitated by loss and improve with time (5). He noted that people with melancholia could become over-talkative and manic but did not adequately explain why this is so (5). Hypothesis People who are depressed (distressed with negative mood and symptoms) are more likely to have more strongly cor- related low and high unstable moods than people who are not depressed. Kraepelin was a notable exception in that he emphasized brief moods swings and rapidly alternating mood symptoms in patients with manic–depressive illness (9). Consistent with his work, recent studies have shown that patients with mood disor- ders report increased affective lability and emotional instability; and this also occurs during euthymic periods in bipolar patients (10–12). Citation: Bowen R, Peters E, Marwaha S, Baetz M and Balbuena L (2017) Moods in Clinical Depression Are More Unstable than Severe Normal Sadness. Front. Psychiatry 8:56. doi: 10.3389/fpsyt.2017.00056 April 2017 \| Volume 8 \| Article 56 Frontiers in Psychiatry \| www.frontiersin.org 1 Bowen et al. Depression More Complicated than Sadness (40). In this study, we investigated how MI might characterize the experience of people who were depressed as compared with those who were not depressed. Other influential writers in psychiatry and psychology have endorsed similar notions. Bowlby compared negative emo- tions in adults to those that occur during separation from the attachment figure in infants (6). Beck attributed depression to dysfunctional negative thoughts of defeat, failure, and rejection (7). He dismissed mood-swings as a normal phenomenon (7). Although Watson paid more attention to mood instability (MI), he also dismissed negative mood variability as mundane and of little importance (8). Participants We used data from participants in four controlled studies (n = 168) that we have published (32, 41–43). All patients had been referred by family physicians for treatment and were all under treatment at the time of the study. In three of the stud- ies, males and females had been referred to general outpatient practices, in one study women had been referred for alcohol abuse, but all had been alcohol free for 3 weeks. All patients completed mood diaries (described in the next section) over a week. All of the studies received approval from the university ethics board. The patient group (n = 104) was assessed with the Mini-International Neuropsychiatric Interview (MINI English Version 5.0.0 for DSM-IV), and 49 met criteria for major depres- sion (44). There was high comorbidity with anxiety disorders. Twenty-two of those with major depression (45%) reported hyperthymic symptoms in the past, which did not meet criteria for hypomania (45). The controls (n = 64) included health-care personnel and 17 graduate students. Depression could be understood as a consequence of loss or stress, but if high moods do occur during a depressive episode, they tend to be ignored (2, 6), dismissed (7, 8), or isolated to separate categories with low prevalences of about 1% such as bipolar mood disorder (13) and borderline personality disorder (14). Recently, DSM-5 has introduced the specifier “with mixed features” to acknowledge manic/hypomanic symptoms, but three out of seven symptoms are required (13). Any attempt to expand the bipolar spectrum to account for brief periods of high mood (15–17) has been vigorously criticized (18–21).h The reliance on retrospective methods for clinical and research interviewing (22) has made it easier to dismiss brief high moods because retrospective recall in depressed people is biased toward the negative. Recalling mood over the previous 2 weeks is more likely to result in smoothing away mood variation (23–25). If so, the raw data for clinically diagnosing major depression are systematically distorted (26). Ecological momentary assessment, which asks patients to record affect at a given moment over a specified duration, provides a fuller picture of mood by capturing variation in addition to severity. As early as 2006, the US Food and Drug Administration recommended that pharmaceutical companies make use of real-time data instead of patient recollec- tion (26). Increasingly, smartphones are being used as a tool for ecological momentary assessment in psychological and clinical studies (27, 28). Frontiers in Psychiatry \| www.frontiersin.org RESULTS In the original group of referred patient participants (N = 104), mean low mood was correlated with low MI; as were mean high mood and high MI (p < 0.001). In the original group of controls (N = 64), mean low mood was correlated with low MI (p < 0.001); as were mean high mood and high MI (p < 0.01). Procedure P MI seems to be the essential component of neuroticism (35, 36) and is an anteced- ent to major depression (37), psychotic symptoms (38), severity of distress (39), suicidal thoughts (35), and self-harm behavior April 2017 \| Volume 8 \| Article 56 2 Depression More Complicated than Sadness Bowen et al. TABLE 1 \| Mood and mood instability (MI) in depressed and non- depressed groups. Non-depressed Depressed t df Sig (two- tailed) N Mean SD N Mean SD – – – Low mood 59 1.50 1.34 78 2.95 2.22 −4.73 129.22 <0.00 High mood 59 4.81 1.69 78 3.05 2.13 5.41 134.68 <0.00 Low MI 59 1.72 1.30 78 2.40 1.49 −2.81 135 <0.01 High MI 59 2.84 1.06 78 2.44 1.60 1.77 132.94 0.08 Low, high mood: mean visual analog scale mood ratings for low and high moods. Low, high MI: mean square successive difference statistic for low mood and high mood. TABLE 1 \| Mood and mood instability (MI) in depressed and non- depressed groups. Non-depressed Depressed t df Sig (two- tailed) N Mean SD N Mean SD – – – Low mood 59 1.50 1.34 78 2.95 2.22 −4.73 129.22 <0.00 High mood 59 4.81 1.69 78 3.05 2.13 5.41 134.68 <0.00 Low MI 59 1.72 1.30 78 2.40 1.49 −2.81 135 <0.01 High MI 59 2.84 1.06 78 2.44 1.60 1.77 132.94 0.08 Low, high mood: mean visual analog scale mood ratings for low and high moods. Low, high MI: mean square successive difference statistic for low mood and high mood. TABLE 1 \| Mood and mood instability (MI) in depressed and non- depressed groups. Mood Instabilityh The depressed group experienced more severe low MI than the non-depressed group, as consistent with the findings of the original individual studies. The difference in high MI between the depressed and non-depressed groups was not significant. Table 2 shows the important finding that low MI and high MI were cor- related in both the depressed and the non-depressed groups, but the magnitude of correlation for the depressed group (r = 0.61) was almost twice that for the non-depressed group (r = 0.31). The difference between these correlations was significant (z = −2.18, p = 0.03, two-tailed). Analysis We used the BDI scores to divide the participants into two distinct groups: those who were within a “normal” range of BDI scores (BDI ≤10) (n = 59) (47) and those with BDI scores ≥18 or who were likely clinically depressed (n = 78) (46, 47). The remaining participants (n = 31) fell outside of these two groups and were excluded from the sample. We used the conventional terms “non-depressed” (BDI ≤10) and “depressed” (≥18) to des- ignate the two groups without assuming that the cut-score of BDI ≥18 defined a latent class of major depression or any particular form of depression. The mean BDI scores of the non-depressed and depressed groups were 4.51 (SD: 2.59) and 20.05 (SD: 13.89), respectively. We used Pearson’s correlation as a measure of association between low and high moods. We then tested whether the correlations between (a) mean low and high mood and (b) low and high MI were different between the non-depressed and depressed groups. This comparison of correlation magnitudes is, in principle, similar to Meehl’s MAXCOV procedure in which the correlation of two variables is compared along successive cuts in a third variable (22). Sample and Group Demographicsh The participants (n = 137) ranged in age from 15 to 64 years (mean age = 30.0 years, SD = 10.9), and 104 (75.9%) were females. The “depressed” group (n = 78) (mean age = 30.62, SD = 11.60; 80.8% females) was composed of 51 patients, 24 volunteers as “controls” in the original studies, and 3 graduate students. The “non-depressed group” (n = 59) (mean age = 29.15 years, SD = 9.66; 69.5% females) included 40 people who volunteered as controls from the original studies, 14 graduate students, and 5 people referred as patients. There was no difference in age or sex distribution between the two groups. In the non-depressed group, the overall means of low mood and high mood were uncorrelated. This supports the observa- tion that in normal people low and high moods are not strongly related and easily distinguished (8). In the depressed group, however, mean low mood and mean high mood were moderately positively correlated. This indicates that the depressed group experienced high moods concurrent with low moods (54, 55). This is contrary to the common-sense view that low mood should not be associated with high moods. Procedure P Participants completed the Beck Depression Inventory-IA (46, 47), which is a 21-item retrospective self-report questionnaire. Statements are presented in the first-person (“I feel sad”), and subjects select the item that best reflects their recent state from a choice of four items. It is reliable and correlates well with other measures of depression but may in part assess a general distress or neuroticism factor (48–50). In a study with undergraduate students, the BDI showed strong latent dimensional structure that is there was no evidence that a cut-score defined a latent class of depression (50). Another way by which recalled moods are distorted is the “common-sense” view that low mood and high mood are mutu- ally exclusive or negatively correlated (8, 29). Since negative affect predominates in depressed people, brief episodes of positive affect tend to be subsumed under overall gloom in patient recollections. There is evidence, however, that positive and negative affect are independent of each other (30) and that people can feel both happy and sad at the same time (31). Participants completed separate visual analog scales (VAS) (8) for low mood and high mood. They rated their moods in the morning after awakening and at night before bedtime, for 7 consecutive days (41, 51). The anchor points were “not at all” and “very much so.”i For clarity, we define “low mood” as participant ratings for “sad/blue” or “depressed” over 1 week (twice daily ratings for a total of 14 ratings). Similarly, “high mood” is defined as partici- pant ratings for “enthusiastic/interested” or “high mood.” “Low MI” refers to the fluctuation of low mood, and “high MI” refers to the fluctuation of high mood over the same period. MI is operationalized as the mean square successive difference (MSSD) statistic across 14 ratings (52). One can think of MSSD as the SD of ratings, taking temporal sequence into account. Among clinical samples, when low and high moods are meas- ured prospectively on separate axes, the rapid cyclic recurrence of high moods with low moods becomes apparent, a phenomenon known as mood instability (11, 32–34). MI has been shown to exist in up to 13.9% of the adult population (34). DISCUSSION On the VAS ratings, the depressed group experienced more severe low moods and less severe high moods than the non-depressed group, as would be expected given the selection criteria. This is consistent with reports of more severe negative emotions and variable positive emotions in ecological momentary assessment studies of patients with major depression (12, 33, 53). Mean Mood Table 1 shows mood scores for both groups. Compared to the non- depressed group, the depressed group experienced more severe low mood (t = −4.73, df = 129, p < 0.001) and less severe high mood (t = 5.41, df = 135, p < 0.001), consistent with the selection into non-depressed and depressed groups. Table 2 shows mean low and mean high mood correlations for both groups. Notably, in the non-depressed group, the correlation between mean low and mean high moods was not significant (r = −0.01), but in the depressed group, mean low and high moods were positively correlated (r = 0.38). The Fisher r-to-z transformation indicated that the difference between these correlations was significant (z = −2.30, p = 0.02, two-tailed). Low MI and high MI were weakly correlated in the non- depressed group. In the depressed group, the correlation was moderate to large, and the difference between these correlations was significant. In other words, in the depressed group, the fluc- tuations of low moods and high moods are more closely related (Table 2). Taken together, these results suggest that in people with depression, mood is a complex combination of rapidly fluctuating seemingly polar opposite emotions. This distinction is more easily understood if MI is considered along with stable low mood (56). Other studies have shown complex emotional patterns in anxiety April 2017 \| Volume 8 \| Article 56 Frontiers in Psychiatry \| www.frontiersin.org 3 Depression More Complicated than Sadness Bowen et al. TABLE 2 \| Correlations.a Non-depressed Depressed n r0 95% confidence limits n r 95% confidence limits Z Sig of the difference in r0 and r (two-tailed) Correlation mean low mood and high mood 59 −0.01 −0.27–0.25 78 0.38b 0.17–0.55 −2.3 0.02 Correlation mean square successive difference (MSSD) (mood instability) low mood and MSSD high mood 59 0.31a 0.06–0.53 78 0.61a 0.45–0.73 −2.18 0.03 aCorrelations between mean low and mean high moods for the non-depressed and depressed groups and MSSD low and MSSD high moods for the non-depressed and depressed groups. bCorrelation is significant at the 0.05 level (two-tailed). and mood disorders (12, 56–58). Two clinical applications are (a) that the usual semi-structured retrospective assessment might provide a limited appreciation of “nuanced” mood symptoms (12) and (b) that attention to mood stabilization might add an extra dimension to treatment (12, 59). Mean Mood In other words, since MI reflects neuroticism (36) that is an antecedent of depression (60), attention to the assessment and treatment of MI in addition to specific symptoms of depression (61, 62) might increase the treat- ment efficacy. in physical parameters such as blood pressure (66) and blood sugar (67) and without assumptions as to cause. MI is consid- ered to be a simpler concept than “affective instability due to a marked reactivity of mood” (13) or emotional dysregulation (68). One question for further studies would be whether MI leads to unstable interpersonal relationships or vice versa. The relationship of MI to interpersonal and social–environmental events is a matter for further research. Instability of low moods is not entirely accounted for by reactivity to negative events (12). Finally, associating MI with neuroticism does not detract from evidence that certain kinds of stress can affect depression (69, 70). The main advantage to this study is the longitudinal collec- tion of data, which is likely to give a more accurate depiction of moods than retrospective studies (23).h fi Our study had several methodological limitations. First, the number of participants was relatively small and from one center. Second, the wording of the questions for low mood and high mood varied slightly between studies, although the words were similar. This might be an advantage by reflecting real-world interviewing conditions. Third, paper and pencil diaries were used, raising the possibility of people retrospectively filling in data. Participants understood that they were to record momentary mood ratings, and the method of calculating MI was not intuitively apparent to the participant (52). Furthermore, all of the individual study results produced clear differences suggesting that the participants understood the instructions. Fourth, choosing graduate students as controls in one of the studies was a convenience sample, but we considered that graduate students would be generally more stable than undergraduates and that they would be older and closer in age to patients. Fifth, for simplicity we considered only low and high moods. Moods in broadly defined depression would likely appear even more complex and distressing if anxiety, irritability, and psychotic symptoms were included (63). Sixth, five people in the “non-depressed” group had been referred as patients. These people may have improved while waiting for treatment or may have had symptoms that were not detected by the way that they completed the BDI. Mean Mood Finally, during the diagnostic interview we did not probe sufficiently for hypomanic symptoms during the depressive episode, so we cannot say how many of the partici- pants would have met criteria for the DSM-5 category of major depression with mixed features (13).h The results show that low and high moods, and low and high MI, are highly correlated in people with depression compared with those who are not depressed. Current psychiatric practice does not assess or treat MI or brief high mood episodes in patients with depression. New models of mood that also focus on MI will need to be developed to address the pattern of mood disturbance in people with depression. ETHICS STATEMENT This study was carried out in accordance with the recommenda- tions of the Canadian Tri-Council Policy Statement for Ethical Conduct in research involving humans with written informed consent from all subjects. All subjects gave written informed con- sent in accordance with the Declaration of Helsinki. The protocol was approved by the University of Saskatchewan Behavioural Ethics Board. AUTHOR CONTRIBUTIONS RB: study conception, drafting the article, revised the article after peer review, and responded to reviewer comments. EP: contribut- ing to the concepts as well as reading and adding to the final ver- sion. SM and MB: contributing to the concepts as well as reading and providing approval to the final version. LB: conception of the work, analysis of the data, and revised the article after peer review. There are also limitations to the conclusions that can be drawn. The correlation between low and high MI might not be the best representation of complex emotions. The data do not address the question of the relative merits of continuous or categorical approaches to classification in psychiatry. 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This is an open-ac- cess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 58. McGrath JJ, Saha S, Al-Hamzawi A, Andrade L, Benjet C, Bromet EJ, et al. The bidirectional associations between psychotic experiences and DSM-IV mental disorders. Am J Psychiatry (2016) 173(10):997–1006. doi:10.1176/appi. ajp.2016.15101293 April 2017 \| Volume 8 \| Article 56 Frontiers in Psychiatry \| www.frontiersin.org 6
https://openalex.org/W4292386161	https://iris.unitn.it/bitstream/11572/352320/3/journal.pbio.3001773.pdf	English	null	Designing and implementing a research integrity promotion plan: Recommendations for research funders	PLoS biology	2,022	cc-by	6,336	CONSENSUS VIEW OPEN ACCESS Citation: Horbach SPJM, Bouter LM, Gaskell G, Hiney M, Kavouras P, Mejlgaard N, et al. (2022) Designing and implementing a research integrity promotion plan: Recommendations for research funders. PLoS Biol 20(8): e3001773. https://doi. org/10.1371/journal.pbio.3001773 * jk.tijdink@amsterdamumc.nl Designing and implementing a research integrity promotion plan: Recommendations for research funders Serge P. J. M. Horbach1, Lex M. Bouter2, George Gaskell3, Maura Hiney4, Panagiotis Kavouras5, Niels Mejlgaard1, Nick Allum6, Noe´mie Aubert Bonn7, Anna- Kathrine Bendtsen1, Costas A. Charitidis5, Nik Claesen8, Kris Dierickx9, Anna Domaradzka10, Andrea Reyes Elizondo11, Nicole Fo¨ger12, Wolfgang Kaltenbrunner11, Teodora Konach12, Krishma Labib7, Ana Marusˇić13, Daniel Pizzolato9, Tine Ravn1, Rea Roje14, Mads P. Sørensen1, Borana Taraj8, Giuseppe A. Veltri15, Joeri K. TijdinkID2,7* 1 Aarhus University, Aarhus, Denmark, 2 Vrije Universiteit Amsterdam, Amsterdam, the Netherlands, 3 London School of Economics and Political Science, London, United Kingdom, 4 Health Research Board, Dublin, Ireland, 5 National Technical University of Athens, Athens, Greece, 6 University of Essex, Colchester, United Kingdom, 7 Department of Ethics, Law and Humanities, Amsterdam University Medical Centers, Amsterdam, the Netherlands, 8 European Association of Research Managers and Administrators, Brussels, Belgium, 9 KU Leuven, Leuven, Belgium, 10 Robert Zajonc Institute for Social Studies, University of Warsaw, Warsaw, Poland, 11 Centre for Science an Technology Studies, Leiden University, Leiden, the Netherlands, 12 Austrian Agency for Research Integrity, Vienna, Austria, 13 University of Split School of Medicine, Split, Croatia, 14 Department of Research in Biomedicine and Health, University of Split School of Medicine, Split, Croatia, 15 University of Trento, Trento, Italy * jk.tijdink@amsterdamumc.nl Published: August 19, 2022 AU : Pleaseconfirmthatallheadinglevelsarerepresentedcorrectly: Various stakeholders in science have put research integrity high on their agenda. Among them, research funders are prominently placed to foster research integrity by requiring that the organizations and individual researchers they support make an explicit commitment to research integrity. Moreover, funders need to adopt appropriate research integrity practices themselves. To facilitate this, we recommend that funders develop and implement a Research Integrity Promotion Plan (RIPP). This Consensus View offers a range of examples of how fund- ers are already promoting research integrity, distills 6 core topics that funders should cover in a RIPP, and provides guidelines on how to develop and implement a RIPP. We believe that the 6 core topics we put forward will guide funders towards strengthening research integrity policy in their organization and guide the researchers and research organizations they fund. Copyright: © 2022 Horbach et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by the European Commission HORIZON 2020 framework programme for Research and Innovation under Grant Agreement number 824481—project "Standard Operating Procedures for Research Integrity—SOPs4RI". The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. To improve research quality and validity, foster responsible research cultures, and maintain public trust in science, various stakeholders have put research integrity high on their agenda. Among them, research funders are increasingly acknowledging their pivotal role in contribut- ing to a culture of research integrity. For example, the European Commission (EC) is mandat- ing research organizations receiving funding from the €95 billion Horizon Europe program to have, at the institutional level, policies and processes in place for research integrity covering the promotion of good practice, prevention of misconduct and questionable practices, and iledforthoseusedthroughoutthetext:Pleaseverifythatallentriesarecorrect: Abbreviations: AU : Anabbreviationlisthasbeenco EC, European Commission; ECoC, European Code of Conduct for Research Integrity; FNR, Fonds National de la Recherche Luxemburg; HRB, Health Research Board; NIH, National Institutes of Health; NSF, National Science Foundation; RCR, Responsible Conduct of 1 / 12 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 PLOS BIOLOGY Research; RIPP, Research Integrity Promotion Plan; SOPs4RI, Standard Operating Procedures for Research Integrity. procedures to deal with breaches of research integrity [1]. To meet these obligations, the EC requires beneficiaries to respect the principles of research integrity as set out in the European Code of Conduct for Research Integrity (ECoC) and suggests that research organizations develop and implement a Research Integrity Promotion Plan (RIPP) [2]. In this Consensus View, we have adopted the World Conference on Research Integrity’s approach to research integrity, by having “research integrity” refer to “the principles and standards that have the purpose to ensure validity and trustworthiness of research” [3]. More specifically, we mostly adhere to the principles outlined in the ECoC: reliability, honesty, respect, and accountability. While many definitions of research integrity exist [4,5], for example, those that distinguish between the integrity of a researcher, integrity of research, and integrity of the research record, the ECoC combines these approaches in a balanced way [1]. We believe that funders are prominently placed to foster a culture of research integrity by requiring that the organizations and individual researchers they support make an explicit com- mitment to research integrity. At the same time, funders need to adopt appropriate research integrity practices themselves. Of late, attention to research integrity among funders has gath- ered pace, as reflected in several initiatives around the globe that demonstrate how funders can support a culture of research integrity. Competing interests: The authors have declared that no competing interests exist. For example, the US National Science Foundation (NSF) [6] requires applicants’ research organizations to provide training and oversight in the responsible conduct of research, designate individuals responsible for research integrity, and have an institutional certification to testify of its commitment. Also, in 2016, 3 Canadian fed- eral funders joined forces to support research integrity in the Canadian Tri-Agency Frame- work: Responsible Conduct of Research–Harmony and Responsibility [7]. The framework was subsequently updated in 2021. This framework sets out responsible practices that research organizations and researchers should follow, including rigor, record keeping, accurate referencing, responsible authorship, and the management of conflicts of interest. It also acknowledges the responsibilities of the funders, including “helping to promote the responsi- ble conduct of research and to assist individuals and institutions with the interpretation or implementation of this Responsible Conduct of Research (RCR) Framework”. It is not only major funding organizations in highly developed research environments that are taking steps. Smaller funders are also acting to mandate compliance with research integrity standards. The constantly growing literature on the topic is another sign of development within this area [2,3]. In the USA, research integrity recently reached the political arena, when, following a call from researchers [8], President Biden’s administration published a memoran- dum on restoring trust [9] that highlights the importance of integrity in research. The memo- randum will be supported by the reintroduction of the Scientific Integrity Act. This act will prohibit research misconduct and the manipulation of research findings. It talks of a “culture of research integrity” and demands that funding agencies adopt and enforce research integrity policies, appoint a research integrity officer, and provide regular research integrity and ethics training. The US are not alone in their endeavors. Governments in other countries are equally gearing up to support the integrity and reproducibility of research [10]. However, so far, there is only limited evidence about the effectiveness of such initiatives, although it is generally accepted that they raise awareness among various stakeholders concerning research integrity challenges, strengthen the sense of responsibility of those stakeholders to address those chal- lenges, and thereby ultimately contribute to fostering a culture of research integrity. In a collective effort to foster research integrity, research organizations and funders have their own, complementary roles. The Standard Operating Procedures for Research Integrity (SOPs4RI) consortium has recommended that both research organizations and funders develop a RIPP. Research; RIPP, Research Integrity Promotion Plan; SOPs4RI, Standard Operating Procedures for Research Integrity. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 Methodology The recommendations in this article are based on the extensive empirical program applied in the context of the SOPs4RI project. In particular, we used a systematic literature review to obtain an overview of existing efforts to foster research integrity [12]. Subsequently, we engaged in consultations with key stakeholders within the funder community to actively solicit their views and interpretations of what constitutes a responsible research culture and how funding organizations, in their local and contextual capacities, can contribute to such a culture. These consultations were conducted using several methodological approaches, including Del- phi surveys [13], expert interviews, focus groups [14], and co-creation workshops [15]. All these consultation efforts have built on each other, with the results of the Delphi study acting as the starting point for prioritizing the topics that were deemed most important for funders, and feeding into the interviews and focus group, which subsequently formed the basis of the co-creation workshops. The 6 topics described in the following section were finally agreed on after intensive deliberations of the author team, based on the empirical material gathered throughout our project. As a final step, we have set up an assessment system to prioritize initia- tives according to a diverse group of stakeholders and project members. These initiatives are collected, divided under the 6 topics, and included in our online toolbox. Competing interests: The authors have declared that no competing interests exist. A RIPP outlines the key responsibilities of an organization concerning research integrity and details methods and procedures to foster it. For example, in the case of PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 2 / 12 PLOS BIOLOGY research organizations, a RIPP should facilitate and stimulate a healthy research environment, proper mentoring and supervision, research ethics structures, research integrity training, high-quality dissemination practices, research collaboration, effective data management, and open and fair procedures to deal with breaches of research integrity [11]. Funders have a dif- ferent role. They can support, safeguard, and incentivise, or even mandate, responsible research practices from research organizations and researchers. Equally important, funders should make sure that their internal processes live up to the highest standards of research integrity. We recognize that funders are many and varied in their scale, portfolio, disciplinary focus, and the extent to which they have procedures and governance arrangements to support research integrity. For all funders, adopting a RIPP will structure and coordinate research integrity practices, giving clarity and transparency to applicant institutions and researchers. In this Consensus View, we highlight examples of best practice of funders worldwide to fos- ter a research integrity culture. With these examples in mind, we suggest guidelines to support funders in taking a leading role in fostering research integrity. In so doing, we acknowledge the local contexts in which funders operate, but we believe that all funders, large and small, in all parts of the world, can and should contribute to improving research validity and building and maintaining trust in science through incentivising and mandating a culture of research integrity. Our core argument is that developing a tailored RIPP will contribute to building an institutional culture of research integrity, both within funding organizations and among the research organizations and individual researchers they fund. Based on empirical work from the SOPs4RI project, we have identified 6 key research integrity topics: researchers’ compli- ance with research integrity standards; expectations for research organizations; selection of grant applications; declaration of interests; monitoring funded research; and dealing with internal integrity breaches (Fig 1). We recommend that these topics should be included in a RIPP and provide guidelines on developing and implementing a RIPP. Recommended topics to be addressed in a RIPP We recommend that funders develop and implement a RIPP that emphasizes the role of research integrity on 2 levels by setting out the expectations of applicants and research organi- zations and by providing guidance on their internal organizational procedures. Drawing on 3 / 12 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 PLOS BIOLOGY Fig 1. Topics to be covered in a RIPP for funders. An overview of the 6 most important topics identified by the SOPs4RI to be included in research funding organization’s RIPP. RIPPAU : AbbreviationlistshavebeencompiledforthoseusedinFigs1 3:Pleaseverifyth , Research Integrity Promotion Plan; SOPs4RI, Standard Operating Procedures for Research Integrity. Fig 1. Topics to be covered in a RIPP for funders. An overview of the 6 most important topics identified by the SOPs4RI to be included in research funding organization’s RIPP. RIPPAU : AbbreviationlistshavebeencompiledforthoseusedinFigs1 3:Pleasever , Research Integrity Promotion Plan; SOPs4RI, Standard Operating Procedures for Research Integrity. https://doi.org/10.1371/journal.pbio.3001773.g001 https://doi.org/10.1371/journal.pbio.3001773.g001 insights from our extensive empirical program, we have identified 6 pivotal topics for a fund- er’s RIPP (Fig 2). In the following paragraphs, we describe the 6 topics and provide examples that illustrate how funders have adopted policies to support research integrity. These examples aim to inspire others to adapt these initiatives to their local context and thereby act as guiding lights to develop their own RIPP. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 Compliance with research integrity standards We recommend that funders ensure that applicants for funding confirm their compliance with the current regulations and codes of conduct for research integrity. For example, the Dan- ish Novo Nordisk Foundation outlines 15 principles [16] that should be adhered to by any applicant it funds. Such policies serve to raise awareness of research integrity in both the authoring of grant applications and the conduct of research. In a similar vein, in its 2009 guidelines on RCR, the US National Institutes of Health (NIH) [17] specifies that principal investigators applying for training grants and career development awards must submit a plan for RCR that includes face-to-face training for research staff and how they will incorporate RCR into daily practices. Designing such plans at the grant writing stage of doing research assists researchers in making responsible research practices part of their daily work. In PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 4 / 12 PLOS BIOLOGY Fig 2. Currently implemented initiatives at funders to address the 6 RIPP topics. An overview of some of the initiatives described in this section, structured around the 6 key topics to be addressed in a RIPP. To find out more about the initiatives, please consult the references added in the main text. RFOAU : PleaseprovidefullspellingforRFOandRPOinFig2abbreviationlistiftheseindeedareabbreviations: , Research Funding Organisation; RIPP, Research Integrity Promotion Plan; RPO, Research Performing Organisation. https://doi.org/10.1371/journal.pbio.3001773.g002 Fig 2. Currently implemented initiatives at funders to address the 6 RIPP topics. An overview of some of the initiatives described in this section, structured around the 6 key topics to be addressed in a RIPP. To find out more about the initiatives, please consult the references added in the main text. RFOAU : PleaseprovidefullspellingforRFOandRPOinFig2abbreviationlistiftheseindeedareabbreviations: , Research Funding Organisation; RIPP, Research Integrity Promotion Plan; RPO, Research Performing Organisation. https://doi.org/10.1371/journal.pbio.3001773.g002 addition, funders such as the Wellcome Trust [18] in the UK are increasingly demanding and enabling the researchers and organizations they fund to comply with standards of open sci- ence. This includes demands to publish open access and the creation of infrastructures to deposit study protocols. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 Expectations for research organizations We recommend funders to, like the Research Foundation Flanders (FWO), mandate research performing organizations, as well as the researchers they employ, to follow a research integrity clause in grant contracts. Furthermore, organizations should meet other expectations includ- ing the provision of research integrity training and the monitoring of supervisors. As is now fairly common among research funders, this could also include mandates with respect to open science practices, including open access publishing, open data, data management plans, and requirements regarding preregistration or protocols for data analysis in research fields where this is appropriate. The requirements of the Canadian Tri-Agency Framework and the Hori- zon Europe program are prime examples of how funders can demand the implementation of research integrity practices in research-performing organizations. The FWO takes this to a next level by providing an innovative example of best practice in their desired profile [19] of potential applicants as well as the applicants’ host institutions. The profile describes in detail what FWO expects of a “good supervisor”. We recommend that funders are also active in requesting detailed information on the research-performing organization’s RIPP and its implementation. The standards and require- ments of such a RIPP for research organizations are outlined in our research organisation guideline [11] and follow the principles of the ECoC [1]. Funders should be informed by research organizations about how the research-performing organization deals with breaches of research integrity. When breaches occur, the funder should be informed promptly about alle- gations and the outcomes of the investigation by research organizations, or if required, insti- gate its own investigation. 5 / 12 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 PLOS BIOLOGY Criteria and processes for selecting grant applications We recommend that the assessment and selection criteria for grant applications is transparent and publicly available. This relates both to the assessment of research proposals and applicants. To ensure optimal fairness in procedures and ultimately encourage integrity of the funded research, funders are expected to adhere to high standards of transparency in their evaluation process, as well as provide the right incentives in the criteria for judging applications. Regard- ing the former, the Norwegian Research Council [20] provides a particularly engaging example of achieving procedural transparency through short videos, infographics, and clear descrip- tions of every step of their assessment process. Regarding the latter, the US NIH’s BioSketch format of grant application [21], established in 2014, emphasizes the quality rather than quan- tity of published research in assessing applicants. More recently, the use of narrative CVs rather than publication lists and quantitative metrics is gaining support from funders includ- ing the Dutch Research Council (now) [22], the Health Research Board (HRB) in Ireland [23], and other signatories of the DORA declaration. These efforts are part of a movement towards responsible assessment criteria that can foster a culture of research integrity, which by now is supported by a substantial evidence base [24]. The Wellcome Trust [25] now restricts its fund- ing to researchers at organizations that have Responsible Assessment Procedures in place. In addition, the Hong Kong Principles [26] describe 6 core principles to help assess researchers’ commitment to responsible research practices, and funders can use these principles to guide their approach to assessing applicants. Alternatively, several widely accepted statements have been released regarding elements to be avoided when evaluating research, including DORA [27] and the Leiden Manifesto [28]. These statements present a warning for the blind use of bibliometric or quantitative indicators, including h-indices, publication and citation counts, in the assessment of researchers and research organizations. Avoiding such purely quantitative assessments is important to allow for more holistic and qualitative evaluations of the merit of researchers and the work they produce. Declaration of interests In 2020, the Fonds National de la Recherche Luxemburg (FNR) published an FNR Ethics Charter and Code of Conduct for Research Assessment [29], establishing a code of conduct for those involved in funding agencies. It stipulates how reviewers and the funder’s staff are expected to behave, with particular attention to impartiality and confidentiality. Similar to the FNR policy, we recommend that RIPPs for funders include procedures for the declaration of interests for funders’ internal staff, members of assessment and selection committees, and peer reviewers of submitted applications. All those involved in funding decisions should declare any financial, professional, or other interests that might be seen to influence a decision or to be affected by the outcome of a decision. Similar to the FNR, the Dutch funders NWO and ZonMw [30] have a set of guidelines relating to the declaration of interests, promoted by short movies and concrete descriptions. This can all help to create awareness of potential conflicts of interest and ways of dealing with them in an accessible way. In a slightly more legalistic fash- ion, the US NIH provides a statement on Integrity and confidentiality in Peer Review [31], outlining prohibitions for reviewers, including potential consequences. Dealing with internal breaches of research integrity We recommend that funders have adequate and transparent procedures in place to manage potential breaches of research integrity standards by their staff, committee and panel members, and peer reviewers. Many funders already have such procedures in place, including established whistleblowing channels and protective mechanisms for both whistleblowers and the accused. The NWO operationalized this in an elaborate manner by setting up the NWO Scientific Integrity reporting center [34] to deal with cases of research integrity breaches both in the projects it funds as well as in its internal procedures. The center features confidential advisors and a scientific integrity committee and acts in accordance with the NWO Scientific Integrity Complaints Procedure and keeps close connections to the National Board for Research Integ- rity in the Netherlands. As such, it provides an inspiring example of how this part of a funder’s RIPP can be designed and implemented. Large funding agencies could have a dedicated ombudsperson or an independent investiga- tory committee, whereas smaller funders could collaborate with other funders or national research integrity boards to make this feasible. Monitoring funded research We recommend funders to establish monitoring procedures, including the monitoring of responsible research practices for the research they fund, for example, including monitoring of open science and FAIR data practices. Most funders already have some monitoring procedures in place. For example, Ireland’s HRB sets out on its website [32] what it will monitor in a 6 / 12 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 PLOS BIOLOGY project through annual and final reports, as well as an end-of-grant evaluation survey. This includes submission of a Data Management Plan as a first output and making all research out- puts openly available. Similarly, the Wellcome Trust [33] states that they will “consider whether researchers have managed and shared their research outputs in line with our require- ments, as a critical part of the end-of-grant reporting process”. Through such monitoring pro- cesses and the transparency in communicating them, funders can guide researchers towards research and publication practices that foster research integrity. Simultaneously, we recom- mend researchers funders to try to avoid unnecessary administrative burdens on researchers, which are in fact considered to be potential drivers of questionable research practices [14]. For compliance with research integrity policies, the US NSF provides a good example of monitoring procedures. They conducted a review of levels of compliance with research integ- rity standards in a sample of 53 research-performing organizations, finding generally high compliance with applicable standards [6]. In their monitoring efforts, funders may distinguish between individual researchers, research consortia, and research organizations. Monitoring aspects could include compliance with good publication and dissemination practices, progress and alignment with the granted application, and the expenditure of funds (for example, to check for gross misuse of funds). While these monitoring endeavours can steer research prac- tices into desirable directions, funders are also recommended to avoid creating an unnecessary bureaucratic burden on the researchers and organisations they fund, since this may be coun- terproductive [14]. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 Guidelines for developing and implementing a RIPP To assist research funders in designing a RIPP and implementing concrete actions that will foster a culture of research integrity, we have created Implementation Guidelines and a RIPP Template that can be accessed online. The guidance to create and implement a RIPP follows the model depicted in Fig 3, which is recommended to be executed in a cyclical manner. As input to the first cycle, a funding agency may use the aforementioned 6 topics to be addressed in a RIPP. The first cycle aims to create and implement a first draft of the RIPP. This RIPP will then be the input for the next cycle. Each cycle consists of 7 distinct steps: diagnosis, assessing readiness, finding the right people, creating/updating a RIPP, executing the imple- mentation plan, assessing change progress and outcomes, and institutionalization (Box 1). PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 7 / 12 PLOS BIOLOGY Fig 3. The implementation model. The model for RIPP implementation consists of 3 phases: Preparation, Execution, and Monitoring. Each of these phases involves tasks that are carried out in multiple steps. Importantly, the model proposes a cyclical format of creating, maintaining, and revising a culture of integrity. RIPP, Research Integrity Promotion Plan. Fig 3. The implementation model. The model for RIPP implementation consists of 3 phases: Preparation, Execution, and Monitoring. Each of these phases involves tasks that are carried out in multiple steps. Importantly, the model proposes a cyclical format of creating, maintaining, and revising a culture of integrity. RIPP, Research Integrity Promotion Plan. https://doi.org/10.1371/journal.pbio.3001773.g003 https://doi.org/10.1371/journal.pbio.3001773.g003 Assessing readiness Assess the readiness of the organization for change, particularly assessing the resource capacity and willingness of the organization to take on the demands that effective change requires. This includes, among others, senior leadership’s capability to guide change, the availability of sufficient resources, and the preparedness for change among the organisa- tion’s members. Diagnosis Gather information to assist in the diagnosis of what change is needed: Which of the RIPP topics are most relevant to the organisation and require the most attention? This also could also include setting an aspirational marker on the horizon, a vision to work towards. Finding the right people Identify the right people to promote and execute the process, forming a change coalition. A change coalition consists of change agents and role models from diverse organiza- tional units that will guide the creation and implementation of a RIPP. Important aspects to take into account comprise the potential need for specific training or preparation for the identified people; inclusion of all relevant types of staff: for example, junior and senior researchers, mid-level management, and people centrally placed in the organisa- tion’s social network and with the right characteristics: trustworthy, supportive, and honest. Creating/updating a RIPP Write the RIPP, specifying goals, relevant stakeholders, the required organizational setup, relevant tools, specific actions and responsibilities, and a set of indicators or 8 / 12 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 PLOS BIOLOGY targets to monitor effectiveness. Specific guidelines, recommendations, and examples from the SOPs4RI toolbox can be used for inspiration or implementation. Executing the implementation plan Properly inform all stakeholders and roll out the change plan. In case the intended change involves a major restructuring of (some) stakeholders’ daily workflow, we sug- gest considering a gradual change process. A gradual process can include several pilot tests, experiments, and local initiatives, which together make complex change easier to implement. At this stage, it is also crucial to allow change recipients to provide feedback and make local adjustments to broader change plans. Assessing change progress and outcomes Perform periodic assessments based on the predetermined set of change indicators to verify whether the planned change is producing anticipated outcomes and/or unin- tended side effects. This also includes an evaluation of the resources required. Institutionalization Integrate the novel procedures into the funder’s workflow, culture and operating sys- tems. Based on the assessment, the RIPP is revisited, resources and responsibilities are allocated for long-term implementation, and the change coalition’s relevant experiences from their organizational unit are implemented into the procedures and policies of the entire organization. For more details and guidance on each step, we refer readers to the Implementation Guidelines created by the SOPs4RI consortium [35]. Some of the 6 topics in a RIPP and some of the 7 steps to create and implement a RIPP may be of greater or lesser relevance to a funder’s local context. The RIPP template and implementation guidelines are there- fore designed to be used flexibly and tailored to the user’s local context and can be used by both organizations that already have research integrity policies in place and organiza- tions that are about to start on their research integrity journey. Based on the monitoring of one cycle, a new diagnosis of the next cycle can be readily performed. We recommend repeating cycles at regular intervals: at least every 3 or 4 years. To avoid additional or redundant administrative workload, we suggest the cycles be coupled with existing evaluation cycles already taking place regularly, such as external or internal audits. Integrating the continuous efforts on the RIPP with existing proce- dures might both reduce administrative burden and allow research integrity to become an integral aspect of the organization’s policies and workflow. Time to follow suit The global research community has seen a considerable increase in attention on research integrity. Researchers across the sciences, social sciences, and humanities and their funders, large and small, must be, and increasingly are, committed to supporting a culture of research integrity. The SOPs4RI project has found examples in many countries of funders that are pick- ing up the challenges posed by research integrity. We are confident that this movement will gather pace over the coming years, supported by contributions made by funders. 9 / 12 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 PLOS BIOLOGY However, we also recognize and acknowledge that we have a long way to go. Best practice initiatives are scattered, fragmented, many are only recently implemented, and implementa- tion often turns out to be challenging. In addition, not all funders are equally well positioned and resourced to enable them to implement all the recommendations and best practices we outline in this Consensus View. We acknowledge that it takes time to change cultures, and we recognize that funders are varied in their aims and the extent to which they have resources available for procedures and governance arrangements to support research integrity. We would also like to emphasize that successful implementation of RIPPs is dependent on local contexts and should take disciplinary differences into account, as successful implementation depends on how practices are taken up by a funder’s main stakeholders. Also, research integ- rity matters do not stand on their own, but rather are related to several other discussions. Reflecting this, funders on their own and collectively are aiming to connect their approaches to addressing related topics; for example, Science Europe have established several working groups on research assessment, open access (publications), open data, and research culture [36]. The SOPs4RI toolbox has been specifically developed to assist funders worldwide in their endeavors to implement research integrity policies and procedures. We believe that this tool- box, consisting of research integrity guidance on all 6 topics, Implementation Guidelines, and a RIPP Template, is an important step in assisting funders to achieve this for their own organi- zation and for the researchers and research organizations they fund. The toolbox can also assist in creating some common standards across funders, leaving enough flexibility for local adapta- tions while harmonizing requirements across different contexts. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 Time to follow suit An additional challenge for the implementation of new policies and procedures is the con- sistent lack of evidence about their effectiveness and wider consequences. Part of the shortage of empirical data on these topics is due to the relatively recent nature of new initiatives and hence the absence of sufficient time to evaluate them. But there are other reasons too. While there has been a drastic shift towards openness and transparency in many aspects of academic practices, for example, journal articles, peer review reports, and research data, the processes at research funding organizations remain largely opaque and unexamined. This lack of evidence creates serious challenges for the research community to properly assess the merits of diverse funding practices and selection procedures. Therefore, in addition to our call to funders to establish a RIPP along the lines described in this Consensus View, we urgently plea for them to follow the global transition towards more transparency in research by collecting and making data on their practices openly available, with the aim to improve them. Particularly, we encour- age funders to be more transparent about their criteria for evaluating proposals, the methods used to select proposals to be funded, and funders’ efforts to avoid or deal with internal breaches of research integrity standards. Among other areas, transparency about these aspects will allow the research community to understand funders’ processes. This should be part of holistic attempts to address research integrity challenges. Only efforts fostering research integ- rity cultures in their broad conception are likely to be effective. In addition to establishing a RIPP, funders can also contribute to research integrity by fund- ing or facilitating research into responsible research or breaches thereof. Such efforts could include the direct funding of studies into research integrity, direct funding of research integrity training, or funding of core facilities that can help with experimental design, research report- ing, and data analysis for individual researchers. This will ultimately contribute to a better understanding of how we can collectively build a research culture that is conducive to research integrity. In this Consensus View, we provided an overview of initiatives that are currently changing the funding landscape towards one that is more conducive to research integrity. References 1. ALLEA. The European Code of Conduct for Research Intregrity—Revised Edition. Berlin, Germany; 2017. 2. European Commission. Horizon Europe (HORIZON) Euratom Research and Training Programme (EURATOM)—General Model Grant Agreement EIC Accelerator Contract Brussels: European Com- mission; 2021 15-12-2021. 3. Foundation WCoRI. What do we mean when we talk about research integrity? 2022. Available from: https://www.wcrif.org/foundation/mission. 4. Horbach SPJM, Halffman W. Promoting Virtue or Punishing Fraud: Mapping Contrasts in the Language of ‘Scientific Integrity’. Sci Eng Ethics. 2017; 23(6):1461–1485. 5. Fanelli D. The black, the white and the grey areas: Towards an international and interdisciplinary defini- tion of scientific misconduct In: Mayer T, Steneck N., editors. Promoting research integrity in a global environment. Singapore: World Scientific Publishing; 2011. p. 79–90. 6. National Science Foundation. Promoting Effectiveness, Efficiency, and Economy AlexandriaN.D. 7. Panel on Responsible Conduct of Research. Tri-Agency Framework: Responsible Conduct of Research 2021. Ottawa; 2021 21-01-2022. 8. Carter JM, Goldman GT, Rosenberg AA, Reed G, Desikan A, MacKinney T. Strengthen scientific integ- rity under the Biden administration. Science. 2021; 371(6530):668–671. https://doi.org/10.1126/ science.abg0533 PMID: 33574196 9. Biden Jr. JR. Memorandum on Restoring Trust in Government Through Scientific Integrity and Evi- dence-Based Policymaking. Washington DC; 2021 27-01-2021. 10. Committee UPRaT. Reproducibility and research integrity London2022. Available from: https:// committees.parliament.uk/work/1433/reproducibility-and-research-integrity/publications/. 11. Mejlgaard N, Bouter LM, Gaskell G, Kavouras P, Allum N, Bendtsen A-K, et al. Research integrity: nine ways to move from talk to walk. Nature. 2020; 586:358–360. https://doi.org/10.1038/d41586-020- 02847-8 PMID: 33041342 12. Sˇ čepanović R, Labib K, Buljan I, Tijdink J, Marusˇić A. Practices for Research Integrity Promotion in Research Performing Organisations and Research Funding Organisations: A Scoping Review. Sci Eng Ethics. 2021; 27(1):4. https://doi.org/10.1007/s11948-021-00281-1 PMID: 33502638 13. Labib K, Roje R, Bouter L, Widdershoven G, Evans N, Marusˇić A, et al. Important Topics for Fostering Research Integrity by Research Performing and Research Funding Organizations: A Delphi Consensus Study. Sci Eng Ethics. 2021; 27(4):47. https://doi.org/10.1007/s11948-021-00322-9 PMID: 34244889 14. Sørensen MP, Ravn T, Marusˇić A, Elizondo AR, Kavouras P, Tijdink JK, et al. Strengthening research integrity: which topic areas should organisations focus on? Humanit Soc Sci Commun. 2021; 8(1):198. 15. Labib K, Pizzolato D, Stappers P-J, Evans N, Lechner I, Widdershoven G, et al. Using co-creation meth- ods for guideline development–How, why and when? OSF Preprints. 2021. 16. Novo Nordisk Foundation. Code of Conduct. Hellerup: Novo Nordisk Foundation; n.d. 17. National Institutes of Health. Requirement for Instruction in the Responsible Conduct of Research. Time to follow suit Through PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 10 / 12 PLOS BIOLOGY empirical work, we identified 6 topics that are crucial in fostering a culture of research integ- rity in funding organizations and have provided guidelines to develop and implement a RIPP. Several funders have taken the first important step by emphasizing the importance of research integrity, requiring that research organizations should have research integrity policies in place, and organizing their own procedures to foster research integrity. We herald these front run- ners, and we believe that now is the time for more funders to take up the challenge to follow suit. Acknowledgments We would like to thank Zoe¨ Hammatt, Susan Zimmerman, Patricia Valdes, Isidoros Karatzas, Lisa Diependaele, and Thed van Leeuwen for their valuable suggestions. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 References Bethesda, Maryland; 2009. 18. Wellcome. Responsible conduct of research London2022. Available from: https://wellcome.org/grant- funding/guidance/responsible-conduct-research. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3001773 August 19, 2022 11 / 12 PLOS BIOLOGY 19. Flanders RF-. Research Integrity within the FWO Brussels2015. Available from: https://www.fwo.be/en/ the-fwo/research-policy/research-integrity/research-integrity-within-the-fwo/. 20. Norway TRCo. The evaluation process Oslon.d. 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https://openalex.org/W2947723618	https://curis.ku.dk/ws/files/215775925/The_joint_effect_of_unemployment_and_cynical_hostility_on_all_cause_mortality.pdf	English	null	The joint effect of unemployment and cynical hostility on all-cause mortality: results from a prospective cohort study	BMC public health	2,019	cc-by	6,503	university of copenhagen The joint effect of unemployment and cynical hostility on all-cause mortality results from a prospective cohort study Kriegbaum, Margit; Lund, Rikke; Schmidt, Lone; Rod, Naja Hulvej; Christensen, Ulla university of copenhagen The joint effect of unemployment and cynical hostility on all-cause mortality results from a prospective cohort study Publication date: 2019 Document version Publisher's PDF, also known as Version of record Document version Publisher's PDF, also known as Version of record Citation for published version (APA): Kriegbaum, M., Lund, R., Schmidt, L., Rod, N. H., & Christensen, U. (2019). The joint effect of unemployment and cynical hostility on all-cause mortality: results from a prospective cohort study. BMC Public Health, 19, [293]. https://doi.org/10.1186/s12889-019-6622-7 Download date: 24. Oct. 2024 university of copenhagen university of copenhagen The joint effect of unemployment and cynical hostility on all-cause mortality results from a prospective cohort study Kriegbaum, Margit; Lund, Rikke; Schmidt, Lone; Rod, Naja Hulvej; Christensen, Ulla The joint effect of unemployment and cynical hostility on all-cause mortality: results from a prospective cohort study Margit Kriegbaum* , Rikke Lund, Lone Schmidt, Naja Hulvej Rod and Ulla Christensen © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: It is hypothesised that hostility accentuates the association between stressful conditions and health. This study aims to test this hypothesis by analysing the joint effect of unemployment and hostility on all-cause mortality among men and women. Methods: The population was 3677 men and 4138 women from the Danish workforce who participated in a survey in 2000. The joint exposure variable was defined as 1) employed, not hostile, 2) unemployed, not hostile, 3) hostile and employed, 4) unemployed and hostile. Outcome was defined as all-cause mortality between 2000 and 2014. Data was analysed with Cox proportional hazards models with age as the underlying time scale. The interaction between unemployment and hostility was studied using the synergy index. Results: Compared to employed non-hostile men, men who were both hostile and unemployed were at markedly higher risk of premature death with a hazard ratio (HR) of 3.19 (95% CI 2.22–4.69). A similar picture was found for hostile and unemployed women, with a HR of 1.97 (95% CI 1.24–3.12). However, the mortality in men and women exposed to both did not exceed what was expected from the combination of their individual effects. Hence, we did not find that hostility enhances the association between unemployment and all-cause mortality. Conclusion: Men and women exposed to both unemployment and hostility were at markedly high risk of premature mortality. However, this study did not support the hypothesis that the deleterious health effect of the combination of unemployment and hostility exceeds their individual effects. Keywords: Hostility, Unemployment, Joint effect, Mortality, Psychosocial vulnerability [9], depression [9, 12, 13], incident cardiovascular dis- ease (CVD) [12–15], and premature death [16–20]. Document license: CC BY Citation for published version (APA): Kriegbaum, M., Lund, R., Schmidt, L., Rod, N. H., & Christensen, U. (2019). The joint effect of unemployment and cynical hostility on all-cause mortality: results from a prospective cohort study. BMC Public Health, 19, [293]. https://doi.org/10.1186/s12889-019-6622-7 Download date: 24. Oct. 2024 Kriegbaum et al. BMC Public Health (2019) 19:293 https://doi.org/10.1186/s12889-019-6622-7 Background Several studies have shown unemployment to be associ- ated with both psychological distress and all-cause and cause-specific mortality in men and women [1–8]. One expression of psychological distress is hostility, a multi- faceted construct defined as an enduring, negative atti- tude towards others involving cognitive, affective, and behavioural components [9]. The relationship between the dimensions of hostility and negative health out- comes is well established. Hostility has been associ- ated with higher symptom load and poorer self-rated health [10, 11], with adverse health-related behaviours Hostility has been related to socioeconomic position and employment status. Hostility was, for example, associ- ated with low educational attainment and low occupa- tional social class in a national sample of American adults aged 18 to 90 years [21]. Also, in the British Whitehall II study, higher scores of hostility have been related to lower occupational grade [22–24]. A similar picture is seen in middle-aged Danish men and women, where the preva- lence of high hostility among men and women receiving benefits, including unemployment, was nearly four times as high as those in the high-occupational social class [10]. In these studies, hostility has mainly reflected a sceptical approach to other peoples’ sincerity, motives, and beliefs and showing a basic mistrust of others. * Correspondence: makr@sund.ku.dk Department of Public Health, University of Copenhagen, Oester Farimagsgade 5, 1014 Copenhagen K, Denmark Kriegbaum et al. BMC Public Health (2019) 19:293 Page 2 of 8 Kriegbaum et al. BMC Public Health (2019) 19:293 Little is known about the health consequences of being exposed to both unemployment and hostility. The psy- chosocial vulnerability model has been proposed as a hypothetical model for the association between hostility and health [11]. According to this model, hostile persons are less likely to engage in social interaction and have a lower stress-buffering potential from interpersonal sup- port, which may predict worse health outcomes when exposed to a stressor such as unemployment [11, 25]. Kivimäki and co-authors have explained the psychosocial vulnerability model as an interaction between hostility and adverse social conditions that leads to an increase in negative health outcomes among hostile individuals be- cause of their lower capacity to benefit from existing psychosocial resources [11]. Few previous studies have investigated the effect modification of hostility on the re- lationship between social conditions and health, and only two studies from Finland [26, 27] have been aimed directly at testing the psychosocial vulnerability hypoth- esis. Data and participants The Danish Longitudinal Study on Work, Unemploy- ment, and Health is a prospective population-based study with a baseline postal survey carried out in 2000. The baseline survey was based on a stratified random sample consisting of two population groups: 1) a group of individuals 40 and 50 years old by 1 October, 1999, (response rate 69%, n = 7588), and 2) a group of 37– 56-year-old individuals who had been unemployed at least 70% of the time during the period 1 October, 1996 to 1 October, 1999, (response rate 57%, n = 2287). The latter group was included to ensure a sufficient number of unemployed which was one of the main interests of the study. In 1999, the unemployment rate was low with 6% unemployed among both the 40- and 50-year-old. Both samples have initially been drawn from the ‘AKF Longitudinal Register’, which is maintained by the National Centre for Social Science Research (previously AKF) and comprises all Danes aged 15 years or older. Data on non-participation was derived from the registers and showed that non-participants were more often men, non-native-born Danes, persons living on transfer in- come, and persons with low educational attainment (non-trained or semi-skilled). Out of 9875 participants in the survey, we were able to link 9870 to register data. The Cause of Death Registry, The Danish National Population Register, The National Patient Registry, and the National Drug Prescription Registry were used to identify mortality and covariates. For the present study, an inclusion criterion was those in the workforce. The workforce is defined as those who are working, plus those who are available for work. From the survey, we selected individuals who were working or unemployed at the time of the survey (8733). Of these, 223 (3%) had missing information on hostility, and 100 (1%) had miss- ing information on education. The final study population was 8426. The details of the population selection are available in Additional file 1. Methodologically, these studies investigated if the as- sociation between social factors and health differed in strata of hostile vs. non-hostile individuals (i.e., effect modification). We suggest that a measure of interaction, which aims to study effects that are due to interaction, that is, cases that would not have occurred if it was not for the presence of both risk factors. Background The psychosocial vulnerability hypothesis was only partly supported in these studies as the moderating ef- fect of hostility seemed to be gender-specific. In one study, there was no interaction between hostility and un- employment on self-rated health among women, but interaction was found among men, that is, hostility ac- centuated the effect of unemployment on self-rated health in men [26]. In the other study conducted over five years with economic decline, hostile women had a higher risk of sickness absence after exposure to stressors such as organisational downsising, job insecur- ity, and a stressful psychosocial work environment. This increased vulnerability was not seen among hostile men exposed to the same stressors [27]. effect of unemployment and hostility on all-cause mortal- ity among a large sample of Danish men and women, followed prospectively in a nationwide mortality register. Data and participants This is relevant in public health because the blocking of pathways to poor health that involves synergistic effects can be powerful because the absolute number of cases that can be pre- vented is higher than the number of cases from individ- ual risk factors. If there is a synergistic effect of being exposed to both unemployment and hostility, interven- tions directed at this group would be highly efficient. Assessment of unemployment and hostility Assessment of unemployment and hostility Information on employment status was based on self-reported information on current labour market par- ticipation in 2000 and grouped as ‘employed’ or ‘un- employed’. Hostility was measured by the eight-item Cynical Distrust Scale, derived from the Cook-Medley Hostility Scale, originally based on the items from the Minnesota Teacher Attitude Inventory [28]. This scale measures a sceptical approach to other peoples’ sincerity, motives, and beliefs and the showing of a basic mistrust While previous studies to some extent support the vulnerability hypothesis, no previous studies have, to our knowledge, focused on how the joint effect of unemploy- ment and hostility affects all-cause mortality. We hypothesised that the joint effects of unemployment and hostility on mortality exceed their individual effects. We aim to test this hypothesis by evaluating the joint Kriegbaum et al. BMC Public Health (2019) 19:293 Page 3 of 8 Page 3 of 8 tool for identification and selection of relevant con- founders in epidemiological studies based on prior sub- stance knowledge. The DAG and overview of included variables are available in Additional file 2. Cohabitation status retrieved from the population registry in 1999 was defined as living with vs. without a partner. Educational attainment was based on self-reported information on the type of vocational training following primary school and categorised into no education (no training—only primary school) or some education (any level beyond primary school). Information on alcohol abuse and de- pression was retrieved from The National Patient Regis- try 1980–1999 and the National Drug Prescription Registry 1995–1999, which contains information about outpatient prescription drug use. We used the Inter- national Classification of Disease (ICD) to identify diag- noses. We included admissions for alcohol, cirrhosis of the liver (571.09; 571.19 ICD 8 /I85.0-I85.9 ICD 10), and Oesophageal varices (456.09; 456.19 ICD 8). Further, a prescription for alcohol dependence (ATC code N07BB) was used to identify alcohol abuse. Depression was de- fined as at admissions with bipolar depression, depres- sion, and recurrent depression (ICD8: 296, 298.09, 298.19; ICD-10: F30-F33.9) and/or prescriptions with an- tidepressants (ATC code N06A). The variable for alcohol abuse was coded as 1 or more indicators of alcohol abuse vs. no indication of alcohol abuse. Similarly, the variable for depression was coded as 1 or more indica- tors of alcohol abuse vs. no indication of depression vs. no indication. of others. Mortality All-cause mortality was assessed in the nationwide Cause of Death Registry between baseline and 2014 and the follow-up time ended at the date of death, or 31 December 2014, whichever came first. Assessment of unemployment and hostility This cynical mistrust dimension of hostility has been most solidly associated with measures of low social position and, consequently, we found it the most relevant measure to include in this specific study. The Cynical Distrust Scale that measured the cognitive com- ponent of hostility was factor-analytically derived from the Cook-Medley Scale by Greenglass and Julkunen [29, 30]. In two separate samples of Canadian and Finnish stu- dents, they demonstrated that the shorter Cynical Distrust Scale is a sufficiently valid, reliable, and specific measure of cynicism and distrust when compared to the full Cook-Medley Scale [29, 30]. In the present study, we used the following eight items as presented in English by Everson et al. [17]: 1) I think most people would lie to get ahead. 2) Most people inwardly dislike putting themselves out to help other people. 3) Most people make friends because friends are likely to be useful to them. 4) It is safer to trust nobody. 5) No one cares much what happens to you. 6) Most people are honest chiefly through fear of being caught. 7) I commonly wonder what hidden rea- sons another person may have for doing something nice to me. 8) Most people will use somewhat unfair means to gain profit or an advantage rather than lose it. We translated the items into Danish and adjusted the wording after a back-translation into English. The Da- nish version was tested in a large-scale pilot study of the entire questionnaire among a study population (n = 993) drawn from the same sampling frame as the main survey [10]. This work was led by the last author of this manu- script. Response options were: completely agree, some- what agree, somewhat disagree, completely disagree. In the analyses, the items were summed to obtain a Cynical Distrust Scale score with a range of 0–24. The internal reliability was tested by Cronbach’s alpha, which at 0.89 was satisfactory. In the literature, there is no established threshold for dichotomisation of the Cynical Distrust Scale. To define the cut point for dichotomy, we divided the scale into quartiles as has been applied in the studies by Everson et al. and Stamatakis et al. [17, 31], and we used separate cut points for men (scoring 9+) and women (scoring 7+). Statistical analysis Cox proportional hazards models with age as the under- lying time scale were used to analyse the data. All variables met the proportional hazards assumption. Initially, we es- timated hazard ratios (HR) and 95% confidence intervals (CI) for all-cause mortality according to hostility, un- employment, and the joint effect of the two. Level of edu- cation and cohabitation status, depression, and alcohol abuse were included in the models and considered as pos- sible confounding factors. By using age as the underlying time scale, we ensured thorough adjustments for age. To study the joint exposure to hostility and unemploy- ment, a new composite variable with four categories was defined: 1) unexposed (reference), 2) unemployed, but not hostile, 3) hostile but not unemployed, 4) unemployed and hostile. The joint reference category allows us to compare each combination of hostility and unemployment level ac- cording to the same baseline hazard. All analyses were conducted using SAS version 9.3. The ‘proc phreg’ procedure was used for survival ana- lysis. The synergy index (SI) was used to assess deviation from additivity, which indicates a causal interaction where some cases of the outcome would not have oc- curred if it had not been for the presence of both Results During 14 years of follow-up, 264 men and 201 women died. Table 1 shows the distribution of hostility, employ- ment, cohabitation status, education, age, alcohol abuse, and depression by vital status. More unemployed men and women died compared to those employed. Also, more hostile men and women died compared to those not hostile. Assessment of covariates Potential confounders for the analyses were identified based on prior knowledge and the method of directed acyclic graphs (DAG) [32]. DAGs provide a graphical Kriegbaum et al. BMC Public Health (2019) 19:293 Kriegbaum et al. BMC Public Health (2019) 19:293 Page 4 of 8 Page 4 of 8 Page 4 of 8 Page 4 of 8 After adjustment for covariates, hostility was associ- ated with higher mortality risk in men, HR 1.47 (95% CI: 1.09–21.99), but not in women, HR 1.17 (95% CI: 0.79– 1.74) (Tables 2 and 3). Unemployment was also associ- ated with a higher risk of premature death in both men, HR 2.32 (95% CI: 1.46–3.70) and women, HR 1.78 (95% CI: 1.16–2.73). Men who were both hostile and un- employed were at higher risk of premature death, HR 3.19 (95% CI: 2.22–4.69) compared to employed and non-hostile men (Table 2). It is similarly, for women, with a HR 1.98 (95% CI: 1.24–3.12) associated with the joint effect of being hostile and unemployed (Table 3). The synergy index was 1.22 (95% CI: 0.69–2.15) for men and 1.17 (95% CI: 0.76–1.81) for women. Hence, the re- sults did not indicate any synergistic effects between un- employment and hostility. hostility and unemployment. The SI calculates the ratio between the combined effect and individual effects of the variables. SI can go from 0 to infinity. SI = 1 means no interaction or exactly additivity, whereas SI > 1 im- plies positive interaction and SI < 1 implies negative interaction. In this study, SI > 1 is interpreted as evi- dence in favour of the psychosocial vulnerability model [33]. We followed the guidelines provided by Anderson et al. to calculate regression coefficients and covariance matrix, which was used as an input to calculate the syn- ergy index and 95% CI in excel [33]. Discussion We found that hostility in men and unemployment in both men and women were associated with all-cause Table 1 Hostility, employment, and covariates by vital status Dec. 31, 2014: 8426 Danish men and women from the Danish Longitudinal Study on Work, Unemployment, and Health Men Women Alive n and row percentage Dead n and row percentage Alive n and row percentage Dead n and row percentage Occupation Employed 3217 (94.7) 179 (5.3) 3668 (96.2) 143 (3.8) Unemployed 386 (82.0) 85 (18.0) 690 (92.2) 58 (7.8) Hostility Not hostile 2321 (94.8) 127 (5.2) 3298 (96.1) 139 (3.9) Hostile 1282 (80.3) 137 (9.7) 1060 (94.4) 63 (5.6) Cohabitation Live with partner 2870 (94.9) 153 (5.1) 3398 (96.1) 139 (3.9) Do not live with partner 733 (86.8) 111 (13.2) 960 (93.9) 62 (6.1) Education Secondary education or more 3187 (93.2) 231 (6.8) 3664 (95.8) 159 (4.2) Primary education 416 (92.7) 33 (7.3) 694 (94.3) 42 (5.7) Age < 40 years 114 (92.7) 9 (7.3) 288 (97.6) 7 (2.4) 41 years 1152 (96.6) 40 (3.4) 1326 (97.4) 36 (2.6) 42–50 years 630 (92.8) 49 (7.2) 936 (96.4) 35 (3.6) 51 years 1186 (93.0) 89 (7.0) 1185 (94.9) 63 (5.1) 52+ years 521 (87.7) 77 (12.9) 624 (91.2) 60 (8.8) Alcohol abuse No 3464 (94.0) 221 (6.0) 4291 (95.7) 195 (4.3) Yes 139 (76.4) 43 (23.6) 67 (91.8) 6 (8.2) Depression No 3426 (93.7) 232 (6.3) 3989 (96.0) 168 (4.0) Yes 177 (84.7) 32 (15.3) 369 (91.8) 33 (8.2) Table 1 Hostility, employment, and covariates by vital status Dec. 31, 2014: 8426 Danish men and women fr Longitudinal Study on Work, Unemployment, and Health and covariates by vital status Dec. 31, 2014: 8426 Danish men and women from the Danish employment, and Health Kriegbaum et al. Methodological considerations Using self-reported psychosocial exposures and subject- ive health outcomes in observational studies has been called into question [37, 38], which points to the issue of reverse causation via the mechanism that perceived poor health also generates hostility [39]. We consider the register-based outcome measure a major strength in this study, as mortality rules out concerns of reverse caus- ation. Furthermore, to reduce confounding by poor health, we excluded all individuals not working due to illness the year before baseline. Most confounders were also measured using register data, which means that in- formation is complete. However, there are also limita- tions. Alcohol abuse and depression are detectable only in registers if the individual had been either hospitalised with a main or contributory diagnosis indicating these or had purchased prescription medicines to treat depres- sion or alcohol dependence. These drugs are not sold over the counter. Hence, all treated individuals are in the registers, but some cases of alcohol abuse may never be treated and represent a source of unmeasured con- founding (see Additional file 1). Hostility was measured once, just before the start of follow-up. However, hostil- ity is considered to be stable from early adulthood [40]. Hostility in early life is likely to influence several Hostility was an independent risk factor in men. It has previously been shown that hostile persons are less likely to engage in social interaction and one of the proposed mechanisms underlying a health consequence of hostility is a lower stress-buffering potential from interpersonal support [11, 25]. Thus, hostility may reduce the health benefits of receiving social support. For example, Holt-Lundstad et al. showed that not only was high hos- tility associated with heightened systolic and diastolic blood pressure during self-disclosure of stressful events, but those high in hostility also perceived their friends to be less friendly and, consequently, they seemed to bene- fit less from social support received during stressful pe- riods [25]. Discussion BMC Public Health (2019) 19:293 Page 5 of 8 Table 2 Separate and joint effect of hostility and unemployment on all-cause mortality among Danish middle-aged men, n = 3867 Separate and joint effects Alive (n and row percentage) Dead (n and row percentage) Model 1a HR 95% CI Model 2b HR 95% CI Employed, not hostile (non- exposed) 2174 (95.5) 103 (4.5) 1 (reference) 1 (reference) Employed, hostile (separate effect) 1043 (93.1) 76 (6.9) 1.59 (CI 1.18–2.15) 1.47 (1.09–1.99) Unemployed, not hostile (separate effect) 147 (85.6) 24 (14.4) 3.31 (CI 2.12–5.17) 2.32 (1.46–3.7) Unemployed, hostile (joint effect) 239 (78.9) 61 (21.1) 5.07 (CI 3.67–7.01) 3.19 (2.22–4.69) aModel 1 unadjusted model bModel 2 adjusted education, cohabitation, alcohol abuse and depression mortality. We found no synergistic effects to support the psychosocial vulnerability hypothesis. This suggests that no special vulnerability stems from being exposed to both. In both men and women, unemployment was related to premature mortality. Unemployment represents a se- vere economically stressful condition, and coping strat- egies are considered a potential modifier of the impact of unemployment on health and well-being [35]. Thus, inadequate coping among unemployed individuals may set these persons at greater risk of the deleterious health impact of unemployment [36]. Few studies have addressed the psychosocial vulner- ability model in relation to hostility and unemployment. Kivimäki et al. found no interaction between hostility and unemployment on self-rated health among women, but interaction was found among men, that is, hostility accentuated the effect of unemployment on self-rated health in men [11]. Another study by the same first au- thor found an increased vulnerability in hostile women to the stressor of organisational downsizing, job insecur- ity, and stressful psychosocial work environment in rela- tion to sickness absence. This vulnerability was not observed in men [27]. These studies, however, differ from the present study with regards to the outcome as well as the concept of interaction. Using the concept of causal interaction, other studies have found support for additional deaths due to exposure to both unemploy- ment and being unmarried [34]. bModel 2 adjusted education, cohabitation, alcohol abuse and depression Methodological considerations Table 3 Separate and joint effect of hostility and unemployment on all-cause mortality among middle-aged Danish women, n = 4559 Separate and joint effects Alive (n and row percentage) Dead (n and row percentage) Model 1a HR 95% CI Model 2b HR 95% CI Employed, not hostile (non-exposed) 2908 (96.5) 107(3.5) 1 (reference) 1 (reference) Employed, hostile (separate effect) 760 (95.5) 36 (4.5) 1.25 (CI 0.85–1.84) 1.17 (0.79–1.74) Unemployed, not hostile (separate effect) 390 (92.7) 31 (7.3) 2.01 (CI 1.32–3.05) 1.78 (1.16–2.73) Unemployed, hostile (joint effect) 300 (91.6) 27 (8.4) 2.34 (CI 1.51–3.62) 1.97 (1.24–3.12) aModel 1 unadjusted model bModel 2 adjusted education, cohabitation, alcohol abuse and depression Kriegbaum et al. BMC Public Health (2019) 19:293 Page 6 of 8 Page 6 of 8 Page 6 of 8 investigate relations between hostility, unemployment, and other social factors. confounding factors throughout the life course such as de- pression and cohabitation (see DAG in Additional file 2). Unemployment was also measured only once. It is likely that, for example, unemployment and alcohol abuse affect each other sequentially over the life course. We adjusted for alcohol abuse and depression which occurred before the time of the survey. In this way, these factors may be considered confounding factors. However, we realise that this does not capture the complexity of associations be- tween covariates and exposures. For simplicity, this was displayed only in the diagram using T1 to indicate time before survey and T2 to indicate time at survey or after survey. Other health behaviours may be on the causal pathway between hostility and unemployment and mortal- ity, for instance, smoking status, which in contrast to alco- hol abuse, is unlikely to affect employment. The relationship between hostility and neuroticism has been shown as possibly two simultaneous dimensions of a disease-prone personality [39, 41] and is thus confounding due to other personality characteristics that may have in- fluenced the relationship between hostility and mortality. Unfortunately, we had no data on other relevant personal- ity factors available in our dataset. To our knowledge, this is the first study focusing on the joint exposure to unemployment and hostility on the risk of premature death. We found that both hostility (in men only) and unemployment were associated with higher mortality risk, and individuals who were both un- employed and hostile were at markedly higher risk of premature death. Conclusions Both hostility and unemployment are risk factors for premature death, especially in men, and those exposed to both unemployment and hostility were at markedly high risk of premature mortality. However, this study did not support the hypothesis that the deleterious health effect of the combination of unemployment and hostility exceeds their individual effects. The study population was restricted to individuals eli- gible for the workforce. Consequently, we cannot general- ise our findings regarding psychosocial vulnerability to individuals who are not part of the workforce. As shown in Additional file 1, a relatively large number had missing information on employment. In a sensitivity analysis, we found that those with missing information on employ- ment more often had a low educational level or missing information on both and that their risk of mortality during follow-up was higher than that of the employed but lower than the mortality of the unemployed and sick (data not shown). We did not study causes of death, but we think that the study does contribute to the knowledge of how unemployment interacts with hostility in a broad popula- tion exposed to unemployment. Abbreviations CHD : Coronary heart disease; CI : Confidence intervals; CVD : Cardiovascular disease; HR : Hazard ratio; SI : Synergy index Our theoretical point of departure was the psycho- social vulnerability model and an assumption that hostil- ity is a stable trait in the adult population, but other models of the relations between hostility, social condi- tions, and health have been suggested, among these the social context model. According to this model, adverse social conditions including unemployment may antecede not only health problems but also hostility [11]. The micro-sociological approach introduced by Thomas Scheff may be of some help in understanding how this causation works. According to Scheff, the individual ex- periences the social world in emotionally loaded categor- ies which have bodily correlates such as pride, shame, anger, and distrust. The way these emotions are experi- enced is not uniform throughout the social structure [42]. Further longitudinal studies are needed to Acknowledgements The authors wish to thank Charlotte Oe. Hougaard for her data management of the Danish Longitudinal Study on Work, Unemployment, and Health. Availability of data and materials Register data used for this study was used under license for this study and may not be transferred outside the environment of Statistics Denmark. Additional files Additional file 1: Flow chart. The file shows the flowchart of selection of study participants. (DOCX 44 kb) Additional file 1: Flow chart. The file shows the flowchart of selection of study participants. (DOCX 44 kb) Additional file 2: DAG and measured variables. The file shows the DAG used for variable selection and an timescale of measurement of included variables. (DOCX 86 kb) Funding g This study was supported by The Danish Research Councils (j.nr.9801268). The funding source had no role in conception and design of the study, interpretation of data, and writing of the manuscript. Methodological considerations This may be due to inadequate coping among hostile, unemployed individuals, but our results did not lend support to the psychosocial vulnerability hypothesis. We did not find indication for causal inter- action, which implies that, while those exposed to both hostility and unemployment represent a group with high mortality risk, which could benefit from health interven- tion, the reduced mortality risk from such intervention is not enhanced by synergistic effects. Competing interests Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. 21. Barefoot JC, Peterson BL, Dahlstrom WG, Siegler IC, Anderson NB, Williams RB. Hostility patterns and health implications: correlates of Cook-Medley hostility scale scores in a national survey. Health Psychol. 1991;10:18–24. References Clemens T, Popham F, Boyle P. What is the effect of unemployment on all- cause mortality? A cohort study using propensity score matching. Eur J Pub Health. 2015;25:115–21. 30. Greenglass ER, Julkunen J. Cook-Medley hostility, anger, and the type a behavior pattern in Finland. 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https://openalex.org/W2952613201	https://revistas.unc.edu.ar/index.php/REM/article/download/22922/22625, http://funes.uniandes.edu.co/17551/1/Miatello2018Matematica.pdf	es		Matemática en azar y juegos	Revista de educación matemática/Revista de educación matemática	2,018	cc-by-sa	6,964	Revista de Educación Matemática Volumen 33, N° 3 (2018), páginas 51 – 67 V Unión Matemática Argentina - Famaf (UNC) Artículo de Matemática MATEMÁTICA EN AZAR Y JUEGOS Roberto J. Miatello, Angel D. Villanueva Resumen. El objetivo de esta nota es aplicar la teoría de probabilidades a diversas situaciones en azar y juegos. Haremos uso de las nociones básicas de la probabilidad discreta y de la combinatoria. Nos interesará especialmente el comportamiento de la probabilidad cuando algunos de los parámetros involucrados crece arbitrariamente. §1. Introducción El concepto de probabilidad surge en los siglos XVI y XVII, en parte ante la necesidad de resolver problemas que se plantean en los juegos de azar (L. Pacioli, Tartaglia, G. Cardano, B. Pascal). Se considera entre los iniciadores a P. Fermat y B. Pascal quienes, con motivo del problema de repartición del premio, alrededor de 1654 fijaron los primeros conceptos. El físico holandés C. Huygens fue uno de los pioneros en el estudio de la probabilidad, tema sobre el que publicó en 1656 el libro De ratiociniis in ludo aleae (Razonamientos sobre los juegos de azar). En él introdujo algunos conceptos importantes en este campo, como la esperanza matemática, y resolvió algunos de los problemas propuestos por Pascal, Fermat y De Méré. Esta obra de Huygens sería estudiada profundamente por Jakob Bernoulli en su Ars conjectandi. Hoy en día la idea de probabilidad (y de su disciplina hermana, la estadística) aparece en muchos campos de la ciencia, en eventos deportivos, juegos de azar, en pronósticos económicos, pronósticos de tiempo, entre tantas otras disciplinas o actividades humanas. Se considera que la teoría ‘moderna’ de probabilidades fue iniciada por el matemático ruso A. Kolmogorov quien en 1933 publicó un libro fundamental sobre el tema. Resumidamente, dado un experimento con una cantidad de resultados posibles se desea saber si un determinado evento puede ocurrir y con qué chances (en inglés ’what are the odds’?). Cuando hay un número finito de resultados posibles y todos ellos son equivalentes, la probabilidad de un evento se calcula dividiendo 51 52 Roberto J. Miatello, Angel D. Villanueva el número de resultados favorables (al evento) por el número total de casos posibles. Cuando hay una infinidad de casos, la situación es más compleja, es necesario dividir la ’medida’ del conjunto de casos favorables por la ’medida’ del conjunto de casos posibles. Obviamente, elegir la manera adecuada de medir depende del problema que se estudie y es un aspecto esencial de la dificultad. De alguna manera, la teoría de probabilidades intenta explicar en conceptos matemáticos cual será el devenir de ciertos acontecimientos. Laplace (1749 – 1827) decía que “la teoría de probabilidades no es más, en el fondo, que el buen sentido reducido al cálculo; ella hace apreciar con exactitud lo que los espíritus justos sienten por una especie de instinto, sin que puedan a menudo darse cuenta de ello”. En estas notas aplicamos conceptos básicos de la teoría de probabilidades a diversas situaciones de azar dentro del primer tipo, esto es, con un número finito de resultados posibles todos ellos equivalentes. En particular analizaremos el problema de repartición del premio, mostraremos que en un curso de 40 estudiantes es muy probable que haya al menos dos que cumplan años el mismo día y explicaremos por qué en un torneo a eliminación directa, aumentan las chances del equipo que es superior si la serie se define en un número creciente de partidos (3, 5 o más) en lugar de en un sólo encuentro. §2. Cálculo de probabilidades Dado un experimento con una cantidad finita de resultados posibles, todos ellos equivalentes, se define la probabilidad de un evento E como el cociente de los casos favorables sobre los casos totales #{resultados favorables} p(E) = , #{resultados posibles} donde el símbolo # denota el número de elementos de un conjunto. Veamos algunos ejemplos para ilustrar esta definición. Ejemplos (1) Si se lanza un dado, la probabilidad de que salga el número 1 es de una (favorable) en 6 (posibles), es decir p({1}) = 16 . (2) Ahora bien, si nuevamente se lanza un dado, la probabilidad de obtener un número par es #{2, 4, 6} = 1. p(par) = #{1, 2, 3, 4, 5, 6} 2 (3) Ahora bien, si se lanzan 2 dados, nos preguntamos cuál es la probabilidad de que salga al menos un 1. Revista de Educación Matemática, Vol. 33 N° 3 – 2018 Matemática en azar y juegos 53 El número de resultados posibles es 6 ⋅ 6 = 36, pues para cada uno de los dados hay 6 posibilidades. Los resultados favorables son los del tipo ⎧ ⎪ ⎪1y (si sale 1 en el primer dado y cualquier número y en el segundo), ⎨ ⎪ ⎪ ⎩x1 (si sale cualquier número x en el primer dado y 1 en el segundo). Notar que hay 6 posibilidades en cada caso y que al 11 lo hemos contado 2 veces, luego hay que restarlo. Luego, los casos favorables son 6 + 6 − 1 = 11 y se tiene p(obtener al menos un 1) = 11 36 . (4) Si se lanzan 3 dados, queremos nuevamente saber la probabilidad de que salga al menos un 1. Los casos posibles son 6 ⋅ 6 ⋅ 6 = 216. Enumeremos los casos favorables. Ellos son del tipo ⎧ ⎪ 1yz hay 6.6 = 36 casos, ⎪ ⎪ ⎪ ⎪ ⎨x1z hay 5.6 = 30 casos, ⎪ ⎪ ⎪ ⎪ ⎪ ⎩xy1 hay 5.5 = 25 casos. Observamos que en la opción x1z, x puede tomar 5 valores (2, 3, 4, 5, 6) pues el caso en que toma el valor 1 ya se contó en el primer combo 1yz. Similar argumento es válido para el combo xy1. De acuerdo a este razonamiento, el conteo de casos favorables da 36 + 30 + 25 = 91, luego 91 . p(salga al menos un 1) = 216 Teniendo en mente estos ejemplos, veremos a continuación que hay un modo más simétrico de contar los casos favorables. Teorema 2.1. (Principio de inclusión-exclusión) Si A1 , . . . , An son conjuntos finitos entonces #(A1 ∪ ⋯ ∪ An ) = ∑ #Ai − ∑ #(Ai ∩ Aj ) 1≤i<j≤n 1≤i≤n + ∑ #(Ai ∩ Aj ∩ Ak ) − ⋯ + (−1)n+1 #(A1 ∩ ⋯ ∩ An ) 1≤i<j<k≤n donde #A denota el cardinal, o sea la cantidad de elementos, de A. Veamos de justificar esta fórmula en los casos n = 2 y n = 3. Si n = 2 tenemos #(A1 ∪ A2 ) = #A1 + #A2 − #(A1 ∩ A2 ), ya que los elementos de A1 ∩ A2 han sido contados dos veces. Revista de Educación Matemática, Vol. 33 N° 3 – 2018 54 Roberto J. Miatello, Angel D. Villanueva Si n = 3 tenemos que #(A1 ∪ A2 ∪ A3 ) =#A1 + #A2 + #A3 − #(A1 ∩ A2 ) − #(A1 ∩ A3 ) − #(A2 ∩ A3 ) + #(A1 ∩ A2 ∩ A3 ) pues, en la primera suma, los elementos de los Ai ∩Aj han sido contados dos veces y entonces hay que restarlos y, por otra parte, los de A1 ∩ A2 ∩ A3 fueron contados tres veces y luego restados tres veces, por lo tanto es necesario sumarlos una vez, como indica la fórmula. Aplicaremos la fórmula del teorema en el caso del ejemplo (4), calculando los casos favorables por medio del principio de inclusión-exclusión. Es decir, contaremos las ternas con al menos un número 1, restaremos las ternas que tienen al menos dos números 1 y finalmente sumaremos las ternas que tienen tres números 1. En la notación del teorema tenemos A1 ∶= {ternas con 1 en la primera posición}, A2 ∶= {ternas con 1 en la segunda posición}, A3 ∶= {ternas con 1 en la tercera posición}. Los casos en que hay al menos un número 1 son ⎧ ⎪ 1yz (esto es, A1 ), ⎪ ⎪ ⎪ ⎪ ⎨x1z (esto es, A2 ), ⎪ ⎪ ⎪ ⎪ ⎪ ⎩xy1 (esto es, A3 ), y en cada caso hay 36 posibilidades. Los casos en que hay al menos dos números 1 son ⎧ ⎪ 11z (esto es, A1 ∩ A2 ), ⎪ ⎪ ⎪ ⎪ ⎨1y1 (esto es, A1 ∩ A3 ), ⎪ ⎪ ⎪ ⎪ ⎪ ⎩x11 (esto es, A2 ∩ A3 ), en cada caso hay 6 posibilidades. Finalmente, el caso en que hay tres números 1 es 111 (esto es, A1 ∩ A2 ∩ A3 ). Por lo tanto, el número de casos favorables es 36 + 36 + 36 − 6 − 6 − 6 + 1 = 91. Antes de discutir algunas aplicaciones introducimos los siguientes conceptos que utilizaremos a lo largo del texto. Revista de Educación Matemática, Vol. 33 N° 3 – 2018 Matemática en azar y juegos 55 El factorial de un entero positivo n se define como el producto de todos los enteros positivos desde 1 hasta n y lo denotamos n! Por ejemplo, 4! = 1 ⋅ 2 ⋅ 3 ⋅ 4 = 24. Por convención, se toma 0! = 1. n ) se define como Dados n ≥ m enteros positivos, el número combinatorio (m n n! ( )= . m (n − m)!m! Este, representa el número total de subconjuntos de m elementos de un conjunto de n elementos. A modo ilustrativo, si tenemos 4 jugadores de tenis A, B, C y D ¿Cuáles son todas las parejas de dobles que podemos armar? Tenemos 4 4! ( )= = 6. 2 2!2! Estas son: AB, AC, AD, BC, BD y CD. §3. Repartición del premio. Un problema que algunos consideran que fue el origen del cálculo de probabilidades es el siguiente. Dos jugadores, A y B, juegan un juego en que ambos tienen las mismas probabilidades de ganar, por ejemplo, tirar una moneda equilibrada: cuando sale cara (C) gana A y cuando sale escudo (E) gana B. El ganador será el primero en ganar (por ejemplo) 6 rondas. La pregunta es ¿Cómo se debería repartir el premio si el juego se interrumpe antes de finalizar cuando un jugador ya ha ganado (por ejemplo) 5 rondas y el otro 3? Este problema fue considerado por matemáticos importantes de la época y tuvo varias soluciones incorrectas. Esto dio origen a una correspondencia entre Pascal y Fermat (aproximadamente 1650) quienes finalmente dieron la solución acertada. Christian Huygens, que tuvo conocimiento de esta correspondencia en 1655 publicó en 1657 el tratado De Ratiociniis in Ludo Aleae (Calculando en juegos de azar) en el que resolvía los problemas sobre probabilidades que circulaban en aquella época. Este tratado se convirtió en el primer tratado publicado sobre cálculo de probabilidades. Según Pascal y Fermat, la repartición del premio debería tener en cuenta lo que puede suceder si el juego continúa. En efecto, si el juego continuara, terminaría como máximo en 3 rondas, si ganara las tres el jugador B y en menos si A ganara alguna. Notar que para que B gane el premio, debería salir tres veces seguidas escudo (E). La probabilidad de que esto suceda es 1 3 1 p(EEE) = ( ) = . 2 8 Revista de Educación Matemática, Vol. 33 N° 3 – 2018 56 Roberto J. Miatello, Angel D. Villanueva Por lo tanto, de 8 casos posibles, hay 7 casos en los que gana el jugador A y un caso en que gana B CCC CCE CEC CEE ECC ECE EEC EEE Notar que el único caso en que gana B es el último (EEE). La conclusión es que corresponde recibir 78 partes del premio al jugador A y 18 parte al jugador B. Más generalmente, consideremos el caso en que varía el número de rondas. Supongamos que B necesita n rondas para vencer y A m rondas, con n > m. En este caso, el juego termina en (n − 1) + (m − 1) + 1 = n + m − 1 rondas y A, para vencer, precisa ganar un número j ≥ m de rondas. Luego 1 n+m−1 n+m−1 n + m − 1 pA = ( ) ). ∑ ( j 2 j=m Por ejemplo, si m = 1 y n = 3 queda pA = 1 3 3 7 1 ∑ (j ) = (3 + 3 + 1) = , 8 j=1 8 8 como hallamos anteriormente. §4. Cumpleaños En una reunión de 30 personas, ¿cuál es la probabilidad de que dos de ellas cumplan años el mismo día? Para determinarlo, es más conveniente calcular la probabilidad de que NO haya dos personas que cumplan años el mismo día. Notar que la primer persona tiene los 365 días del año disponibles, pero la segunda persona sólo dispone de 364 días, y así hasta la persona número 30 que tiene disponibles 336 días. Luego, en un grupo de 30 personas, la probabilidad de que todas ellas cumplan años en distintos días es 365 ⋅ 364 ⋯ 337 ⋅ 336 p(cumplan días distintos) = ∼ 0, 29. 36530 Dejamos la verificación del cálculo al lector. Revista de Educación Matemática, Vol. 33 N° 3 – 2018 Matemática en azar y juegos 57 Por lo tanto, la probabilidad de que haya al menos 2 personas que cumplan el mismo día, es p(dos cumplen el mismo día) = 1 − p(todos cumplen en días distintos) = 1 − 0, 29 = 0, 71 ∼ 71 %. Uno puede hacer otro cálculo y comprobar que en un grupo de 23 personas o más resulta más probable que haya al menos 2 que cumplan años el mismo día, a que no los haya. Asimismo, se puede ver que en un grupo de 60 personas, la probabilidad de que haya dos que cumplan años el mismo día es realmente muy grande, de aproximadamente 99 %. §5. Dígitos en un número natural La pregunta es ahora estimar la probabilidad pn de que un dígito fijo (digamos 7) aparezca en la expresión de un número natural y analizar el comportamiento de pn cuando n tiende a infinito. Supondremos que n tiene a lo sumo h cifras. Si h = 1, entonces n puede tomar todos los valores entre 0 y 9 salvo 0 y 7 (ya que 0 no es natural), es decir hay 8 posibilidades. Si h=2, la primera cifra tiene 8 valores posibles y la segunda 9 pues 0 ahora está permitido. Similarmente, si n posee h cifras, todas las cifras salvo la primera tienen 9 valores posibles. Esto nos dice que entre 0 y n = 10h − 1 el número de casos desfavorables es 9h − 1 = 9h − 1. 9−1 Luego, la probabilidad de que no aparezca el dígito 7 es 8(1 + 9 + 92 + ⋯ + 9h−1 ) = 8 9 h 1 h 9h − 1 = ( ) − ( ) , 10h 10 10 cantidad que tiende a 0 si h (o n) tiende a infinito. Esto nos dice que la probabilidad de que el dígito 7 no aparezca en un número natural n cuando n es muy grande tiende a 0. Sin embargo, el comportamiento de esta sucesión de números es muy lento. En efecto, se puede calcular que pn = h = 2, h = 3, h = 4, h = 6, h = 9, 81 − 1 = 0,8 100 729 − 1 p= = 0,728 1000 6561 − 1 p= = 0,656 10000 53145 − 1 p= = 0,531 100000 38742705 − 1 p= = 0,429 . 100000000 p= Revista de Educación Matemática, Vol. 33 N° 3 – 2018 58 Roberto J. Miatello, Angel D. Villanueva Esto dice en particular que aún para números n con un millón de cifras, la probabilidad de que no aparezca el dígito 7 es elevada y mayor que 12 . Sin embargo, cuando n tiende a infinito, la probabilidad de que aparezca el dígito 7 tiende a 1. §6. Torneos y competencias Consideremos ahora un torneo de eliminación simple, es decir, aquél en el cual se enfrentan dos rivales y el vencedor avanza de fase y el perdedor queda eliminado. Por ejemplo los torneos de tenis, el mundial de fútbol a partir de los octavos de final, los play-offs en la NBA, etc. 6.1. Eliminación simple. Supongamos que un jugador (o un equipo) A llega a los octavos de final y que la probabilidad de vencer a cualquier otro es de 23 . Surge la pregunta natural ¿Cuál es la probabilidad de que A sea campeón? La probabilidad de que A gane los octavos de final es de 32 . Los octavos y los cuartos de final son eventos independientes entre sí, por lo tanto las probabilidades de vencer en cada uno se multiplican, luego la probabilidad de que A llegue a semifinales, es decir, de que gane octavos y cuartos de final es 2 2 4 2 2 p(semi final) = ⋅ = ( ) = = 0, 44 . . . ∼ 44 %. 3 3 3 9 Observamos que aún siendo A superior a cualquiera de sus rivales la probabilidad de llegar a semifinales ya es menor al 50 %. Finalmente, la probabilidad de que A salga campeón es 2 2 2 2 2 4 16 p2/3 (A campeón) = ( ) ⋅ ( ) ⋅ ( ) ⋅ ( ) = ( ) = = 0, 1975 . . . ∼ 19, 75 % < 20 %. 3 3 3 3 3 81 ● Hagamos la misma pregunta suponiendo que A tiene una mayor superioridad sobre sus contrincantes. Por ejemplo, supongamos que la probabilidad de vencer a cualquier otro equipo sea de 43 . Entonces, 3 3 3 3 3 4 81 p3/4 (A campeón) = ( ) . ( ) . ( ) . ( ) = ( ) = = 0, 316 . . . ∼ 31, 6 %. 4 4 4 4 4 256 Como vemos la chance de ser campeón es aún pequeña, pese a la superioridad sobre cada rival. 6.2. Definiciones al mejor de 3, 7 ó una cantidad arbitraria de partidos Revista de Educación Matemática, Vol. 33 N° 3 – 2018 Matemática en azar y juegos 59 Al mejor de 3. Ahora nos preguntamos qué sucede si la serie se juega al mejor de 3 partidos, es decir, se enfrentan 3 veces los mismos rivales y el que haya ganado más partidos avanza de fase. Sea p la probabilidad que tiene A de vencer a sus rivales. Las formas que tiene A de pasar de fase son (a) ganando los tres partidos, (b) ganando dos partidos y perdiendo uno. La probabilidad de que A gane los tres partidos es p(A gana tres) = p ⋅ p ⋅ p = p3 . El evento de que A gane dos partidos y pierda uno puede darse de tres maneras distintas: ● A gana los primeros dos partidos y pierde el último, ● A gana el primer y tercer partido y pierde el segundo, ● A pierde el primer partido y gana los últimos dos. Cada una de estas situaciones tiene probabilidad p2 (1 − p). Entonces p(A gana dos) = 3p2 (1 − p). Luego, p(A gana al mejor de tres) = p3 + 3p2 (1 − p) = 3p2 − 2p3 = p2 (3 − 2p). En consecuencia, si la probabilidad de que A venza a cualquier rival es de 32 , entonces la probabilidad de que A pase de fase al mejor de tres partidos es 2 2 2 20 p(A gana al mejor de tres) = ( ) (3 − 2 ⋅ ) = = 0, 74 . . . ∼ 74 %. 3 3 27 Luego, la probabilidad de que A salga campeón jugando cada fase al mejor de tres partidos es p2/3 (A campeón) = ( 20 20 20 20 20 4 ) ⋅ ( ) ⋅ ( ) . ( ) = ( ) = 0, 30 . . . ∼ 30 %. 27 27 27 27 27 Haciendo el mismo cálculo pero suponiendo que la probabilidad que tiene A de vencer a cualquier rival es de 43 , resulta que la probabilidad de que A pase de fase al mejor de tres partidos es 3 2 3 27 p3/4 (gana A al mejor de tres) = ( ) . (3 − 2 ⋅ ) = ∼ 84, 4 %. 4 4 32 y de que A salga campeón con este sistema p3/4 (A campeón) = ( 27 27 27 27 27 4 ) ⋅ ( ) ⋅ ( ) . ( ) = ( ) ∼ 50, 7 %. 32 32 32 32 32 Revista de Educación Matemática, Vol. 33 N° 3 – 2018 60 Roberto J. Miatello, Angel D. Villanueva Al mejor de 7. Ahora nos preguntamos qué sucede si la serie se juega al mejor de 7 partidos. Sea p la probabilidad que tiene A de vencer a cualquiera de sus rivales. Las formas que tiene A de pasar de fase son (a) ganando los siete partidos, (b) ganando seis partidos y perdiendo 1, (c) ganando cinco partidos y perdiendo 2, (d) ganando cuatro partidos y perdiendo 3. Con el mismo razonamiento que en la subsección anterior, tenemos p(caso a) = p7 , 7 p(caso b) = ( )p6 (1 − p), 6 7 p(caso c) = ( )p5 (1 − p)2 , 5 7 p(caso d) = ( )p4 (1 − p)3 . 4 Luego 7 7 7 p(A pasa) = p7 + ( )p6 (1 − p) + ( )p5 (1 − p)2 + ( )p4 (1 − p)3 6 5 4 7 6 5 2 4 = p + 7p (1 − p) + 21p (1 − p) + 35p (1 − p)3 . Si suponemos que la probabilidad que tiene A de vencer a cualquier rival es p = 32 (1 − p = 31 ) entonces la probabilidad de que A pase la serie al mejor de 7 es 2 6 1 2 5 1 2 2 4 1 3 1808 2 7 ∼ 82, 7 %. p2/3 (A pasa) = ( ) + 7( ) ( ) + 21( ) ( ) + 35( ) ( ) = 3 3 3 3 3 3 3 2187 Luego, la probabilidad de que A sea campeón con este sistema es p2/3 (A campeón) = ( 1808 4 ) ∼ 46, 7 %. 2187 Si suponemos que la probabilidad que tiene A de vencer a cualquier rival es p = 34 (1 − p = 14 ) entonces la probabilidad que tiene A de pasar la serie al mejor de 7 es 3 7 3 6 1 3 5 1 2 3 4 1 3 15.228 p3/4 (A pasa) = ( ) + 7( ) ( ) + 21( ) ( ) + 35( ) ( ) = ∼ 92, 94 %. 4 4 4 4 4 4 4 16.384 Luego, la probabilidad de que A sea campeón con este sistema es p3/4 (A campeón) = ( 15.228 4 ) ∼ 74, 62 %. 16.384 Estos cálculos muestran, como era de esperar, que la probabilidad de A aumenta si el número de partidos disputados en la serie crece. Revista de Educación Matemática, Vol. 33 N° 3 – 2018 Matemática en azar y juegos 61 Al mejor de n. Ahora generalizaremos el problema anterior, cuando el enfrentamiento se decide con n partidos. Nos interesa especialmente ver lo que sucede cuando n → ∞. Haremos uso de las siguientes identidades entre números combinatorios válidas con las convenciones de que (nj) = 0 si j < 0 y (n0 ) = 1. ( (6.1) 2n + 3 2n + 2 2n + 2 ) =( )+( ) j j j−1 2n + 1 2n + 1 2n + 1 2n + 1 =( )+( )+( )+( ) j j−1 j−1 j−2 2n + 1 2n + 1 2n + 1 =( ) + 2( )+( ). j j−1 j−2 Sea p2n+1 la probabilidad de A de vencer en 2n + 1 partidos. Usando (6.1) establecemos una relación entre p2n+3 y p2n+1 . Tenemos 2n+3 2n + 3 j ) p (1 − p)2n+3−j . j j=n+2 p2n+3 = ∑ ( Luego, 2n+3 p2n+3 = 2n+3 2n + 1 j 2n + 1 j ) p (1 − p)2n+3−j + 2 ∑ ( ) p (1 − p)2n+3−j j j − 1 j=n+2 j=n+2 ∑ ( 2n+3 2n + 1 j ) p (1 − p)2n+3−j j=n+2 j − 2 + ∑ ( 2n+1 = ∑ (( j=n+2 2n + 1 j )p (1 − p)2n+1−j ) (1 − p)2 j 2n+1 2n+1 2n + 1 j ) p (1 − p)2n+1−j + p2 ∑ pj (1 − p)2n+1−j j j=n+1 j=n +2p(1 − p) ∑ ( = p2n+1 (1 − p)2 − ( +( 2n + 1 n+1 ) p (1 − p)n+2 + p2n+1 (2p(1 − p) + p2 ) n+1 2n + 1 n+2 ) p (1 − p)n+1 , n de donde p2n+3 = p2n+1 + ( 2n + 1 n+1 ) (p(1 − p)) (2p − 1). n Revista de Educación Matemática, Vol. 33 N° 3 – 2018 62 Roberto J. Miatello, Angel D. Villanueva Esto muestra que si p > 12 el segundo sumando a la derecha es positivo, luego la probabilidad, de 2n + 1 juegos a 2n + 3 juegos, ha aumentado. Además se tiene p2n+3 = (p2n+3 − p2n+1 ) + (p2n+1 − p2n−1 ) + ⋯ + (p3 − p1 ) 2n+1 = (2p − 1) ∑ ( j=0 2j + 1 ) (p(1 − p))j+1 + p. j Se sabe que, para n ∈ N, vale la identidad √ n 1 − 1 − 4x 2j + n j 1 ( ) 2∑( )x = √ 2x j 1 − 4x j=0 ∞ (ver por ejemplo Anexo: series matemáticas en Wikipedia). Usando esto con n = 1 sale que 2j + 1 )(p(1 − p))j j j=0 ∞ lı́m p2n+1 = p + (2p − 1)p(1 − p) ∑ ( n→∞ √ 1 − 1 − 4p(1 − p) = p + (2p − 1) √ . 2 1 − 4p(1 − p) El lector interesado puede encontrar el límite anterior en Wikipedia. Ahora ponemos p = 12 + t. Luego, 4p(p − 1) = 4t2 − 1 y obtenemos que el límite anterior es igual a 1 − 2t p + (2p − 1) = p + 21 − t = 1. 4t 1 Este cálculo verifica que si p > 2 la función p2n+1 tiende a 1 cuando n crece arbitrariamente, es decir que el jugador que tiene alguna superioridad, aunque sea pequeña, vence con probabilidad aproximada a 1 si se juega al mejor de un número suficientemente grande de partidos. Por otra parte el argumento también prueba que pn nunca es igual a 1, salvo que se parta inicialmente de p = 1. §7. Regalos Ahora planteamos el siguiente problema. Supongamos que hay un grupo de n compañeros de trabajo y cada uno compra un regalo para repartirse entre ellos de manera aleatoria. ¿Cuál es la probabilidad de que a ninguno le toque el regalo que el mismo compró? Analicemos la situación. Supongamos que el grupo es de 2 personas: A1 y A2 . Hay 2 situaciones posibles: que se intercambien los regalos o que a cada uno le toque el que compró: p(ninguno recibe su propio regalo) = 1 = 50 %. 2 Revista de Educación Matemática, Vol. 33 N° 3 – 2018 Matemática en azar y juegos 63 Supongamos ahora que el grupo es de 3 personas: A1 , A2 , A3 . Hay 3! = 6 formas posibles de repartir los regalos: A1 A2 A3 , A1 A3 A2 , A2 A1 A3 , A2 A3 A1 , A3 A2 A1 , A3 A1 A2 . En el primer caso a cada uno le toca su propio regalo; en el segundo caso a A1 le tocó su propio regalo, a A2 el regalo que compró A3 y a A3 el que compró A2 , etc. En el cuarto y en el sexto caso nadie recibe el regalo que compró. Luego, p(ninguno recibe su propio regalo) = 2 1 = ∼ 33, 3 %. 6 3 Nos gustaría poder deducir qué ocurre en un grupo con un número arbitrario n de personas. Etiquetamos a las personas de a1 a an . Utilizaremos el principio de inclusiónexclusión para contar los casos en que hay al menos una persona que recibe su propio regalo. Para eso, primero sumamos los casos en que a1 recibe su propio regalo, mas los casos en que a2 recibe su propio regalo, siguiendo así hasta sumar los casos en que an recibe su propio regalo n n ∑ #{casos que ai recibe su propio regalo} = ( )(n − 1)! 1 i=1 ya que hay (n1 ) formas de elegir a la persona que recibe su propio regalo y para cada una de esas elecciones hay (n − 1)! formas de repartir los regalos entre las n − 1 personas restantes. Notar que los casos en que hay dos personas que reciben su propio regalo los hemos contado en más de una oportunidad. Por lo tanto, debemos sustraer: #{casos que a1 y a2 reciben su propio regalo} + ⋯ + #{casos que a1 y an reciben su propio regalo} + ⋯ + #{casos que a2 y a3 reciben su propio regalo} + ⋯ + #{casos que a2 y an reciben su propio regalo} + ⋯ n + #{casos que an−1 y an reciben su propio regalo} = ( )(n − 2)! 2 pues tenemos (n2 ) formas de elegir a las 2 personas que reciben su propio regalo y para cada una de esas elecciones hay (n − 2)! formas de repartir los regalos entre las n − 2 personas restantes. Notar que los casos en que exactamente un mismo grupo de 3 personas reciben su propio regalo ha sido sumado primero 3 veces y luego sustraido 3 veces. Revista de Educación Matemática, Vol. 33 N° 3 – 2018 64 Roberto J. Miatello, Angel D. Villanueva Entonces tenemos que sumar: #{casos que a1 , a2 y a3 reciben su propio regalo}+ #{casos que a1 , a2 y a4 reciben su propio regalo} + ⋯+ n #{casos que an−2 , an−1 y an reciben su propio regalo} = ( )(n − 3)! 3 Procedemos con el mismo razonamiento, sumando y restando los casos siguientes para concluir que la cantidad de casos desfavorables, es decir, aquellos en que al menos una persona recibe su propio regalo, es n n n n n ( )(n−1)!−( )(n−2)!+( )(n−3)!−⋯+(−1)n ( )(n−(n−1))!+(−1)n+1 ( )(n−n)! 1 2 3 n−1 n Aplicamos esta fórmula cuando el grupo es de 3 personas: 3 3 3 #(casos desfavorables) = ( )2! − ( )1! + ( ) 1 2 3 3! 3! 1 1 1 3! 2! − 1! + = 3! ( − + ) = 4. = 1!2! 2!1! 3! 1! 2! 3! Es decir, los casos favorables son 6−4 = 2 como habíamos verificado anteriormente. Supongamos ahora que el grupo es de 4 personas, entonces tenemos 4 4 4 4 #(casos desfavorables) = ( )3! − ( )2! + ( )1! − ( ) 1 2 3 4 4! 4! 4! 4! 1 1 1 1 3! − 2! + 1! − = 4! ( − + − ) = 15. = 1!3! 2!2! 3!1! 4! 1! 2! 3! 4! Los casos totales son 4! = 24, entonces los favorables son 9 y p(ninguno recibe su propio regalo) = 9 3 = = 0, 375 ∼ 37, 5 %. 24 8 Finalmente, cuando el grupo es de n personas se tiene n n n #(casos desfavorables) = ( )(n − 1)! − ( )(n − 2)! + ⋯ + (−1)n+1 ( ) 1 2 n n! n! n! = (n − 1)! − (n − 2)! + ⋯ + (−1)n+1 1!(n − 1)! (n − 2)!2! n! 1 1 1 = n! ( − + ⋯ + (−1)n+1 ) . 1! 2! n! Como #(casos favorables) = #(casos totales) − #(casos desfavorables) Revista de Educación Matemática, Vol. 33 N° 3 – 2018 Matemática en azar y juegos 65 se tiene que 1 1 1 − + ⋯ + (−1)n+1 ) 1! 2! n! 1 1 1 1 = n! [1 − ( − + + ⋯ + (−1)n+1 )] 1! 2! 3! n! 1 1 1 = n! [1 − + + ⋯ + (−1)n ] . 1! 2! n! #(casos favorables) = n! − n! ( Luego, n! [1 − 1!1 + 2!1 + ⋯ + (−1)n n!1 ] p(nadie recibe su propio regalo) = n! 1 1 1 = 1 − + + ⋯ + (−1)n 1! 2! n! y esta suma es la expansión en serie del inverso de un número muy importante: e ≈ 2, 718281828459 . . . Tenemos que (−1)n 1 1 (−1)n =1− + −⋯+ +⋯ n! 1! 2! n! j=0 ∞ e−1 = ∑ y se puede verificar usando el teorema de Taylor que a partir de n = 5 la diferencia entre e−1 y la suma 1 − 1!1 + 2!1 + ⋯ + (−1)n n!1 es menor que 0, 01. Luego, obtenemos que la probabilidad de que en un grupo de n personas, con n ≥ 5, a una persona le toque el regalo que compró es aproximadamente 1 = 0, 36787944117144 . . . ∼ 36, 78 %. e §7. El mono y la máquina de escribir Este es un problema clásico que plantea calcular la probabilidad de que un mono tipee al azar, idealmente, una obra de la literatura, por ejemplo ‘El Quijote’, si lo intenta un número suficiente de veces. El planteo es que el mono comienza a tipear, en una computadora con M teclas, los caracteres de la obra, digamos que sean T en total. Se supone que hace un intento y otro sucesivamente repitiéndolo una y otra vez indefinidamente. Cada T intentos el mono retoma la acción sin saber si ya cumplió su cometido. En el desarrollo no tenemos en cuenta el tiempo que le lleva hacer cada intento y se incluyen entre los caracteres, los espacios, acentos y otros símbolos necesarios en la obra. Claramente, la probabilidad de acertar el primer carácter es p1 = 1/M , la de acertar los dos primeros es p1,2 = 1/M 2 y así sucesivamente la probabilidad de acertar de entrada los T caracteres de la obra es p1,2,...T = 1/M T . Revista de Educación Matemática, Vol. 33 N° 3 – 2018 66 Roberto J. Miatello, Angel D. Villanueva Para cada número natural n, dividamos en segmentos de longitud T los intentos del mono, es decir, primero el segmento 1, 2, . . . , T , luego T + 1, T + 2, . . . , 2T , y, en el paso n-ésimo, (n − 1)T + 1, . . . , nT . Supongamos hipotéticamente que el mono erra en alguno de sus primeros T intentos de reproducir la obra y también en alguno de sus segundos T y en alguno de los n-ésimos T y acierta en todo el siguiente, el N + 1-ésimo segmento. La probabilidad de que esto suceda es N (8.1) pN = ∑ (1 − M −T )n M −T n=0 ya que erra en los primeros N intentos (con probabilidad (1 − M −T )) y acierta en el intento N + 1. Denotaremos desde ahora p = M −T , por simplicidad. Entonces N N n pN ∶= ∑ (1 − M −T ) M −T = p ∑ (1 − p)n = n=0 n=0 p(1 − (1 − p)n+1 ) = 1 − (1 − p)N +1 . 1 − (1 − p) Esta es una serie geométrica de razón 1 − p < 1, luego su suma cuando N → ∞ es 1 = 1, 1 − (1 − p) n=0 lo que nos dice que en tiempo indefinido, con probabilidad 1, el mono logrará reproducir toda la obra. ∞ p ∑ (1 − p)n = p Este resultado si bien es teóricamente correcto, carece de valor práctico pues el tiempo necesario para lograr el objetivo puede ser muy grande, incluso comparable a la edad de la tierra (unos 4.500 millones de años) o de la del universo, estimada en 13.700 millones de años. Como vemos, es imprescindible, en todo cálculo probabilístico con un número creciente indefinido de intentos, poder estimar cuan rápido el valor parcial se aproxima a su valor límite. Comentarios Cursillo dictado por los autores el 9/10/18 en el Instituto de Enseñanza Superior Simón Bolívar, Córdoba, destinado a estudiantes de Profesorado en Matemática. Agradecimientos. Los autores desean agradecer a Juan Pablo Rossetti por la lectura completa del trabajo y por enviar algunas correcciones y varias sugerencias que ayudaron a mejorar la exposición del mismo. Revista de Educación Matemática, Vol. 33 N° 3 – 2018 Matemática en azar y juegos 67 Referencias Kisbye, Patricia, y Miatello, Roberto. (2004). Álgebra I – Matemática Discreta I. Trabajos de Matemática, Serie C, FaMAF. Santaló, Luis A. (1955). La probabilidad y sus aplicaciones. Editorial Iberoamericana, Buenos Aires. Székely, Gábor J. (2001). Paradoxes in Probability Theory and Mathematical Statistics . Mathematics and its Applications. Reidel 1986, Springer Verlag 2001. Roberto J. Miatello Facultad de Matemática, Astronomía, Física y Computación (FaMAF) – Universidad Nacional de Córdoba (UNC) ( ) miatello@famaf.unc.edu.ar Angel D. Villanueva Facultad de Matemática, Astronomía, Física y Computación (FaMAF) – Universidad Nacional de Córdoba (UNC) ( ) villanueva@famaf.unc.edu.ar Recibido: 22 de mayo de 2018. Aceptado: 25 de julio de 2018. Publicado en línea: 21 de diciembre de 2018. Revista de Educación Matemática, Vol. 33 N° 3 – 2018 ¿Sabías que... por Leandro Cagliero y Ricardo Podestá el área de las cuatro lúnulas rosadas es igual al área del cuadrado celeste? Figura 1 Este bonito y sorprendente resultado es conocido como la cuadratura de las lúnulas de Hipócrates. Contemos algo de su historia y veamos por qué es cierto. Se conoce como la cuadratura del círculo al famoso problema que consiste en construir con regla y compás un cuadrado de la misma área que un círculo dado. Este problema tuvo ocupados a los matemáticos desde siempre, hasta que en el siglo XIX por fin se pudo demostrar que esto es imposible. Resulta que para lograr “cuadrar” el círculo de radio 1 √ es necesario construir con regla y compás el número π, lo cual equivale a construir con regla y compás el número π, y esto es imposible. ¿Por qué? En 1882 ya se sabía, desde hacía mucho tiempo, que si un número real α se puede construir con regla y compás, entonces hay un polinomio p(x) con coeficientes enteros tal que α es raíz de p(x), es decir que p(α) = 0. En ese año, Lindemann y Weierstrass obtienen un resultado que implica que no existe ningún polinomio p(x) con coeficientes enteros tal que p(π) = 0, con lo que queda probado que es imposible construir con regla y compás el número π, y por lo tanto es imposible cuadrar el círculo de radio 1. El Teorema de Lindemann y Weierstrass es profundo y un poco difícil. Aprovechamos para recordar que los números reales que son raíces de polinomios con coeficientes enteros se llaman números algebraicos, mientras que los que no lo son, se llaman números trascendentes. El argumento anterior se resume en: (1) Todo número construíble con regla y compás es algebraico. (2) En 1982 Lindemann y Weierstrass demuestran que π es trascendente. (3) Es imposible cuadrar el círculo de radio 1 pues si se pudiera π sería algebraico. Por otro lado, Hipócrates de Quíos, intentando resolver la cuadratura del círculo, descubrió que las lúnulas se pueden “cuadrar”. Éste, fue autor de un libro llamado Elementos, del cual no se conservan copias aunque otros documentos históricos (v. gr. Eudemo) dan cuenta de él. Se piensa que éste, y otros libros, han sido modelos de lo que luego fue el famoso ‘Elementos’ de Euclides. En dicho libro, Hipócrates demuestra un teorema que implica que la lúnula se puede cuadrar. Una versión muy elegante de éste es como sigue. Revista de Educación Matemática. Vol. 33, N◦ 3 – 2018 68 ¿Sabías qué...? 69 Teorema. En la Fig. 1, las cuatro lúnulas tienen la misma área que el cuadrado. La demostración es muy elegante y sólo utiliza el Teorema de Pitágoras que, en una de sus versiones, dice que dado un triángulo rectángulo, el área del semicírculo cuyo diámetro es la hipotenusa es igual a la suma de las áreas de los semicírculos cuyos diámetros son cada uno de los catetos. Figura 2. Interpretación gráfica del Teorema de Pitágoras usando círculos. Utilizando esto, la prueba del teorema es inmediata. Demostración. = = − + − = ¡¡Sin palabras!! Bellísima demostración que no utiliza palabras y nos deja sin ellas. Hipócrates de Quíos fue un matemático, geómetra y astrónomo griego que vivió entre 470 – 410 a. C., aproximadamente 100 años antes de Euclides. De hecho, se lo considera el geómetra mas importante del siglo V a. C. Escribió una obra de carácter enciclopédico titulada Elementos, en el que expone teoremas a partir de unos axiomas y postulados (los cuales fueron incluidos por Euclides en su famosa obra del mismo nombre). Éste no debe ser confundido con su contemporáneo Hipócrates de Cos, el famoso médico de Antigua Grecia autor del Juramento Hipocrático. Revista de Educación Matemática. Vol. 33, N◦ 3 – 2018 70 Leandro Cagliero y Ricardo Podestá Revista de Educación Matemática. Vol. 33, N◦ 3 – 2018
https://openalex.org/W2137104359	https://nhess.copernicus.org/articles/8/763/2008/nhess-8-763-2008.pdf	English	null	Using stochastic space-time models to map extreme precipitation in southern Portugal	Natural hazards and earth system sciences	2,008	cc-by	7,262	Using stochastic space-time models to map extreme precipitation in southern Portugal A C Costa1 R Dur˜ao2 M J Pereira2 and A Soares2 A. C. Costa1, R. Dur˜ao2, M. J. Pereira2, and A. Soares2 1ISEGI, Universidade Nova de Lisboa, Lisbon, Portugal 2CERENA, Instituto Superior T´ecnico, Lisbon, Portugal Abstract. The topographic characteristics and spatial cli- matic diversity are signiﬁcant in the South of continental Por- tugal where the rainfall regime is typically Mediterranean. Direct sequential cosimulation is proposed for mapping an extreme precipitation index in southern Portugal using eleva- tion as auxiliary information. The analysed index (R5D) can be considered a ﬂood indicator because it provides a measure of medium-term precipitation total. The methodology ac- counts for local data variability and incorporates space-time models that allow capturing long-term trends of extreme pre- cipitation, and local changes in the relationship between ele- vation and extreme precipitation through time. Annual grid- ded datasets of the ﬂood indicator are produced from 1940 to 1999 on 800 m×800 m grids by using the space-time rela- tionship between elevation and the index. Uncertainty eval- uations of the proposed scenarios are also produced for each year. The results indicate that the relationship between el- evation and extreme precipitation varies locally and has de- creased through time over the study region. In wetter years the ﬂood indicator exhibits the highest values in mountainous regions of the South, while in drier years the spatial pattern of extreme precipitation has much less variability over the study region. The uncertainty of extreme precipitation esti- mates also varies in time and space, and in earlier decades is strongly dependent on the density of the monitoring stations network. The produced maps will be useful in regional and local studies related to climate change, desertiﬁcation, land and water resources management, hydrological modelling, and ﬂood mitigation planning. Natural Hazards and Earth System Sciences Natural Hazards and Earth System Sciences Nat. Hazards Earth Syst. Sci., 8, 763–773, 2008 www.nat-hazards-earth-syst-sci.net/8/763/2008/ © Author(s) 2008. This work is distributed under the Creative Commons Attribution 3.0 License. 1 Introduction Elevation of the study region in the South of Portugal and stations’ locations. Fig. 2. Distribution of the number of available stations by year. Fig. 2. Distribution of the number of available stations by year. Fig. 1. Elevation of the study region in the South of Portugal and stations’ locations. Fig. 2. Distribution of the number of available stations by year. Fig. 1. Elevation of the study region in the South of Portugal and stations’ locations. ﬁeld (Willmott and Matsuura, 2006). Unlike traditional in- terpolation methods (e.g., cokriging), geostatistical simula- tion procedures aim at reproducing the spatial uncertainty of the attribute under study. The series of simulated maps can be post-processed and the spatial uncertainty summarized (Goovaerts, 1997). For example, the uncertainty at an un- sampled location can be evaluated through spread measures, such as the variance, derived from the corresponding local histogram. The number of studies on the interpolation of extreme pre- cipitation indicators is limited. The works of Faulkner and Prudhomme (1998) and Prudhomme and Reed (1998, 1999) focused on an index of extreme rainfall, named RMED – me- dian of the annual maximum rainfall, while Hundecha and B´ardossy (2005) and Perry and Hollis (2005) describe the mapping of several extreme precipitation indices. Faulkner and Prudhomme (1998), Prudhomme and Reed (1999), and Perry and Hollis (2005) used techniques that combine dis- tance weighting methods and regression to produce gridded datasets of extreme precipitation indices, whereas Hundecha and B´ardossy (2005) used kriging with external drift to inter- polate daily precipitation observations on a 5 km×5 km grid and calculated the extreme precipitation indices, afterwards, on derived grids of 5, 10, 25 and 50 km2. Interpolation usually leads to a smoothing of the distribution inferred by the observations and thus to a loss of variance. For exam- ple, it is well known that kriging is locally accurate in the minimum error variance sense, but does not provide repre- sentations of spatial variability given the “smoothing” effect of kriging (Goovaerts, 1997). The smoothing effect in pre- cipitation data is undesirable considering the modelling of ﬂoods or other extreme hydrological processes (Haberlandt, 2007). To overcome this limitation, geostatistical stochas- tic simulation has become a widely accepted procedure to reproduce the spatial distribution and uncertainty of highly variable phenomena in geosciences (e.g., Franco et al., 2006; Bourennane et al., 2007). Nat. Hazards Earth Syst. Sci., 8, 763–773, 2008 1 Introduction Information on the spatial variability of extreme precipita- tion is important for river basins management, ﬂood hazards protection, studies related to climate change, erosion mod- elling and other applications for hydrological impact mod- elling. It is long recognized that topography and other geo- graphical factors are responsible for considerable spatial het- erogeneity of the precipitation distribution at the sub-regional scale (e.g., Mart´ınez-Cob, 1996; Daly, 2006). A comprehen- sive review on the complex relationship between precipita- tion, airﬂow and physiographic features of mountainous re- gions is presented by Johansson and Chen (2003), and Smith and Barstad (2004). Accordingly, it is commonly accepted that interpolation techniques that make use of the relation- ship between existing station data and explanatory physio- graphic variables (e.g., elevation or distance to the coast- line) have the potential to better represent the actual climate spatial patterns, especially in mountainous areas and in re- gions with complex atmospheric inﬂuences (Prudhomme and Reed, 1998; Daly, 2006). Over the past two decades, efforts have been undertaken by many authors to incorporate elevation and other phys- iographic features into the spatial interpolation of rainfall ﬁelds. Some examples are multivariate geostatistics such as kriging with external drift or cokriging (Mart´ınez-Cob, 1996; Goovaerts, 2000; Diodato, 2005; Lloyd, 2005), tech- niques combining distance weighting methods and regres- sion (Faulkner and Prudhomme, 1998; Prudhomme and Reed, 1999; Perry and Hollis, 2005), splines (Hutchinson, 1995, Boer et al., 2001), and local regressions (Daly et al., 1994; Brunsdon et al., 2001). Most studies do not model simultaneously the rainfall space-time patterns, but rather focus on the generation of surfaces of long-term averaged precipitation (e.g., Mart´ınez-Cob, 1996; Diodato, 2005), or independently derived surfaces for yearly and monthly data (e.g., Lloyd, 2005; Perry and Hollis, 2005). Correspondence to: A. C. Costa (ccosta@isegi.unl.pt) ocus o t e precipitation independent (e.g., Lloyd, Published by Copernicus Publications on behalf of the European Geosciences Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. A. C. Costa et al.: Space-time mapping of extreme precipitation A. C. Costa et al.: Space-time mapping of extreme precipitation 764 A. C. Costa et al.: Space-time mapping of extreme precipitation Fig. 2. Distribution of the number of available stations by year. Fig. 1. A. C. Costa et al.: Space-time mapping of extreme precipitation 765 Fig. 3. Spatial and temporal components of the experimental semivariogram of the 1960s decade data of R5D with the exponential model ﬁtted. Fig. 3. Spatial and temporal components of the experimental semivariogram of the 1960s decade data of R5D with the exponential model ﬁtted. Geostatistical estimators, known as kriging, provide sta- tistically unbiased estimates of surface values from a set of observations at recorded locations, using the estimated spa- tial (and temporal) covariance model of the observed data. detected in southern Portugal (Corte-Real et al., 1998). Ac- cordingly, the methodology not only accounts for local data variability by using stochastic simulation procedures, but also incorporates space-time models that allow capturing long-term trends of extreme precipitation, and local corre- lations between elevation and precipitation through time. Consider the two dimensional problem of estimating a pri- mary variable z at an unsampled location u0. Let {z(uα), α=1, ..., n} be the set of primary data measured at n loca- tions uα. Most of geostatistics is based on the assumption that the set of unknown values is a set of spatially dependent random variables, hence each measurement z(uα) is a partic- ular realization of the random variable Z(uα). Kriging uses a linear combination of neighbouring observations to estimate the unknown value at the unsampled location u0. This prob- lem can be expressed in terms of random variables as: The current work has three objectives: (i) to assess the space-time relationship between elevation and extreme pre- cipitation in southern Portugal; (ii) to use this relationship to produce annual gridded datasets of a ﬂood indicator (R5D) from 1940 to 1999; and (iii) to provide an uncertainty evalu- ation of the produced scenarios. The R5D index was computed using high quality daily pre- cipitation observations measured at 105 monitoring stations with data within the period 1940/1999. The direct sequen- tial cosimulation was performed for generating one map per year, using 800 m×800 m grids and elevation as exhaustive secondary information. ˆZ (u0) = n X α=1 λαZ (uα). (1) (1) The optimal kriging weights λα are determined by solving the kriging equations that result from minimizing the estima- tion variance while ensuring unbiased estimation of Z(u0) by ˆZ (u0). The methodology is brieﬂy introduced in Sect. 2, and the study region and data are described in Sect. 3. The main results are presented and discussed in Sect. A. C. Costa et al.: Space-time mapping of extreme precipitation 4, including the relationship between elevation and the ﬂood indicator, the space-time patterns of the index in 1940/1999, and the un- certainty evaluation of the produced maps. Finally, Sect. 5 states the major conclusions. Kriging methods require a stationarity assumption, ex- pressed in two parts. First, the mean of the process is as- sumed constant and invariant with spatial location (ﬁrst order stationarity). Second, the variance of the difference between two values is assumed to depend only on the distance h be- tween the two points, and not on their location u (second order stationarity). Stationarity assumptions on kriging are traditionally accounted for by using local search neighbour- hoods so that the dependence on stationarity becomes local (Goovaerts, 1997). 1 Introduction Geostatistical simulation methods describe local data variability based on many, equally proba- ble, realizations of the phenomenon, consistent with the data and its statistical characteristics. As other southern European regions, the rainfall regime in southern Portugal is Mediterranean, and so highly vari- able in both the spatial and temporal dimensions. One par- ticularly relevant feature of the rainfall regime in this area is the occurrence of short but very intensive rainfall events that may lead to signiﬁcant damages, by causing ﬂash ﬂoods that affect small drainage basins (Ramos and Reis, 2002). Fragoso and Gomes (2008) concluded that the most southern region (Algarve) is the one where episodes of heavy rainfall are most frequent and which exhibits the strongest torrential character. In this work, the R5D index was chosen to characterize the extreme precipitation regime. This indicator is deﬁned as the highest consecutive 5-day precipitation total (in millimetres) per year, and so provides a measure of medium-term precipi- tation total. For the interpolation and uncertainty assessment of extreme precipitation in the southern region of continen- tal Portugal, we explore the application of direct sequential cosimulation, which allows incorporating covariates such as elevation. The choice of cosimulation follows the premises that ele- vation and precipitation may interact differently in space and during drier and wetter periods (Goovaerts, 2000). Further- more, there are evidences of a trend towards a drier climate in southern Europe as a result of increased evapotranspira- tion and a relatively slow decrease of rainfall amounts and precipitation frequency (Cubasch et al., 1996; Kostopoulou and Jones, 2005; Vicente-Serrano and Cuadrat-Prats, 2007). Moreover, a negative trend in March precipitation has been The accuracy and uncertainty of gridded data sets is dif- ﬁcult to assess because the ﬁeld that is being estimated is unknown between data points. Spatial interpolation errors are interdependent functions of the station-network distribu- tion, the efﬁcacy of the interpolation procedure, and the real (but unknown) spatial distribution of the underlying climatic Nat. Hazards Earth Syst. Sci., 8, 763–773, 2008 www.nat-hazards-earth-syst-sci.net/8/763/2008/ Nat. Hazards Earth Syst. Sci., 8, 763–773, 2008 2.3 Direct sequential cosimulation 2.3 Direct sequential cosimulation Soares (2001) describes the sequence of the direct sequen- tial simulation (DSS) algorithm of a continuous variable as follows: where N(h) is the number of pairs of data locations a vec- tor h apart. In bounded models (e.g., spherical and exponential), var- iogram functions increase with distance until they reach a maximum, named sill, at an approximate distance known as the range. The range is the distance h at which the spatial (or temporal) correlation vanishes, i.e. observations separated by a distance larger than the range are spatially (or temporally) independent observations. 1. Randomly select the spatial location of a node z(u0) in a regular grid of nodes to be simulated; 2. Estimate local mean and variance identiﬁed with simple kriging estimator (Eq. 3) and kriging variance. Sample from the global histogram a value zs(ui) centred in the estimated local mean and variance. 2 Methods This section brieﬂy introduces the kriging techniques and de- scribes the reasoning of the geostatistical stochastic simula- tion algorithms implemented. Interested readers should refer to geostatistical textbooks (e.g., Isaaks and Srivastava, 1989; Goovaerts, 1997) for detailed descriptions of univariate and multivariate geostatistical methods. For a thorough descrip- tion of the direct sequential simulation, and cosimulation, al- gorithms the reader is referred to Soares (2001). When developing the kriging equations the model of spa- tial covariances, or variogram (inverse function of the spatial covariances), is assumed known. This is a key function of geostatistics and characterizes the variability of the spatial (and temporal) patterns of physical phenomena. Typically, a Nat. Hazards Earth Syst. Sci., 8, 763–773, 2008 www.nat-hazards-earth-syst-sci.net/8/763/2008/ A. C. Costa et al.: Space-time mapping of extreme precipitation A. C. Costa et al.: Space-time mapping of extreme precipitation A. C. Costa et al.: Space-time mapping of extreme precipitation 766 Fig. 4. Plot of elevation against R5D values calculated within 1940/99. Fig. 5. Regional correlation between elevation and R5D values by year. Fig. 5. Regional correlation between elevation and R5D values by year. 2.2 Collocated cokriging 2.2 Collocated cokriging Fig. 4. Plot of elevation against R5D values calculated within 1940/99. Consider now the situation where the set of primary data {z(uα), α=1, ..., n} is complemented by secondary data available at all estimation grid nodes and denoted by y(u). The collocated cokriging estimate is (Goovaerts, 2000): mathematical variogram model is selected from a small set of authorised ones (e.g. exponential or spherical) and is ﬁtted to experimental semivariogram values calculated from data for given angular and distance classes. ˆzCoK (u0) = n(u) X α=1 λCoK α (u0) z (uα) + λCoK (u0) [y (u0) −mY + mZ] (4) The experimental semivariogram ˆγ (h) is computed as half the average squared difference between data pairs belonging to a certain angular and distance class: where mZ and mY are the global means of the primary and secondary variables, Z(u) and Y(u), respectively. Note that only the secondary datum collocated at the location u0 being estimated is retained for estimation. ˆγ (h)= 1 2N(h) N(h) X α=1 [z(uα) −z(uα + h)]2 (2) (2) (2) Nat. Hazards Earth Syst. Sci., 8, 763–773, 2008 www.nat-hazards-earth-syst-sci.net/8/763/2008/ 2.1 Simple kriging 3. Return to step 1) until all nodes have been visited by the random path. The simple kriging estimate of z(u0) is: The simple kriging estimate of z(u0) is: ˆzSK(u0)−m= n(u) X α=1 λSK α (u0) [z(uα)−m] (3) Soares (2001) also extended the DSS algorithm for the joint simulation of different variables, thus named direct se- quential cosimulation (coDSS) algorithm. Instead of simu- lating all variables simultaneously, it simulates each variable in turn conditioned to the previous simulated variable. (3) where m is the stationary mean of Z(u), and λSK α (u0) is the weight assigned to datum z(uα) within a search neigh- bourhood that comprises n(u) samples. The coDSS algorithm uses collocated simple cokriging to estimate local means and variances, incorporating the sec- ondary information and the relationship between secondary and primary variables. In this study, the collocated cokriging was applied with a Markov-type approximation (Goovaerts, 1997) for cross-continuity model. Hence, only the primary Nat. Hazards Earth Syst. Sci., 8, 763–773, 2008 www.nat-hazards-earth-syst-sci.net/8/763/2008/ A. C. Costa et al.: Space-time mapping of extreme precipitation 767 n 767 Fig. 7. Equiprobable simulated realizations of R5D for 1945 and 1949. A. C. Costa et al.: Space time mapping of extreme precipitation 767 Fig. 6. Local correlation models between elevation and R5D values for each decade Fig. 7. Equiprobable simulated realizations of R5D for 1945 and 1949. possible long-term trends or ﬂuctuations in extreme rainfall, and for changes in the relationship between elevation and ex- treme precipitation through time. Alternative physiographic variables, such as slope or distance from the coastline, could have been used as secondary information. The relationship between elevation and extreme precipi- tation, described by correlation models, was also assessed locally to allow accounting for changes in correlation across the study area. First, for each decade, local correlations were Fig. 6. Local correlation models between elevation and R5D values for each decade. Fig. 7. Equiprobable simulated realizations of R5D for 1945 and 1949. possible long-term trends or ﬂuctuations in extreme rainfall, and for changes in the relationship between elevation and ex- treme precipitation through time. Alternative physiographic variables, such as slope or distance from the coastline, could have been used as secondary information. The relationship between elevation and extreme precipi- tation, described by correlation models, was also assessed locally to allow accounting for changes in correlation across the study area. 2.1 Simple kriging First, for each decade, local correlations were calculated using a search neighbourhood centred at each sta- tion’s location (further details from this stage are described in Sect. 4.2). To reproduce the spatial distribution of the re- lationship between elevation and extreme precipitation, the second stage used the DSS algorithm to interpolate the local correlations. In this stage, 50 equiprobable simulated real- izations were generated through the DSS algorithm for each decade on 800 m×800 m grids. The correlation models used later with the coDSS algorithm were determined by comput- ing the mean of the distribution of the 50 simulated values at each grid node, by decade. Fig. 6. Local correlation models between elevation and R5D values for each decade. variable variogram model and a correlation model between primary and secondary data were required. To reproduce the spatial distribution and uncertainty of the ﬂood indicator, 100 equiprobable simulated realizations were generated through the coDSS algorithm on 800 m×800 m grids, one for each year, using different space-time continu- ity and correlation models for each decade. These models are brieﬂy described in the following section and further detailed in Sect. 4. Elevation was used as secondary information. 3 Study region and data Simulated images were generated by the coDSS algorithm using for each decade a different space-time variogram model of the primary variable (R5D index), and a differ- ent correlation model between primary and exhaustive sec- ondary data (elevation). This strategy allows accounting for The study domain refers to the South of continental Portu- gal (Fig. 1) and includes the Algarve region, in the far South, and most of the Alentejo region (limited in the North by the Tejo River). The study region has different physiographic Nat. Hazards Earth Syst. Sci., 8, 763–773, 2008 www.nat-hazards-earth-syst-sci.net/8/763/2008/ 768 Fig. 8. Scenarios of extreme precipitation (R5D index). Decade Spatial range (m) Temporal range Sill (years) 1940/49 85 000 4.5 2923.364 1950/59 100 000 1 2075.247 1960/69 70 000 4 1263.25 1970/79 70 000 5 1543.205 1980/89 150 000 4.5 2075.301 1990/99 165 000 1.3 2803.646 Soares, 2006; Costa et al., 2008). The length of the series is highly variable. The extreme precipitation index (R5D) was calculated for each station using all quality data avail- able within the 1940/99 period (Fig. 2). The selected stations have less than 16% of the days missing in each year and most stations’ data do not have any missing records. The R5D in- dex is deﬁned as the highest consecutive 5-day precipitation total (in millimetres) per year, and can be considered a ﬂood indicator (Frich et al., 2002). Elevation data were taken from a digital elevation model (DEM) with a grid resolution of 20 m×20 m and resampled to an 800 m×800 m grid mesh. The topographic variable de- rived is deﬁned as the elevation of the nearest grid point to the meteorological station location, sometimes named smoothed elevation. Nat. Hazards Earth Syst. Sci., 8, 763–773, 2008 www.nat-hazards-earth-syst-sci.net/8/763/2008/ 4.2 Relationship between extreme precipitation and eleva- tion An exploratory evaluation of the relationship between the ex- treme precipitation index and elevation was made by aver- aging the R5D index at each station, and by computing af- terwards the Pearson’s correlation coefﬁcient between those values and elevation, not only measured by the actual sta- tions altitude, but also by the stations grid point elevation (smoothed elevation). This regional analysis shows that the correlation is slightly stronger for the smoothed elevation (0.49) than the actual one (0.43). Other studies on this sub- ject concluded likewise (e.g., Diodato, 2005). A plot of elevation against R5D index values is given in Fig. 4. The coefﬁcient of determination, r2, is small (0.10) and so there is little evidence of a (global) linear relationship between elevation and precipitation, as expected (e.g., Lloyd, 2005). Moreover, the correlation for elevation against R5D index values is not constant through time (Fig. 5), but rather shows a negative trend during the study period, although not statistically signiﬁcant. The number of stations used in the computation of these correlations ranges from 19 (in 1940) to 93 (in 1991 and 1992), and is always less than 40 before 1980. Because of the sparse coverage of meteorological sta- tions in some areas, especially until the 1980s decade (Ta- ble 2), the local relationship between elevation and extreme precipitation was assessed by decade. Fig. 9. Uncertainty of the scenarios of extreme precipitation (R5D index). ond, the DSS algorithm was applied to interpolate the lo- cal correlations by decade. This procedure used a single space-time spherical variogram model of the local correla- tions, where the spatial dimension was modelled as isotropic, the estimated range of the spatial dimension was 110 000 m, the range of the temporal dimension was 6 decades, and the estimated sill was 0.053. Finally, the correlation models were determined by computing the mean of the distribution of 50 simulated values at each grid node, by decade (Fig. 6). The coDSS algorithm uses a different correlation model between the R5D index and elevation within each decade. In order to determine these models, ﬁrst, the relationship be- tween elevation and precipitation was assessed locally by computing, for each decade, Pearson’s correlation coefﬁ- cients using stations’ data falling within a circle centred at each station’s location. As in earlier decades meteorological stations are scarce, larger radii were used (Table 2). 4.1 Space-time continuity of extreme precipitation Distribution of weather stations (with records within each decade) by elevation classes, and radii of the search neighbourhoods used to calculate the local correlations. Table 2. Distribution of weather stations (with records within each decade) by elevation classes, and radii of the search neighbourhoods used to calculate the local correlations. Decade Elevation (m) Radius (m) <140 140–280 281–420 421–560 561–700 1940/49 9 13 2 2 1 65 000 1950/59 16 17 3 2 1 50 000 1960/69 16 20 2 2 1 50 000 1970/79 17 18 2 2 1 40 000 1980/89 31 43 17 1 2 35 000 1990/99 31 44 16 1 2 35 000 no relevant tendencies. However, the range of the models’ spatial component shows a strong increase in the spatial con- tinuity of the ﬂood indicator on the last two decades. For illustration purposes, the exponential model ﬁtted to the ex- perimental semivariogram of the 1960s decade is shown in Fig. 3. 4.2 Relationship between extreme precipitation and eleva- tion 4.1 Space-time continuity of extreme precipitation Variograms were calculated according to Eq. 2. The vari- ogram models are chosen and their parameters are estimated taking into account the experimental data values of vari- ograms. The objective is to capture the spatial pattern of physical phenomenon in the variogram model rather than getting a best ﬁt of a second moment (Goovaerts, 1997). In this study, we chose exponential models that capture the ma- jor spatial features of the attribute under study within each decade. The selected models were subjectively ﬁtted to the experimental semivariogram values, not only by giving more importance to the smaller lags and the ones computed from more data pairs, but also by taking into account physical knowledge of the area and phenomenon. Considering the re- sults of a thorough analysis on directional variograms, only the omnidirectional semivariograms for the spatial dimen- sion were retained. Consequently, the spatial variability is assumed to be identical in all directions (i.e. isotropic) within each decade. Fig. 8. Scenarios of extreme precipitation (R5D index). characteristics. In the far South, the relief is dominated by the two main Algarve’s mountains: Monchique on the West, and Caldeir˜ao on the East. In contrast, the Alentejo region is characterized mainly by vast ﬂat to rolling country, the pene- plain, where the average altitude is approximately 200 m. The S˜ao Mamede mountain ridge, the highest in the Alen- tejo region with an altitude of 1000 m, lies in the extreme North-East. Daily rainfall series from 105 stations distributed irregu- larly over the study region were collected (Fig. 1). Most of them were extracted from the National System of Wa- ter Resources Information (SNIRH – Sistema Nacional de Informac¸˜ao de Recursos H´ıdricos) database (http://snirh. inag.pt), and three of them were compiled from the Euro- pean Climate Assessment dataset (http://eca.knmi.nl). Each station series data was previously quality controlled and stud- ied for homogeneity (e.g. Wijngaard et al., 2003; Costa and The parameters for each exponential variogram of the R5D index, used in the coDSS algorithm, are summarized in Ta- ble 1. In what concerns the temporal component, there are www.nat-hazards-earth-syst-sci.net/8/763/2008/ www.nat-hazards-earth-syst-sci.net/8/763/2008/ Nat. Hazards Earth Syst. Sci., 8, 763–773, 2008 A. C. Costa et al.: Space-time mapping of extreme precipitation 769 Fig. 9. Uncertainty of the scenarios of extreme precipitation (R5D index). Table 2. Nat. Hazards Earth Syst. Sci., 8, 763–773, 2008 4.3 Extreme precipitation scenarios Using the coDSS algorithm, a set of 100 equiprobable sim- ulated realizations of the R5D index was computed at each simulated grid node, by year. For illustration purposes, two equiprobable realizations, for 1945 and 1949, are shown in Fig. 7. The space-time inference was performed by means of computing the mean of those distributions. Uncertainty was assessed by means of computing the standard-deviation (STD) and the coefﬁcient of variation (CV) of the distribu- tion of the 100 simulated values at each simulated grid node, by year. For illustration purposes, the ﬂood indicator maps of six years are shown in Fig. 8, while Fig. 9 presents their un- certainty evaluation measured by the coefﬁcient of variation (CV). Although the correlation model for the 1940s decade seemed unrealistic, a visual inspection of all produced sce- narios for those years reveals a rather realistic spatial pat- tern of extreme precipitation. On the other hand, the lack of precipitation data on the north-eastern area makes the esti- mates more uncertain. For that reason, the extreme precipita- tion values were possibly less accurately estimated over that area in several years. In fact, as expected, one of the regions where the distribution of extreme precipitation shows greater variability, thus more uncertainty, is in the northern part of the maps, corresponding to regions less densely sampled in most years. This is especially evident in the maps of the standard-deviation. Moreover, the scenarios for the last two decades of the twentieth century show less uncertainty than the previous ones because the availability of stations over the study region is considerably greater (Fig. 2). Fig. 10. Probability of the uncertainty of the R5D index scenarios, measured by the coefﬁcient of variation, to be greater than or equal to 25%. stations. Accordingly, the variability associated with esti- mated correlations over this area is also high. Nevertheless, they correspond to moderate values of approximately 0.50, and so the contribution of elevation to the estimation of the ﬂood indicator will also be moderate. The correlation model of the 1950s decade exhibits a more realistic pattern, with correlations ranging from −0.17 to 0.67. The model of the 1960s decade also shows a realistic pattern of correlations, although the West and North-East areas present high vari- ability. Similarly, the 1970s decade model is realistic but with very high variability within the North-East region. A. C. Costa et al.: Space-time mapping of extreme precipitation 0 Fig. 10. Probability of the uncertainty of the R5D index scenarios, measured by the coefﬁcient of variation, to be greater than or equal to 25%. Nat. Hazards Earth Syst. Sci., 8, 763–773, 2008 4.2 Relationship between extreme precipitation and eleva- tion Sec- The estimated correlation between elevation and the R5D index ranges from very weak (−0.21) to moderately strong (0.72) across the region and through decades. In the 1940s decade, correlations range from −0.18 to 0.65 across the study region. The correlation model of this decade shows an unrealistic pattern of correlations on the West of the region, North of Algarve, caused by the scarce number of available www.nat-hazards-earth-syst-sci.net/8/763/2008/ Nat. Hazards Earth Syst. Sci., 8, 763–773, 2008 770 A. C. Costa et al.: Space-time mapping of extreme precipitation www.nat-hazards-earth-syst-sci.net/8/763/2008/ 4.3 Extreme precipitation scenarios Be- cause of the higher density of stations, the correlation models of the 1980s and 1990s decades exhibit much less variability than the previous ones. Fig. 10 shows estimated local probabilities of the coefﬁ- cient of variation of the scenarios produced for R5D to be greater than or equal to 25%. These probabilities were eval- uated as the proportion of the sixty estimated values of CV (computed at each grid cell throughout the years) that exceed that threshold. Only a few stations are located at medium (>400 m) and high elevations (Table 2), thus greater uncertainty would be expected at those regions. However, the uncertainty in the mountainous regions of the South is often small (Fig. 10), because of the use of elevation as secondary exhaustive in- formation in the spatial interpolation procedure of R5D. These results suggest that using elevation as a secondary variable in estimation will increase the accuracy of estimates in some locations, i.e. those mountainous areas where cor- relations are large (e.g. on the West of Algarve and on the North-East of the study region). In contrast, univariate in- terpolators (e.g., DSS) are likely to provide estimates as ac- curate as those provided by coDSS, with less computational effort, in places where correlations are small. Other common spatial patterns can be observed. In wetter years, the ﬂood indicator exhibits the highest values in moun- tainous regions of Algarve, especially over the Monchique mountains, due to the greater inﬂuence of altitude there than in the rest of the study domain. For this reason, high values also appear over North-East areas in several years. In drier years, the spatial pattern of extreme precipitation is much smoother as estimates have less variability over the study do- main. As expected from the space-time continuity analysis (Sect. 4.1), the spatial patterns of extreme precipitation are becoming more homogenous over time. This is especially Nat. Hazards Earth Syst. Sci., 8, 763–773, 2008 www.nat-hazards-earth-syst-sci.net/8/763/2008/ A. C. Costa et al.: Space-time mapping of extreme precipitation 771 Fig. 11. Probability maps of extreme precipitation (R5D index). noticeable in the maps of the last two decades of the twenti- eth century. 4.3 Extreme precipitation scenarios The precipitation regimes are of a different nature in north- ern and southern regions of Portugal: in the north, the precip- itation regime has an orographic origin, whereas in the south it is mainly associated with vertical motions induced by cy- clogenic activity (Trigo and DaCamara, 2000). In southern Portugal, summer precipitation, almost close to zero during this season, is sometimes associated with local convective activity. These storms can occur with a large degree of in- dependence from the weather circulation type that character- izes the Iberian circulation for that speciﬁc day (Trigo and DaCamara, 2000). Changes in the North Atlantic Oscillation (NAO) are likely to be responsible for much of the observed change in the frequency of above 90th percentile winter pre- cipitation between the 1960s and 1990s over Europe (Scaife et al., 2008). Moreover, the NAO–rainfall relationships tend to be stronger during the wet seasons of the last decades of the twentieth century in southern Portugal (Goodess and Jones, 2002; Trigo et al., 2004). Accordingly, changes in the NAO are likely to be responsible for the increase of the spatial continuity of R5D in southern Portugal, which is es- pecially pronounced during the last two decades of the twen- tieth century. Fig. 11. Probability maps of extreme precipitation (R5D index). tion data to inform estimation will also vary locally. There- fore, using elevation data in the estimation process is likely to be beneﬁcial, especially in mountainous areas. The esti- mates accuracy also varies in time, and in earlier decades is strongly dependent on the density of the stations network. Nevertheless, the ﬂood indicator values at locations be- tween meteorological stations have been estimated to a good degree of accuracy in most years, producing detailed and rep- resentative maps of extreme precipitation space-time patterns over the South of Portugal for the 1940/1999 period. There- fore, the produced gridded datasets provide a consistent set of data that allows further investigations on the spatial and temporal variability and trends in the South Portugal climate, over 60 years. In fact, the usefulness of the datasets was il- lustrated by probability maps of extreme precipitation. To illustrate the usefulness of the annual gridded datasets produced for the R5D index, probability maps of extreme precipitation were computed as follows. First, the regional histogram and its basic statistics were calculated using the R5D values from maps corresponding to the climate normal 1961/1990. 4.3 Extreme precipitation scenarios For example, the median was equal to 105 mm, the third quartile was 125 mm, and the 95th percentile was 160 mm. Second, the probability is approximated by the rel- ative frequency computed at each grid node for 1940/1999. Frequencies correspond to the number of times that R5D values are equal or over a ﬁxed threshold. For example, the probability map corresponding to the 105 mm threshold (Fig. 11) shows that the mountainous regions of Algarve and North-East, and also the West coast have high probability of extreme rainfall events. On the other hand, the probability map for 125 mm (Fig. 11) shows that the heaviest medium- term rainfall events occur at the Algarve region, especially over the Monchique mountains, as expected. Probability maps such as these might be useful to identify regions at risk of erosion caused by extreme precipitation events. The methodology considered here has clear theoretical ad- vantages over alternative ones, although no single method is optimal for all regions and time periods (e.g., Isaaks and Srivastava, 1989; Lloyd, 2005). Among the sequential al- gorithms of stochastic simulation, one advantage of direct sequential simulation and cosimulation is the use of original variables instead of transformed ones: Gaussian (sequential Gaussian simulation) or indicator (sequential indicator sim- ulation). The direct sequential cosimulation proved to be a valuable technique to deepen the knowledge on the space- time patterns of extreme precipitation over the study domain, because it allowed incorporating elevation information into prediction and also used all quality data available for every station. 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POCI/CLI/56371/2004), CIDmeg – Construction of a Desertiﬁcation Susceptibility In- dex for the Left Margin of Guadiana (FCT/MCTES, contract no. POCI/CLI/58865/2004) and SADMO – Syst`eme d’´Evaluation et Contrˆole de la D´esertiﬁcation dans la M´editerran´ee Occidentale The study was performed in the South of continental Portugal where the topographic characteristics and spatial climatic di- versity are signiﬁcant, although the region’s relief is not very complex if compared to other European study regions (e.g., Prudhomme and Reed, 1999; Perry and Hollis, 2005). The correlation maps of elevation and R5D values indicate that, as the relationship varies locally, the beneﬁts in using eleva- Nat. Hazards Earth Syst. Sci., 8, 763–773, 2008 www.nat-hazards-earth-syst-sci.net/8/763/2008/ 772 A. C. Costa et al.: Space-time mapping of extreme precipitation A. C. Costa et al.: Space-time mapping of extreme precipitation (Programme Interreg IIIB, MEDDOC, convention no. 2005-05-4.4- P-105). 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https://openalex.org/W4249758152	https://www.researchsquare.com/article/rs-37055/latest.pdf	English	null	Epitope-based Immunoinformatics Approach on Nucleocapsid Protein of Severe Acute Respiratory Syndrome-Coronavirus-2	Research Square (Research Square)	2,020	cc-by	10,808	Epitope-based Immunoinformatics Approach on Nucleocapsid Protein of Severe Acute Respiratory Syndrome-Coronavirus-2 Ahmed Rakib Department of Pharmacy, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh Saad Ahmed Sami Department of Pharmacy, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh Md. Ashiqul Islam Department of Pharmacy, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh Shahriar Ahmed Department of Pharmacy, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh Farhana Binta Faiz Department of Pharmacy, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh Bibi Humayra Khanam Department of Pharmacy, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh Kay Kay Shain Marma Department of Pharmacy, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh Maksuda Rahman Department of Pharmacy, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh Mir Muhammad Nasir Uddin Department of Pharmacy, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh Talha Bin Emran Drug Discovery, GUSTO A Research Group, Chittagong-4000, Bangladesh Jesus Simal-Gandara (  jsimal@uvigo.es ) University of Vigo https://orcid.org/0000-0001-9215-9737 1. Introduction The present world has witnessed the outbreak of many life-threatening human pathogens including Ebola, Chikungunya, Zika, Severe Acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) in the 21st century. More recently in late December 2019, a cluster of pneumonia cases was reported in the city of Wuhan, Hubei province, China which was of unknown cause. Later it was confirmed that these pneumonia cases were due to a novel coronavirus named SARS-CoV–2 (previously named as 2019-nCoV) and the disease condition of this virus is referred to as COVID–19 (1–3).On March 11, 2020, the World Health Organization (WHO) has assessed that COVID–19 can be characterized as a pandemic. The current COVID–19 pandemic is a global concern and is spreading at an alarming rate and as of April 12, 2020, more than 1.6 million cases and over 105,000 deaths have been reported globally (4). Coronaviruses (CoVs) are phenotypically and genotypically diverse group viruses that can adapt to the new environment through mutation and recombination, probably even more than influenza.Coronaviruses often infect mammals, birds and can transmit to humans. Six strains of coronaviruses were found in the last few decades but this is a completely new strain and of zoonotic origin. COVID–19 virus belongs to the Coronaviridae family of the Genus Betacoronavirus, pleomorphic or spherical particles, 150 to 160 nm in size, associated with positive single-strand RNA (ssRNA) which is surrounded by crown-shaped, club-like spikes projection on the outer surface. Among all RNA viruses, Coronaviruses have the largest genome typically ranging from 27 to 32 kb. After the two previously reported coronavirus-SARS-CoV and MERS-CoV, this is the third coronavirus that has already infected humans and the preliminary investigations revealed that some environmental specimens of the Huanan seafood market in Wuhan were positive for COVID–19 (3). Although the seafood market was reckoned positive for COVID–19, no specific association with an animal is confirmed yet based on the WHO report. Researchers are working to establish a possible animal reservoir for COVID–19 (5). As COVID–19 is mainly a respiratory disease, in most cases it might affect the lungs only. The primary mode of infection is human-to-human transmission through close contact, which occurs via spraying droplets from the infected individual through their cough or sneeze. The symptoms of this coronavirus can be mild to moderate or severe including, fever, cough, and shortness of breath or pneumonia. Research Article DOI: https://doi.org/10.21203/rs.3.rs-37055/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 Internationa Version of Record: A version of this preprint was published at Molecules on November 2nd, 2020. See the published version at https://doi.org/10.3390/molecules25215088. Page 1/23 Page 1/23 Abstract With an increasing fatality rate, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has emerged as a promising threat to human health worldwide. SARS-CoV-2 is a member of the Coronaviridae family, which is transmitted from animal to human and because of being contagious, further it transmitted human to human. Recently, the World Health Organization (WHO) has announced the infectious disease caused by SARS-CoV-2, which is known as coronavirus disease-2019 (COVID-2019) as a global pandemic. But, no specific medications are available for the treatment of COVID-19 so far. As a corollary, there is a need for a potential vaccine to impede the progression of the disease. Lately, it has been documented that the nucleocapsid (N) protein of SARS-CoV-2 is responsible for viral replication as well as interferes with host immune responses. We have comparatively analyzed the sequences of N protein of SARS-CoV-2 for the identification of core attributes and analyzed the ancestry through phylogenetic analysis. Subsequently, we have predicted the most immunogenic epitope for T-cell as well as B- cell. Importantly, our investigation mainly focused on major histocompatibility complex (MHC) class I potential peptides and NTASWFTAL interacted with most human leukocyte antigen (HLA) that are encoded by MHC class I molecules. Further, molecular docking analysis unveiled that NTASWFTAL possessed a greater affinity towards HLA and also available in a greater range of the population. Our study provides a consolidated base for vaccine design and we hope that this computational analysis will pave the way for designing novel vaccine candidates. 1. Introduction Activated CD8+ T cells then recognize the infected cells and clear them. T-cell immunity also depends strictly on the MHC-peptide complexes which are similar to the antigen-antibody association. MHC proteins are encoded by human leukocyte antigen (HLA) which is located among the most genetically variable regions on the human genome. Each HLA allele can only present a certain set of peptides that can be presented on the infected cell surface and recognized by T cells are called T-cell epitopes. For a vaccine, it is essential to identify T-cell epitopes that originate from conserved regions of the virus T cell responses against the S and N proteins have been reported to be the most dominant and long-lasting (14). To develop effective diagnostic tests and vaccine, the identification of B-cell and T-cell epitopes for SARS-CoV–2 proteins are critical especially for structural N and S proteins. Both humoral immunity and cellular immunity provided by B-cell antibodies and T-cells respectively are essential for effective vaccines (15,16). Although humans may mount an antibody response against viruses normally, only neutralizing antibodies can block the entry of viruses into human cells completely (17). Antibody binding site’s location on a viral protein strongly affects the body’s ability to produce neutralizing antibodies(18). It is important to understand whether SARS-CoV–2 has potential antibody binding sites (B-cell epitopes) near their interacting surface with its known human entry receptor, ACE2. Besides neutralizing antibodies, human bodies also depend on cytotoxic CD8+ T-cells and helper CD4+ T-cells to clear viruses completely from the body. For anti-viral T-cell responses, presentation of viral peptides by human MHC class I and class II is essential (19). MHC-I analysis includes common alleles for HLA-A, HLA-B, and HLA-C. Multiple investigations have indicated that antibodies generated against the N protein of SARS-CoV are highly immunogenic and abundantly expressed protein during infection (20). Our group is targeting for immunoinformatics-based vaccine design using bioinformatics and immunoinformatics tools by utilizing different protein sequences of SARS-CoV–2. Recently, we have already established two epitopes with a greater candidacy profile using the S protein of SARS-CoV–2 (21). Moreover, other published work also utilized the S protein of SARS- CoV–2 for epitope-based vaccine design (22). The purpose of our present study is to promote the designing of a vaccine against COVID–19 using in silico methods, considering SARS-CoV–2 N protein. 1. Introduction Respiratory, hepatic and neurological complications can be seen in case of severe cases that can lead to death. It seems that the severity and fatality rate of COVID–19 is milder than that of SARS and MERS. Although diarrhea was presented in about 20–25% of patients with SARS and MERS, intestinal symptoms were rarely reported in patients with COVID–19 (6–8).Multi-organ failure, especially in elderly people and people with underlying health conditions such as hypertension, cardiovascular disease and diabetes, are exhibiting a higher mortality rate in COVID–19. Page 2/23 Page 2/23 Interestingly, SARS-CoV–2 has 82% similarity with the original SARS-CoV virus attributed to the outbreak in 2003 (9). A mature SARS-CoV–2 virus generally has a polyprotein (the open reading frame 1a and 1b, Orf1ab), four structural proteins such as envelope (E) protein; membrane (M) protein; nucleocapsid (N) protein; spike (S) protein and five accessory proteins (Orf3a, Orf6, Orf7a, Orf8, Orf10), and particularly, SARS-CoV–2 encodes an additional glycoprotein having acetyl esterase and hemagglutination (HE) attributes, which identified it distinct than its two predecessors (10). The functions of accessory proteins may include signal inhibition, apoptosis induction and cell cycle arrest (11).The S protein on the surface of the viral particle enables the infection of host cells by binding to the host cell receptor angiotensin-converting enzyme 2 (ACE2), utilizing the S- protein’s receptor-binding domain (RBD). The N protein binds to the RNA genome of the COVID–19 and creates a shell or capsid around the enclosed nucleic acid. N protein is involved in viral RNA synthesis and folding which interacts with the viral membrane protein during viral assembly affects host cell responses including cell cycle and translation. An epitope-based peptide vaccine has been raised in this aspect. The core mechanism of the peptide vaccine is based on the chemical method to synthesize the recognized B-cell and T-cell epitopes that can induce specific immune responses and are immune-dominant. T-cell epitopes are short peptide fragments (8– 20 amino acids) while the B-cell epitopes can be proteins (12,13). Once a mutated virus infects the host cells by escaping the antibodies, it then relies upon the T-cell mediated immunity to fight against the virus. Viral proteins are processed into short peptides inside the infected cells and then loaded onto major histocompatibility complexes (MHC) proteins. After that, the MHC-peptide complexes are presented on the infected cell surface for recognition by specific T cells. 2.4 Protein secondary and tertiary structure prediction The secondary structure of the SARS-CoV-2 N protein was predicted by using CFSSP (Chou & Fasman Secondary Structure Prediction) because the antigenic part of the protein is more likely to belong to the β-sheet region (30). Also, we predicted the 3D structure of the protein using EasyModeller, a graphical user interface (GUI) version of MODELLER, where we designed the three- dimensional structure of the SARS-CoV-2 N protein using template proteins from Protein Data Bank. The model was validated using PROCHECK and PROSA web servers (31–34). 2.2 Sequence analysis The understanding of the features, function, structure, and evaluation is mainly based on the process of sequence analysis which depicts the process of subjecting DNA, RNA, or peptide sequences to wide ranges of analytical methods. We implied NCBI BLAST (Basic Local Alignment Search Tool) (25) that screens homologous sequences from its database and selects those sequences that are more similar to our SARS-CoV-2 N protein; we also performed multiple sequence alignment (MSA) using the ClustalW web server with default settings, and a phylogenetic tree was assembled using MEGA6 software and a web logo was also generated for the conserved peptide sequences using WebLogo based on this alignment (25–27). 2. Materials And Methods The methodologies used for peptide vaccine development for SARS-CoV-2 N protein are shown in Figure 1. 2.1 Protein sequence retrieval The SARS-CoV-2 N protein sequence was extracted from NCBI (National Center for Biotechnology Information) protein database (Accession no.: QIC53221.1, GI: 1811294683) in FASTA format. 1. Introduction The reason for focusing particularly on the epitopes in the N structural proteins is due to their dominant and long-lasting immune response which was reported against SARS-CoV previously (23). Besides, it has been reported that N protein of many viruses are highly conserved and immunogenic, which expressed extensively in the course of infection (24). For the identified T-cell epitopes, we incorporated the information on the associated MHC alleles so that we can provide a list of epitopes that seek to maximize population coverage globally. Therefore, we designed an epitope-based peptide vaccine to potentially narrow down the search for potent targets against SARS-CoV–2 using the computational approach with an expectation that the wet laboratory research will validate our result. Page 3/23 Page 3/23 2.3 Protein antigenicity and toxicity prediction To determine the potent antigenic protein of the SARS-CoV-2 N protein, we used the online server VaxiJen v2.0, with a default threshold value (28). All the antigenic proteins of SARS-CoV-2 N protein with their respective scores were obtained then sorted in Notepad++. A single antigenic protein with maximum antigenicity scores was selected for further evaluation. The toxicity of epitopes was analyzed using the Toxinpred web server (29). 2.8.3 Molecular docking analysis Molecular docking analysis was performed using Autodock vina in PyRx 0.8, by considering the HLA-A68:02 molecule as receptor protein and identified epitopes as ligand molecule (41). Firstly, we used the protein preparation wizard of UCSF Chimera (Version 1.11.2) to prepare the protein for docking analysis by deleting the attached ligand, adding hydrogens and Gasteiger– Marsili charges (42,43). The prepared file was then added to the Autodock wizard of PyRx 0.8 and converted into pdbqt format. The energy form of the ligand was minimized and converted to pdbqt format by OpenBabel (44). The parameters used for the docking simulation were set to default. The size of the grid box in AutoDock Vina was kept at 50.183 × 50.183 × 50.183 Å respectively, for X, Y, and Z-axis. AutoDock Vina was implemented via the shell script offered by AutoDock Vina developers. Docking results were observed by the negative score in kcal/mol, as the binding affinity of ligands (45). 2.5.1 CD8+ T-cell epitope prediction For the de novo prediction of T-cell epitope, NetCTL 1.2 server was used in this experiment, using a 0.95 threshold to maintain sensitivity and specificity of 0.90 and 0.95, respectively. The tool expands the prediction for 12 MHC-I supertypes and integrates the prediction of peptide MHC-I binding, proteasomal C-terminal cleavage with TAP transport efficiency. These predictions were performed by an artificial neural network, weighted TAP transport efficiency matrix and a combined algorithm for MHC-I binding and proteasomal cleavage efficiency was then used to determine the overall scores and translated into sensitivity/specificity. Based on this overall score, five best peptides (epitopes) were selected for further evaluation. For the prediction of peptides binding to MHC-I, we used a tool from the Immune Epitope Database (IEDB) and calculate IC50 values for peptides binding to specific MHC-I molecules (35). For the binding analysis, all the frequently used alleles were selected with a word length of nine residues and binding affinity < 200 nm for further analysis. Another tool (named as MHC-NP) Page 4/23 Page 4/23 Page 4/23 provided by the IEDB server was used to assess the probability that a given peptide was naturally processed and bound to a given MHC molecule (36). provided by the IEDB server was used to assess the probability that a given peptide was naturally processed and bound to a given MHC molecule (36). 2.8.1 Epitope model generation The 3D structures of the selected epitopes were predicted by PEP-FOLD, a web-based server (40). For each sequence, the server predicted five probable structures. The energy of each structure was determined by SWISS-PDB VIEWER and the structure with the lowest energy was chosen for further analysis. 2.6 Epitope conservancy and immunogenicity prediction The degree of similarity between the epitope and the target (i.e. given) sequence is elucidated by epitope conservancy. This property of epitope gives us the promise of its availability in a range of different strains. Hence for the analysis of the epitope conservancy, the web-based tool from IEDB analysis resources was used (37). Immunogenicity prediction can uncover the degree of influence (or efficiency) of the respective epitope to produce an immunogenic response. The T-cell class I pMHC immunogenicity predictor at IEDB, which uses amino acid properties as well as their position within the peptide to predict the immunogenicity of a class I peptide MHC (pMHC) complex (38). 2.8.2 Retrieval of HLA allele molecule The three-dimensional structure of the HLA-A68:02 (PDB ID: 4I48) was retrieved from Prot The three-dimensional structure of the HLA-A68:02 (PDB ID: 4I48) was retrieved from Protein Data Bank (RCSB-PDB). 2.7 Prediction of population coverage and allergenicity assessment The population coverage tool from IEDB was applied to determine the population coverage for every single epitope by selecting HLA alleles of the corresponding epitope. Allergenicity of the predicted epitope was calculated using AllerTop v2.0 (39), which is an alignment-free server, used for in silico based allergenicity prediction of a protein-based on its physiochemical properties. 3.1 Sequence retrieval and analysis We retrieved the SARS-CoV-2 N protein sequence from the NCBI database (Accession No.: QIC53221.1). Then we performed BLASTp using NCBI-BLAST for the nucleocapsid protein of SARS-CoV-2. We searched for a total of 100 homologs with > 60% identical sequences. Multiple sequence alignment was then performed (Supplementary data 1), and a phylogenetic tree was constructed (Supplementary Figure S1). From multiple sequence alignment, it has been confirmed that the protein sequences have a close relationship. A web logo was designed using the WebLogo server to demonstrate the conserved region (Figure 2). 3.2 Antigenic protein prediction The most potent antigenic protein of SARS-CoV-2 N protein was predicted by VaxiJen v2.0, which is based on the auto-cross covariance transformation of protein sequences into uniform vectors of principal amino acid properties. The overall antigen prediction score was 0.5002 (probable antigen) at 0.4 threshold value. 3.3 Toxicity prediction The toxicity of the selected peptide sequences was assessed using the ToxinPred web server. The results from the ToxinPred server represented that all of our probable epitopes were found non-toxic (Table 1). The toxicity of the selected peptide sequences was assessed using the ToxinPred web server. The results from the ToxinPred server represented that all of our probable epitopes were found non-toxic (Table 1). TABLE 1. Toxicity prediction of the selected epitopes by ToxinPred Server TABLE 1. Toxicity prediction of the selected epitopes by ToxinPred Server Peptide Sequence SVM Score Prediction Hydrophobicity Hydropathicity Hydrophilicity Charge Mol wt LSPRWYFYY -1.08 Non-Toxin -0.06 -0.57 -1.26 1 1294.59 GTTLPKGFY -1.13 Non-Toxin -0.01 -0.27 -0.49 1 983.26 DLSPRWYFY -1.18 Non-Toxin -0.14 -0.81 -0.67 0 1246.5 SSPDDQIGY -0.33 Non-Toxin -0.2 -1.21 0.3 -2 981.1 LLNKHIDAY -0.81 Non-Toxin -0.09 -0.17 -0.28 0.5 1086.39 GTDYKHWPQ -0.29 Non-Toxin -0.29 -2.11 -0.04 0.5 1131.34 SPDDQIGYY -0.54 Non-Toxin -0.17 -1.27 0.01 -2 1057.2 NTASWFTAL -1 Non-Toxin 0.08 0.4 -1 0 1010.23 2.9 B-cell epitope identification The prediction of B-cell epitopes was performed to find the potential antigen that assures humoral immunity. To detect B-cell epitope, various tools from IEDB were used to identify the B-cell antigenicity, together with the Emini surface accessibility Page 5/23 Page 5/23 prediction, Kolaskar and Tongaonkar antigenicity scale, Karplus and Schulz flexibility prediction, Bepipred linear epitope prediction analysis and since antigenic parts of a protein belonging to the beta-turn regions, the Chou and Fasman beta-turn prediction tool was also used (46–51). 3.4 Protein structure prediction and validation The secondary of a protein describes the α-helix, β-sheets, and random coil. Our SARS-CoV-2 N protein has 419 residues, of which 213 residues (50.8%) from the helix, 187 residues from sheets (44.6%) and 66 (15.8%) residues from the coil (Figure 3). For 3D structure, we built a model using EasyModeller, which was validated using PROCHECK and PROSA web server. The discrete optimized protein energy (DOPE) score was calculated -19585.62653. PROSA predicts the z-score of the model which was depicted 0.56 and the PROCHECK server was used for Ramachandran plot calculations. Ramachandran plot of the model Page 6/23 Page 6/23 protein indicated that 76% of residues in the most favorable region, 22% in the allowed region and 0.4% in the disallowed region (Figure 4). 3.6 Population coverage The cumulative amount of the population coverage was obtained for the predicted epitope NTASWFTAL Results from the population coverage demonstrated that with 57.16% coverage, East Asia found the highest coverage region. The results of the population coverage were shown in Table 3 and Supplementary Figures S2-S5. The cumulative amount of the population coverage was obtained for the predicted epitope NTASWFTAL Results from the population coverage demonstrated that with 57.16% coverage, East Asia found the highest coverage region. The results of the population coverage were shown in Table 3 and Supplementary Figures S2-S5. TABLE 3. Analysis of the population coverage for the proposed epitope against SARS-Cov-2 3. Analysis of the population coverage for the proposed epitope against SARS-Cov-2 nalysis of the population coverage for the proposed epitope against SARS-Cov-2 Population Coverage(%)a Average hitb PC90c Central Africa 35.31 0.40 0.15 East Africa 39.25 0.45 0.16 East Asia 57.56 0.71 0.24 Europe 42.95 0.50 0.18 North Africa 42.15 0.49 0.17 North America 45.32 0.53 0.18 Northeast Asia 48.11 0.55 0.19 Oceania 31.43 0.34 0.15 South Africa 33.91 0.38 0.15 South America 38.66 0.44 0.16 South Asia 36.53 0.41 0.16 Southeast Asia 49.45 0.57 0.20 Southwest Asia 28.53 0.32 0.14 West Africa 56.22 0.67 0.23 West Indies 12.89 0.13 0.11 ion coverage for the proposed epitope against SARS-Cov-2 Population Coverage(%)a Average hitb PC90c Central Africa 35.31 0.40 0.15 East Africa 39.25 0.45 0.16 East Asia 57.56 0.71 0.24 Europe 42.95 0.50 0.18 North Africa 42.15 0.49 0.17 North America 45.32 0.53 0.18 Northeast Asia 48.11 0.55 0.19 Oceania 31.43 0.34 0.15 South Africa 33.91 0.38 0.15 South America 38.66 0.44 0.16 South Asia 36.53 0.41 0.16 Southeast Asia 49.45 0.57 0.20 Southwest Asia 28.53 0.32 0.14 West Africa 56.22 0.67 0.23 West Indies 12.89 0.13 0.11 Notes: a Projected population coverage. Notes: a Projected population coverage. b Average number of epitope hits/HLA combinations recognized by the population. b Average number of epitope hits/HLA combinations recognized by the population. c Minimum number of epitope hits/HLA combinations recognized by 90% of the population. 3.5 CD8+ T-cell epitope identification Based on high combinatorial and MHC binding, the top eight epitopes were predicted by the NetCTL server from the selected protein sequence were selected for further analysis. Using the MHC-I binding prediction tool, which is based on stabilized matrix method (SMM), we selected those MHC-I alleles for which the epitopes showed the highest affinity (IC50 < 200 nm). Proteasomes play an important role in cleaving the peptide bond, resulting in the conversion of protein into the peptide. The peptide molecules that are homogeneous to class I MHC molecules and the peptide-MHC molecule after the proteasomal cleavage were presented as T-helper cells after the transportation into the cell membrane. The total score of each epitope-HLA interaction was taken into consideration and higher processing efficiency was meant by obtaining a higher score. The epitope NTASWFTAL interacted with most of the MHC-I alleles including, HLA-A68:02, HLA-C16:01, HLA-C03:03, HLA-C03:04, HLA-C12:03, HLA-A02:06, HLA- C03:02, HLA-A26:01 and HLA-C14:02 (Table 2). Moreover, the MHC-NP prediction tool was used to find the highest probable score of our predicted epitope NTASWFTAL, with a score of 1.11, for HLA-A68:02. Furthermore, all the predicted epitopes had a maximum identity for conservancy hit and 100% maximum identity was found (Table 2). Also, the I-pMHC immunogenicity prediction analysis of the epitope NTASWFTAL was found 0.22775 (Table 2). TABLE 2. The potential CD8+ T-cell epitopes along with their interacting MHC class I alleles and total processing score, epitopes conservancy_hits and pMHC-I immunogenicity score Epitopes NetCTL Combined score Epitope Conservancy Hit (MAX. Identity %) MCH-I interaction with an affinity of IC50 < 200 and the total score (proteasome score, TAP score, MHC-I score, processing score) pMHC-I immunogenicity score LSPRWYFYY 2.3408 100 HLA-A29:02 (1.32), HLA-A30:02 (0.8), HLA-A01:01 (0.66), HLA-C16:01 (0.26) 0.35734 NTASWFTAL 0.9521 100 HLA-A68:02 (1.11), HLA-C16:01 (0.18), HLA-C03:03 (0.12), HLA-C03:04 (0.12), HLA-C12:03 (0.10), HLA-A02:06 (0.04), HLA-C03:02 (-0.07), HLA-A26:01 (-0.13), HLA-C14:02 (-0.34) 0.22775 DLSPRWYFY 1.4994 100 HLA-A29:02 (0.99) 0.25933 SPDDQIGYY 1.1404 100 HLA-B35:01 (0.52) 0.06844 SSPDDQIGY 0.6895 100 - 0.0634 TABLE 2. The potential CD8+ T-cell epitopes along with their interacting MHC class I alleles and total processing score, epitopes conservancy_hits and pMHC-I immunogenicity score Page 7/23 3.7 Allergenicity assessment The AllerTop server was used for the identification of the allergic reaction caused by a vaccine in an individual which might be harmful or life-threatening. The allergenicity of the selected epitope was calculated using the AllerTop tool and predicted as probable non-allergen. 3.8 Molecular docking analysis for HLA and epitope in Page 8/23 In this study, the verification of the interaction between the HLA molecules and our predicted potential epitope was done by molecular docking simulation using Autodock Vina in PyRx 0.8 software. Among all the MHC class I alleles, only HLA-A68:02 had a maximum probable score for our most potent epitope NTASWFTAL. Therefore, we carried out the molecular docking study using HLA-A68:02 (PDB ID: 4I48). The three-dimensional structure of the predicted epitope, NTASWFTAL and HLA-A68:02 molecules are represented in Figure 5. We found that our predicted epitope NTASWFTAL interacted with HLA-A68:02 with strong binding affinities of -9.4 kcal/mol (data not shown). The selected epitope interacted with Arg6, Ser4, Ser2, Asp30 residues of chain-A and Lys59, Asp60, Ser58, Gly30 of chain-B through hydrogen bonding (H-bond), whereas Lys7 residue of chain-B form bonds as a result of sharing electrons (which may happen as a result of charge distribution) (Figure 6). had a maximum probable score for our most potent epitope NTASWFTAL. Therefore, we carried out the molecular docking study using HLA-A68:02 (PDB ID: 4I48). The three-dimensional structure of the predicted epitope, NTASWFTAL and HLA-A68:02 molecules are represented in Figure 5. We found that our predicted epitope NTASWFTAL interacted with HLA-A68:02 with strong binding affinities of -9.4 kcal/mol (data not shown). The selected epitope interacted with Arg6, Ser4, Ser2, Asp30 residues of chain-A and Lys59, Asp60, Ser58, Gly30 of chain-B through hydrogen bonding (H-bond), whereas Lys7 residue of chain-B form bonds as a result of sharing electrons (which may happen as a result of charge distribution) (Figure 6). 4. Discussion As yet, it has been reported that the reproduction rate of SARS-CoV-2 is greater than SARS and MERS and the symptoms of the COVID-19 infection include fever with more than 38 °C body temperature along with alveolar edema, leading to difficulty in breathing, whereas mild symptoms perhaps not engender high fever (52). Surprisingly, with a high fatality rate, the severity of the infection was found more than the infection caused by both SARS and MERS, with multiple organ damage, which was reported not long ago (53). At present, researchers are examining repurposed compounds from other viral infections to treat SARS-CoV-2. For example, both lopinavir and ritonavir are HIV protease inhibitors but in a lopinavir-ritonavir clinical trial report, the treatment benefit derived was dubious (54). From recovering patients, several convalescent immunoglobulins are derived which is currently investigated as a potential treatment for the disease (55). As there have been no approved treatments for COVID-19 that exists until now, these treatments are the best hope for striving to keep the mortality rate low before vaccines become widely available. Despite many potential challenges, vaccine development is a crucial factor in modern biotechnology as vaccines are the most important prerequisites for defending the burden of diseases over the world (56). With the divulgement of sequence-based technology in genomics and proteomics, enough pieces of information are available regarding different eukaryotic and prokaryotic organisms including viruses. Therefore, utilizing various bioinformatics tools, it is possible to design peptide-based vaccines through comprehensibly studying the epitopes and several studies suggested epitope-based vaccines against different diseases including dengue, chikungunya, Saint Louis encephalitis virus (53–55). Although epitope-dependent vaccine design is quite familiar, a little research works are done in the case of SARS-CoV-2. Being an RNA virus, SARS-CoV-2 is different from the DNA virus as well as the rate of mutation is higher than the DNA viruses and according to various research, it can be assumed that the mutations might occur in the N protein (60). Recently, N proteins of SARS-CoV-2 are regarded as a primary target for vaccine development as its function includes viral replication and directly associated with the infection process, as a consequence related to the pathogenesis of COVID-19 (61). Previous research works have already established that N proteins of several viruses as well as SARS are considered as a potential target for the development of vaccines (62–65). 3.9 B-cell epitope prediction The Parker Hydrophilicity Prediction tool predicts the hydrophilicity of SARS-CoV-2 N protein with an average score of 2.80, a minimum of 0.874 and the region 235-241 possessed a score of 5.943, which found moderately similar with the maximum value. For predicting the surface ability, this study includes the Emini surface accessibility prediction method. The average surface accessibility was 1.0 and a minimum 0.050. In alignment with the previous B-cell epitope results, we predicted the peptide sequence from 237 to 242 had the better surface ability. 3.9 B-cell epitope prediction In this study, using the amino acid scale-based method, we predicted the B-cell epitope identification. Different analysis methods were used for the prediction of continuous B-cell epitope. The results of the B-cell predictions were shown in Table 4 and Figures 7-9. TABLE 4 Combined B-cell linear epitope prediction Method Region Residues Length Score Max. Avg. Min. Bepipred Linear Epitope Prediction 232- 269 SKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYN 38 2.416 0.813 -0.001 Chou & Fasman Beta-Turn Prediction 233- 239 KMSGKGQ 7 1.439 1.070 0.610 Emini Surface Accessibility Prediction 237- 242 KGQQQQ 6 7.006 1.000 0.050 Karplus & Schulz Flexibility Prediction 238- 244 GQQQQGQ 7 1.161 1.035 0.885 Kolaskar & Tongaonkar Antigenicity 243- 249 GQTVTKK 7 1.197 0.988 0.874 Parker Hydrophilicity Prediction 235- 241 SGKGQQQ 7 6.871 2.800 -5.971 TABLE 4 Combined B-cell linear epitope prediction Firstly, Bepipred linear epitope prediction was used, which is regarded as the best single method for predicting linear B-cell epitopes using a Hidden Markov model. Our analysis revealed that the peptide sequences from 232 to 269 amino acid residues were able to induce the desired immune response as B-cell epitopes. The β-turns were predicted by Chaus and Fasman β-turn prediction method. The region 233-239 residues were predicted as a β- turn region with a score of 1.164, which was higher than the average score. For antigenicity prediction, the Kolaskar and Tongaonkar antigenicity prediction methods were implied. The method evaluates the antigenicity based on the physicochemical properties of amino acids and their abundances in experimentally known epitopes. The average antigenic propensity of our SARS-CoV-2 N protein was 0.988 with a maximum of 1.197 and a minimum of 0.874. Region 243-249 residues were found prone to antigenicity. Also, the average flexibility of 1.035 and a minimum of 0.874 Page 9/23 Page 9/23 was predicted by the Karplus and Schulz flexibility prediction method. The residues from 238 to 244 were found to be most flexible with the highest score of 1.161. The Parker Hydrophilicity Prediction tool predicts the hydrophilicity of SARS-CoV-2 N protein with an average score of 2.80, a minimum of 0.874 and the region 235-241 possessed a score of 5.943, which found moderately similar with the maximum value. was predicted by the Karplus and Schulz flexibility prediction method. The residues from 238 to 244 were found to be most flexible with the highest score of 1.161. 4. Discussion In this experiment, we determined the binding (presence of the antigen on the surface) affinity of the predicted epitope using molecular docking analysis and demonstrated that NTASWFTAL interacted with HLA-A68:02 and found a binding affinity of -9.4 kcal/mol, which depicted a greater interaction with the epitope and the HLA molecule as the more negative energy implied to more binding affinity (69). The results from the molecular docking studies also revealed that epitope NTASWFTAL formed H- bond with both chain-A and chain-B of the HLA molecule and attractive charges were also responsible for the binding. Another factor that is considered as the most prominent one during the process of vaccine development is population coverage, as the distribution of HLA varies according to ethnicity and geographical region. Our experiment showed that the epitope NTASWFTAL covered almost all regions of the world, where the highest coverage was observed in East Asia, where COVID-19 first reported. Interestingly, our findings indicated that our predicted epitope specifically binds with the widespread HLA molecules and the vaccine will be easily employed. Importantly, the accurate prediction of T-cell epitopes along with B-cell epitopes is a crucial challenge for immunoinformatics study and it is unlikely that different HLAs are expressed at different frequencies amongst the ethnic groups. But substantial research in several in silico markers including, matrix-based profile, regular expressions in immunoinformatics study provide a cogent way for prediction of several immunobiological phenomena, for instance, the prediction of subcellular localization (SCL) of a protein is identified by several computational tools. Similarly, T-cell epitope identification has been undergone by implying numerous computational methods and various research areas, including cancer therapy and other infections, T-cell epitope identification is presently apparent (70–72). Also, experimental methods established for the calculation of binding interaction between MHC molecules and an antigenic protein is complicated and time-consuming. Hence, several computational tools have been introduced concerning simulation of the experimental methods, and the methods of the MHC binder prediction are based on motifs, quantitative matrices (QM), ab initio prediction, machine-learning techniques, DiscoTope, etc (73). Several algorithms including, PePSSI (peptide-MHC prediction of structure through solvated interfaces), PREDEP (prediction of MHC class I epitopes) are implemented for the structural prediction and side-chain orientation of the binding proteins. 4. Discussion Moreover, we have already mentioned the detrimental role of SARS-CoV-2 in host-cell responses. This aspect leads us to conduct in silico experiments for designing a peptide-based vaccine against the novel SARS-CoV-2. Earlier, it has been thought that vaccine development primarily relies on B-cell immunity, but recent discovery unveiled that T-cell epitopes are more propitious as a result of more long-lasting immune response mediated by CD8+ T-cells and due to the antigenic drift, by which an antibody is not able to respond against an antibody (66). In this study, focusing on MHC class I potential peptide epitopes, we predicted T-cell as well as B-cell epitopes which were able to show immune responses in various ways. Many characteristics including antigenicity, toxicity need to take into consideration for identifying a protein sequence- based epitope into a vaccine candidate and the predicted eight epitopes were fulfilled the entire criterion. However, only five potent epitopes have been predicted from the NetCTL 1.2 server and the epitopes were further taken for the progressive analysis. Besides, all peptides except SSPDDQIGY were able to interact with the MHC class I alleles, and NTASWFTAL interacted with the most MHC class I alleles. Amongst them, HLA-A68:02 possessed the highest probable score. Further, the conservancy of the epitopes which was predicted by the IEDB conservancy analysis tool delineated that all of our predicted epitopes had the maximum identity of 100%. Therefore, we have taken the epitope NTASWFTAL for further analysis due to its maximum interaction with MHC class I alleles and the highest conservancy. Page 10/23 Allergenicity is regarded as one of the most noteworthy obstacles in vaccine development. Importantly, T-cells not CD4+ T-cells are involved in an allergic reaction and an allergic reaction is stimulated by type 2 T helper cell along with immunoglobulin E (67). In this experiment, we assessed the allergenicity using AllerTop 2.0, which is well recognized for its high sensitivity, and able to identify structurally diverse allergens in comparison with the known allergens. AllerTop predicted our selected epitope as non- allergen. It has been proposed that the T-cell epitopes bind with the MHC molecules and MHC class I molecules generally presented short peptides that are 8-11 amino acid long, whereas MHC class II molecules present longer peptides with 13-17 amino acid residues (68). 5. Conclusion The advancement in immunoinformatics has now emerged as a potential field for the prediction of epitope-based vaccines. As viruses can delineate both T-cell and humoral immunity, our predicted epitope might suggest enhancing the immunity against SARS-CoV-2. The assumption is based on the basic principles of immunity, which confers the attachment of virus with the host cell, evoking immune responses and transfers the information to a broad spectrum of T cells and B cells. Our investigated epitopes mimic the interaction to CD8 cells antigen presentation using computational approaches. However, our study is an introductory design to predict epitope-based vaccine against SARS-CoV-2 and we hope that this predicted epitope will assist the further laboratory analysis for designing and predicting novel candidates against COVID-19. 4. Discussion On the other hand, our study was more specific than some similar studies, for example, a study from Khan et al had selected MHC-I alleles for which the epitopes representing higher affinity (IC50 < 500 nm), but in our study, we showed that epitopes for MHC I alleles showing higher affinity (IC50 < 200 nm), as peptides with minimum IC50 values, exhibited greater inhibition (85,86). In addition, we assessed immunogenicity, allergenicity, and toxicity of the selected epitopes. Moreover, B-cell epitopes can pave the way for experimental epitope mapping and also crucial concerning the interpretation of results from several experiments, including ELISA, radioimmunoassay, and western blotting. Recently, immunoinformatics-aided vaccine design has received experimental validation, which targeted multi-epitope protein clusters from Mycobacterium tuberculosis that interacted with HLA class I and II molecules and their prediction was experimentally validated through in vitro studies (84). On the other hand, our study was more specific than some similar studies, for example, a study from Khan et al had selected MHC-I alleles for which the epitopes representing higher affinity (IC50 < 500 nm), but in our study, we showed that epitopes for MHC I alleles showing higher affinity (IC50 < 200 nm), as peptides with minimum IC50 values, exhibited greater inhibition (85,86). In addition, we assessed immunogenicity, allergenicity, and toxicity of the selected epitopes. Moreover, B-cell epitopes can pave the way for experimental epitope mapping and also crucial concerning the interpretation of results from several experiments, including ELISA, radioimmunoassay, and western blotting. Of course, we understand that this research work does not claim to be exhaustive and all-inclusive as it is true that in silico works have its advantages and limitations. But, recently immunoinformatics is regarded as a new branch of computational biology techniques and effective in quest of new immunotherapeutics, amalgamating bioinformatics techniques to figure out several unique problems of vaccinology and immunology (87). Epitope prediction can be regarded as a high parameter in immunoinformatics investigation, and immunoinformatics calculations are considered as the high frontier to develop effective vaccines true of practical value. However, the experimental validations of the underlying approaches are required to establish a predicted epitope into a vaccine candidate. The accuracy of the predicted computational analysis should be corroborated by more accessible and robust laboratory experiments. 4. Discussion In the current study, the prediction of MHC-I binding with T-cell antigenic peptides from the SARS-CoV-2 N protein sequences was done through SMM algorithm, which incorporates proteasomal cleavage, TAP transport, and MHC class I affinity into the final output and recent studies suggested that, SMM is more established than other algorithms such as EpiJen, MAPPP (74–76). Recently, other research works have suggested vaccine design from antigenic protein sequences of SARS-CoV-2 through utilizing in silico immunoinformatics-based methodologies. A study from Lee et al reported a comprehensive list of antigenic peptides for vaccine development against SARS-CoV-2 (77). But, the findings of the research work represented that the N protein patterns retained from SARS-CoV-2 were unable to interact with HLA alleles. Several other studies also delineated the high binding affinity of predicted epitopes towards HLA-A24:02 and HLA-A02:01 alleles respectively (78,79). Conversely, in the current research work, our predicted epitope NTASWFTAL exhibited greater affinity towards HLA-A68:02, predicted by NetCTL 1.2 server. Besides, molecular docking simulation unveiled the greater interaction between the predicted epitope and HLA-A68:02 molecules. Moreover, our current study is in alignment with previous research work which depicted peptide-based sequence against S protein of human coronavirus (71). However, we cannot rule out the role of MHC class II peptides during the design of epitope- based vaccine, as it plays a phenomenal role in humoral immunity through helping B-cells. In addition, the B-cell epitope provides a strong immune response without causing any adverse effects. Generally, B-cell epitopes are either linear (continuous) or conformational (non- continuous) (80). In this experiment, we also calculated the B-cell epitope prediction and found that the protein sequences from 232 to 249 amino acid residues as B-cell epitope. The identified region Page 11/23 Page 11/23 might be able to stimulate the desired immune response and also important for developing a vaccine. Previously, several studies have reported the characterization of B-cell epitope from the N protein of many viruses from humans and animals (24,81–83). might be able to stimulate the desired immune response and also important for developing a vaccine. Previously, several studies have reported the characterization of B-cell epitope from the N protein of many viruses from humans and animals (24,81–83). Recently, immunoinformatics-aided vaccine design has received experimental validation, which targeted multi-epitope protein clusters from Mycobacterium tuberculosis that interacted with HLA class I and II molecules and their prediction was experimentally validated through in vitro studies (84). Declarations Conflict of interest The authors report no conflicts of interests in this work. Author contributions Author contributions Study concept and design: A.R., S.A.S., M.A.I., T.B.E., and J.S.-G. Acquisition of data: A.R., S.A.S., and M.A.I. Analyses and interpretation of data: A.R., M.A.I., K.K.S.M., M.R., T.B.E., and J.S.-G. Drafting the manuscript: A.R., S.A.S., T.B.E., and J.S.-G. Critical revision of the manuscript for important intellectual content: S.A., F.B.F., B.H.K., T.B.E., and J.S.-G. Technical or material support: M.M.N.U. Study supervision: T.B.E. Study concept and design: A.R., S.A.S., M.A.I., T.B.E., and J.S.-G. Acquisition of data: A.R., S.A.S., and M.A.I. Analyses and interpretation of data: A.R., M.A.I., K.K.S.M., M.R., T.B.E., and J.S.-G. Drafting the manuscript: A.R., S.A.S., T.B.E., and J.S.-G. Critical revision of the manuscript for important intellectual content: S.A., F.B.F., B.H.K., T.B.E., and J.S.-G. Technical or material support: M.M.N.U. Study supervision: T.B.E. Study concept and design: A.R., S.A.S., M.A.I., T.B.E., and J.S.-G. Acquisition of data: A.R., S.A.S., and M.A.I. Analyses and interpretation of data: A.R., M.A.I., K.K.S.M., M.R., T.B.E., and J.S.-G. Drafting the manuscript: A.R., S.A.S., T.B.E., and J.S.-G. Critical revision of the manuscript for important intellectual content: S.A., F.B.F., B.H.K., T.B.E., and J.S.-G. Technical or material support: M.M.N.U. Study supervision: T.B.E. Data availability statement The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. Funding This work is conducted with the individual funding of all authors. The authors would like to thank Cambridge Proofreading®️ & Editing LLC. (https://proofreading.org/) for editing a draft of this manuscript. The authors would like to thank Niaz Uddin Mahmud, Data Analyst at the University of Nevada, Reno, USA for kind Page 12/23 Page 12/23 ssistance with the statistical analysis, and for critical comments on the manuscript. assistance with the statistical analysis, and for critical comments on the manuscript. assistance with the statistical analysis, and for critical comments on the manuscript. References SARS coronavirus accessory proteins. Virus Res. 2008; 11. Narayanan K, Huang C, Makino S. SARS coronavirus accessory proteins. Virus Res. 2008;133(1):113–21. 11. Narayanan K, Huang C, Makino S. 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Nucleic Acids Res. 2008;36(suppl_2):W509-- W512. 87. Flower DR. Immunoinformatics and the in silico prediction of immunogenicity. In: Immunoinformatics. Springer; 2007. p. 1– 15. 87. Flower DR. Immunoinformatics and the in silico prediction of immunogenicity. In: Immunoinformatics. Springer; 2007. p. 1– 15. 87. Flower DR. Immunoinformatics and the in silico prediction of immunogenicity. In: Immunoinformatics. Springer; 2007. p. 1– 15. 15. Figures Figures Page 16/23 Figure 1 Workflow of the methodologies used in peptide vaccine design. Figure 1 Workflow of the methodologies used in peptide vaccine design. Workflow of the methodologies used in peptide vaccine design. Workflow of the methodologies used in peptide vaccine design. Page 17/23 Figure 2 Logo of the conserved peptide sequence. Figure 3 Composition of secondary structure from amino acid residues of SARS-CoV-2 nucleocapsid protein. Only 44.6% residues form sheet, 50.8% form helices and 15.8% residues form the turn region. Figure 2 Logo of the conserved peptide sequence. Figure 3 Composition of secondary structure from amino acid residues of SARS-CoV-2 nucleocapsid protein. Only 44.6% residues form sheet 50 8% form helices and 15 8% residues form the turn region Logo of the conserved peptide sequence. Logo of the conserved peptide sequence. (A) Three-dimensional representation of the predicted epitope, NTASWFTAL. (B) Three-dimensional representation of HLA- A68:02 molecule. Figure 3 Figure 3 Composition of secondary structure from amino acid residues of SARS-CoV-2 nucleocapsid protein. Only 44.6% residues form sheet, 50.8% form helices and 15.8% residues form the turn region. Page 18/23 Figure 4 (A) The three-dimensional structure of the protein prepared using EasyModeller; (B) using PROCHECK web server; (C) z-score predicted by PROSA server. Figure 4 Figure 4 (A) The three-dimensional structure of the protein prepared using EasyModeller; (B) Ramachandran plot analysis of th using PROCHECK web server; (C) z-score predicted by PROSA server. Figure 4 (A) The three-dimensional structure of the protein prepared using EasyModeller; (B) Ramachandran plot analysis of the protein using PROCHECK web server; (C) z-score predicted by PROSA server. Page 19/23 Page 19/23 Page 19/23 Page 19/23 Figure 5 (A) Three-dimensional representation of the predicted epitope, NTASWFTAL. (B) Three-dimensional representation of HLA- A68:02 molecule. Page 20/23 Figure 6 Three-dimensional representation of molecular docking studies representing the binding affinity of the predicted epitope, NTASWFTAL to the groove of the HLA-A68:02. The selected epitope, NTASWFTAL interacted with Arg6, Ser4, Ser2, Asp30 residues of chain-A and Lys59, Asp60, Ser58, Gly30 of chain-B through hydrogen bonding and charge distribution was depicted during bonding with Lys7 residue of chain-B. Figure 7 Combined B-cell linear epitope prediction showed the region from 232 to 249 amino acid residues had the highest antigenic propensity for B-cell linear epitopes. Surrounded by six differently coloured lines, which cover the region 232–269 amino acid residues in SARS-CoV-2 nucleocapsid pritein, each line indicating different analysis methods with the maximum scores. p p p g propensity for B-cell linear epitopes. Surrounded by six differently c residues in SARS-CoV-2 nucleocapsid pritein, each line indicating d Figure 8 Figure 8 Combined B-cell linear epitope prediction using (A) Bepipred linear epitope prediction, (B) Chou and Fasman beta-turn prediction, (C) Emini surface accessibility prediction methods. Figure 6 Three-dimensional representation of molecular docking studies representing the binding affinity of the predicted epitope, NTASWFTAL to the groove of the HLA-A68:02. The selected epitope, NTASWFTAL interacted with Arg6, Ser4, Ser2, Asp30 residues of chain-A and Lys59, Asp60, Ser58, Gly30 of chain-B through hydrogen bonding and charge distribution was depicted during bonding with Lys7 residue of chain-B. Three-dimensional representation of molecular docking studies representing the binding affinity of the predicted epitope, NTASWFTAL to the groove of the HLA-A68:02. The selected epitope, NTASWFTAL interacted with Arg6, Ser4, Ser2, Asp30 residues of chain-A and Lys59, Asp60, Ser58, Gly30 of chain-B through hydrogen bonding and charge distribution was depicted during bonding with Lys7 residue of chain-B. Page 21/23 Page 21/23 Combined B-cell linear epitope prediction using (A) Bepipred linear epitope prediction, (B) Chou and Fasman beta-turn prediction, (C) Emini surface accessibility prediction methods. Figure 9 Combined B-cell linear epitope prediction using (A) Karplus and Schulz flexibility prediction, (B) Kolaskar and Tongaonkar antigenicity, (C) Parker hydrophilicity prediction methods. Combined B-cell linear epitope prediction using (A) Karplus and Schulz flexibility prediction, (B) Kolaskar and Tongaonkar antigenicity, (C) Parker hydrophilicity prediction methods. Figure 8 Combined B-cell linear epitope prediction using (A) Bepipred linear epitope prediction, (B) Chou and Fasman beta-turn prediction, (C) Emini surface accessibility prediction methods. Combined B-cell linear epitope prediction using (A) Bepipred linear epitope prediction, (B) Chou and Fasman beta-turn prediction, (C) Emini surface accessibility prediction methods. Combined B-cell linear epitope prediction using (A) Bepipred linear epitope prediction, (B) Chou and Fasman beta-turn prediction, (C) Emini surface accessibility prediction methods. Page 22/23 Figure 9 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryFigures.docx SupplementaryMaterial.pdf Page 23/23 Page 23/23
https://openalex.org/W2606067123	https://asa.scitation.org/doi/pdf/10.1121/1.4979257	English	null	Laser-driven resonance of dye-doped oil-coated microbubbles: A theoretical and numerical study	The Journal of the Acoustical Society of America/The journal of the Acoustical Society of America	2,017	cc-by	18,208	Laser-driven resonance of dye-doped oil-coated microbubbles: A theoretical and numerical study Guillaume Lajoinie; Erik Linnartz; Pieter Kruizinga; Nico de Jong; Eleanor Stride; Gijs van Soest; Michel Versluis J. Acoust. Soc. Am. 141, 2727–2745 (2017) https://doi.org/10.1121/1.4979257 J. Acoust. Soc. Am. 141, 2727–2745 (2017) https://doi.org/10.1121/1.4979257 Articles You May Be Interested In Pieter Kruizinga and Nico de Jonga) [http://dx.doi.org/10.1121/1.4979257] 24 October 2024 05:24:01 Pages: 2727–2745 Pages: 2727–2745 [JFL] technology to superﬁcial or catheter-based tissue imaging. Diffusive scattering and absorption of both the incident light and subsequent acoustic emissions prevent adequate signal- intensities from being obtained beyond a depth of a few mm.1 A possible solution to this problem is to use contrast agents, in the form of dyes or nanoparticle suspensions, to increase optical absorption at the site of interest and thereby increase the amplitude of the acoustic emissions from that region.2,3 Metallic nanoparticles in particular have been shown to offer considerable improvement in photo- acoustic contrast.4 By exploiting the plasmon resonance phe- nomena, their optical absorption at a given wavelength can be much greater than that available from, e.g., hemoglobin.5 Nanoparticle agents have been designed with multiple shapes and sizes to tune the absorption wavelength and to add speciﬁc functionalities.5–7 a)Also at: Acoustical Waveﬁeld Imaging, TU Delft, Delft, The Netherlands. b)Electronic mail: m.versluis@utwente.nl Pieter Kruizinga and Nico de Jonga) Biomedical Engineering, Thoraxcenter, Erasmus MC, Rotterdam, the Netherlands Eleanor Stride Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Old Road Campus, Oxford OX3 7DQ, United Kingdom Eleanor Stride Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Old Road Campus, Oxford OX3 7DQ, United Kingdom Eleanor Stride Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Old Road Campus, Oxford OX3 7DQ, United Kingdom Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Old Road Campus, Oxford OX3 7DQ, United Kingdom Gijs van Soest Biomedical Engineering, Thoraxcenter, Erasmus MC, Rotterdam, the Netherlands Biomedical Engineering, Thoraxcenter, Erasmus MC, Rotterdam, the Netherlands Michel Versluisb) Physics of Fluids Group, MIRA Institute for Biomedical Technology and Technical Medicine and MESAþ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE Enschede, the Netherlands (Received 22 September 2016; revised 11 March 2017; accepted 14 March 2017; published online 19 April 2017) Microbubbles are used to enhance the contrast in ultrasound imaging. When coated with an optically absorbing material, these bubbles can also provide contrast in photoacoustic imaging. This multimodal aspect is of pronounced interest to the ﬁeld of medical imaging. The aim of this paper is to provide a theoretical framework to describe the physical phenomena underlying the photoacoustic response. This article presents a model for a spherical gas microbubble sus- pended in an aqueous environment and coated with an oil layer containing an optically absorb- ing dye. The model includes heat transfer between the gas core and the surrounding liquids. This framework is suitable for the investigation of both continuous wave and pulsed laser excitation. This work utilizes a combination of ﬁnite difference simulations and numerical inte- gration to determine the dependancy on the physical properties, including composition and thickness of the oil layer on the microbubble response. A normalization scheme for a linear- ized version of the model was derived to facilitate comparison with experimental measure- ments. The results show that viscosity and thickness of the oil layer determine whether or not microbubble resonance can be excited. This work also examines the use of non-sinusoidal excitation to promote harmonic imaging techniques to further improve the imaging sensitivity. V C 2017 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Articles You May Be Interested In The effect of size range on ultrasound-induced translations in microbubble populations J. Acoust. Soc. Am. (May 2020) Numerical analyses of nonlinear behavior of microbubble contrast agents in ultrasound field and effective parameters J. Acoust. Soc. Am. (April 2018) Laser-driven resonance of dye-doped oil-coated microbubbles: Experimental study J. Acoust. Soc. Am. (June 2017) The effect of size range on ultrasound-induced translations in microbubble populations Numerical analyses of nonlinear behavior of microbubble contrast agents in ultrasound field and effective parameters Laser-driven resonance of dye-doped oil-coated microbubbles: Experimental study 24 October 2024 05:24:01 24 October 2024 05:24:01 Laser-driven resonance of dye-doped oil-coated microbubbles: A theoretical and numerical study Guillaume Lajoinie and Erik Linnartz Physics of Fluids Group, MIRA Institute for Biomedical Technology and Technical Medicine and MESAþ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE Enschede, the Netherlands A. Physical problem and analytical derivation In this paragraph, we give a summary of the theoretical derivation containing the main steps of the reasoning and of the derivation. All details can be found in Appendix A. The microbubble system (Fig. 1) consists of three domains: a gas core, an oil layer and the surrounding liquid and is assumed to remain spherically symmetric. In each domain, three inter- related physical processes are to be evaluated. First, the ther- mal diffusion-convection problem will deﬁne the heat transfer and the instantaneous temperature in each domain. In spherical coordinates, it obeys the relation, Dj @2 @r2 ~T þ rBj qjcpj ¼ @ ~T @t ; (1) (1) Previous work has examined the case of a microbub- ble exposed to a laser pulse.16 The purpose of that study, was to compare the relative efﬁciency of different types of PA contrast agents according to their geometrical arrangement of the various components. The effects of heat transfer between the microbubble coating, gas core and surrounding liquid were neglected, for simplicity. In this paper, we show that these effects are in fact impor- tant for PA generation by light absorbing microbubbles. We propose a revised theoretical description of a spheri- cal bubble consisting of a gas core surrounded by an optically absorbing layer suspended in an aqueous envi- ronment. The absorbing layer considered here consists of an oil in which a dye of speciﬁc optical properties can be dissolved.17 We show that through an appropriate selection of materials, a strong microbubble resonance can be excited. We also study the inﬂuence of the different micro- bubble and laser light exposure parameters upon the ampli- tude and frequency spectrum of the acoustic emissions and demonstrate the potential for utilizing a harmonic imaging technique to achieve further improvement in imaging sensitivity. where T denotes the temperature ﬁeld, r the radial coordinate and ~T ¼ rT. The density of the ﬂuid is qj, cpj is the speciﬁc heat capacity, Dj is the heat diffusivity ½Dj ¼ kj=ðqjcpjÞ, Bj is the thermal power deposition density (W/m3) and t is the time variable. The subscript letter j refers to one of the three domains. Second, the gas equation of state determines the relation between the temperature and the pressure in the gas core. Considering low Mach numbers (Ma < 0.01) the pressure can be considered homogeneous in the gas core. II. THEORY The biological safety of nanoparticle agents, however, remains uncertain, motivating the scientiﬁc community to look for alternatives. It has recently been proposed that even greater contrast enhancement in photoacoustic imaging could be achieved through the use of volatile droplets whose vaporization is triggered by light.2,8,9 However, the neces- sary phase change consumes energy under the form of latent heat, that cannot then participate in the acoustic generation, which makes the use of the available energy inherently suboptimal. Stable microbubbles modiﬁed with the addition of an optically absorbing coating have been proposed as a potential solution to this problem.10,11 This is an attractive approach since gas bubbles are well established as contrast agents for ultrasound imaging.12–15 In this case, the micro- bubble oscillations are stimulated by the heating and cooling of the coating and subsequently of the gas core upon optical irradiation. I. INTRODUCTION technology to superﬁcial or catheter-based tissue imaging. Diffusive scattering and absorption of both the incident light and subsequent acoustic emissions prevent adequate signal- intensities from being obtained beyond a depth of a few mm.1 A possible solution to this problem is to use contrast agents, in the form of dyes or nanoparticle suspensions, to increase optical absorption at the site of interest and thereby increase the amplitude of the acoustic emissions from that region.2,3 Metallic nanoparticles in particular have been shown to offer considerable improvement in photo- acoustic contrast.4 By exploiting the plasmon resonance phe- nomena, their optical absorption at a given wavelength can be much greater than that available from, e.g., hemoglobin.5 Nanoparticle agents have been designed with multiple shapes and sizes to tune the absorption wavelength and to add speciﬁc functionalities.5–7 Ultrasound imaging is a safe, fast, and relatively cheap imaging modality that offers high resolution images deep inside the human body. However, ultrasound imaging lacks the speciﬁcity of other techniques such as photoacoustic imaging. Contrast in photoacoustic (PA) imaging results from variations in the absorption of pulsed or modulated light and the subsequent propagation of sound in tissue.1 The amplitude of the emitted acoustic signal is unique for every tissue type which makes PA imaging a very attractive clini- cal imaging modality. A major limitation of PA imaging, however, is its limited penetration depth that restricts the 0001-4966/2017/141(4)/2727/19 2727 V C Author(s) 2017. J. Acoust. Soc. Am. 141 (4), April 2017 A. Physical problem and analytical derivation lw is the dynamic viscosity of water and its temperature depen- dence can be written as18 (8) lw ¼ 2:414 105 10247:8=ðT140Þ Here k is the thermal conductivity, the subscript eq refers to the equilibrium state and Ba,av (in W/m3) is the average ther- mal power deposited by the laser. Over the course of each oscillation, the temperature has to change not only in the oil layer but also in a layer of water corresponding to the thermal diffusion radius over half of the excitation period: Rd ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ pDw=2flas p with flas the laser driving frequency. Here, we are primarily interested in frequencies in the MHz range, corresponding to commonly used ultrasound imaging fre- quencies. Higher frequencies offer higher resolution whereas lower frequencies offer deeper penetration. To a ﬁrst approx- imation, the thermal diffusion radius is then equal to rd ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ pDw=2flas p 0:1 lm with a corresponding volume Vw0.1. The heat capacity of the gas, i.e., the thermal energy that can be stored in the gas is negligible as compared to the speciﬁc heat of the oil and the water and can therefore be omit- ted. The heat diffusion in the gas is rapid when the condition given by Eq. (6) is satisﬁed. Thus, the temperature variation in the gas can be estimated by considering the change in enthalpy of the system following a change in the temperature, for the water with T the temperature of the water at the water-oil interface. rwo is the oil/water interfacial tension and ro is the gas/oil interfacial tension. The subscripts o, w, and g refer to the oil, the water and the gas, respectively. The boundary conditions are inserted into the integrated momentum equation, leading to Pg P1 ¼ €Ri R2 i Re qw qo ð Þ þ qoRi þ _R 2 i Ri qo 3 2Ri 2 Re þ 1 2 R3 i R4 e ! þ qw Re 2 1 2 R3 i R3 e ! " # þ4lo _Ri Ri _RiR2 i R3 e " # þ 4 _RiR2 i R3 e lw þ 2row Re þ 2ro Ri ; (5) (5) where P1 is the pressure far away from the bubble. In prin- ciple, this derivation is identical to that of the Rayleigh- Plesset equation,19 but now including multiple domains. A. Physical problem and analytical derivation Here we chose the ideal gas law as the state function for the gas core in the considered temperature range (between 20 C and 100 C) and pressures (around ambient pressure 105 Pa) thus, 24 October 2024 05:24:01 4p 3 PgRi 3 ¼ nKTg; (2) (2) where Pg is the gas pressure, Ri is the bubble radius and in our case also the inner oil radius, K is the universal gas constant, Tg is the gas temperature and n is the number of moles of the gas that is assumed constant. We neglect vaporization and molecu- lar diffusion phenomena in this derivation. Finally, writing the momentum equation in both the oil and the water phase will determine the dynamic behavior of the system by relating the motion of the ﬂuids to the inner gas pressure. Because of the low Mach number in the water and the oil (Ma < 5 103), the radial momentum equation can be derived by integration of the Navier-Stokes equation in potential ﬂow in each domain. The boundary conditions at the interfaces are obtained by bal- ancing the normal stress tensors on each side of the interface. For the gas/oil interface that provides FIG. 1. (Color online) Schematic of the microbubble system with the three domains and the corresponding physical parameters. Pg P Rþ i ¼ 4lo _Ri Ri þ 2ro Ri : (3) (3) For the oil/water interface, we obtain P Rþ e P R e ð Þ ¼ 4 _RiR2 i R3 e lo lw ð Þ 2rwo Re : (4) (4) Here i refers to the gas/oil interface and e refers to the oil/water interface. R is the radius of the interface (gas/oil interface and oil/water) and the superscripts þ and refer to FIG. 1. (Color online) Schematic of the microbubble system with the three domains and the corresponding physical parameters. 2728 J. Acoust. Soc. Am. 141 (4), April 2017 Lajoinie et al. Lajoinie et al. Ri;eq ¼ Ri;0 Tg;eqP0 T0 P1 þ 2rgo Ri;eq þ 2row Re;eq 2 64 3 75 1=3 : (8) the outer and inner side of the interface, respectively. B. Governing differential equation A number of simpliﬁcations can be made to the descrip- tion of the physical system presented above to derive an ana- lytical solution. First, the microbubble resonance frequency is assumed to lie in the same range as its acoustical resonance frequency [fr(MHz) R0(lm) 3.3 (Ref. 20) veri- ﬁed a posteriori]. During fast thermal processes (in the tran- sient regime), the thermal boundary layer in the gas will obey d ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ pDgt p where Dg is the thermal diffusivity of the gas and t is the time. The temperature in the gas can then be considered constant when the establishment of the thermal boundary layer is faster than the variations in deposited heat by the modulated laser beam. This holds for bubbles smaller than P0R3 i;0 T0R3 i ðt 0 Ba tð ÞdtVoil qoVoilcpo þ qwVw0:1cpw 2 64 þ Ba;av 3ko 1:5R2 i;eq þ R3 i;eq Re;eq 1 ko kw þ R2 e;eq 0:5 þ ko kw þ Troom 3 75 P1 ¼ €Ri R2 i Re qw qo ð Þ þ qoRi þ _R 2 i Ri qo 3 2Ri 2 Re þ 1 2 R3 i R4 e ! þ qw Re 2 1 2 R3 i R3 e ! " # þ 4lo _Ri Ri _RiR2 i R3 e " # þ 4 _RiR2 i R3 e lw þ 2row Re þ 2ro Ri : (10 R ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃ pDg 6:6 r 11 lm: (6) R ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃ pDg 6:6 r 11 lm: (6) This limit comprises the range of bubble sizes relevant for medical use. Thus, we consider both the pressure and tem- perature to be homogeneous in the gas core. Therefore the bubble is considered to oscillate around its equilibrium state given by the solution of the static heat diffusion equation. One then easily obtains This limit comprises the range of bubble sizes relevant for medical use. Thus, we consider both the pressure and tem- perature to be homogeneous in the gas core. Therefore the bubble is considered to oscillate around its equilibrium state given by the solution of the static heat diffusion equation. One then easily obtains DTg;eq ¼ Ba;avRi;eq2 3ko 3 2 þ Ri;eq Re;eq 1 ko kw þ Re;eq2 Ri;eq2 1 2 þ ko kw ; (10) A. Physical problem and analytical derivation The above equations can be discretized and used in a ﬁnite differ- ence model (FDM). dTg dt ¼ Ba tð ÞVoil qoVoilcpo þ qwVw0:1cpw ; (9) (9) 24 October 2024 05:24:01 with Voil the volume of the oil layer. This temperature varia- tion can then be inserted into the equation of state of the gas together with the equation for the equilibrium radius [Eq. (8)] to obtain the gas pressure as a function of the system parameters and as a function of the initial and equilibrium bubble radii. In turn, the gas pressure can be replaced in the modiﬁed Rayleigh-Plesset equation [Eq. (5)] to give C. Linearization and resonance behavior (11) then becomes z ¼ 1 2 d ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ b 2 þ f ð Þ p lo þ x3 lw lo ð Þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Pg;eq qo þ x qw qo ð Þ ð Þ p 2ﬃﬃﬃ 3 p Ri : (14) ri ¼ a= 2 þ f ð Þ jx 1 þ jw d 2 þ f x2 b 2 þ f Ba: (12) (12) 24 October 2024 05:24:01 Thus, the damping coefﬁcient depends on the oil and water viscosities, oil and water densities, as well as the bubble size. Here x is the angular frequency and the underline refers to the complex notation. A consequence of the term 1/jx (that has a p/2 phase) in Eq. (12) is a p phase at res- onance instead of p/2 for an acoustically driven bubble. This is a consequence of the necessary integration of the heat deposition as the energy is the quantity driving the system. One notices that the direct inﬂuence of the laser intensity appears only in the gain of this transfer function. Equation (12) leads to the undamped natural frequency of the system, Here x is the angular frequency and the underline refers to the complex notation. A consequence of the term 1/jx (that has a p/2 phase) in Eq. (12) is a p phase at res- onance instead of p/2 for an acoustically driven bubble. This is a consequence of the necessary integration of the heat deposition as the energy is the quantity driving the system. One notices that the direct inﬂuence of the laser intensity appears only in the gain of this transfer function. Equation (12) leads to the undamped natural frequency of the system, C. Linearization and resonance behavior (7) We can now differentiate Eq. (10) to suppress the integral and approximate Ri to Ri,eq in the non-linear products giving J. Acoust. Soc. Am. 141 (4), April 2017 Lajoinie et al. 2729 Lajoinie et al. 2729 Lajoinie et al. 2729 similar nature of the volumetric oscillations in both cases. For comparison, if the bubble is acoustically driven, Ri,eq ¼ Ri,0, Re,eq ¼ Re,0, Pg,eq ¼ Pg,0 and the term f becomes f ¼ 3jðPg;0=Ri;0Þ where j is the polytropic exponent of the gas. j is then also the corrective factor of the description of the thermal behavior of the gas between the acoustically and thermally driven bubbles. Neglecting the surface tension terms for the larger bubbles, the resonance frequency for both driving modes will differ by the quantity ﬃﬃﬃj p . For the smaller bubbles where the surface tension dominates, both expressions become identical. As a remark, both reso- nance frequencies for an acoustically driven and a thermally driven bubble become identical when j ¼ 1, i.e., in the iso- thermal case. aBa f _Ri ¼ Ri ::: b þ 2c €Ri _Ri þ d €Ri þ 2 _Ri; (11) where where a ¼ P0R3 i;0 T0R3 i;eq qocpo þ qw Vw0:1 Voil cpw ; f ¼ 3 P0Tg2 T0 R3 i;0 R4 i;eq ¼ 3 Pg;eq Ri;eq ; b ¼ R2 i;eq Re;eq qw qo ð Þ þ qoRi;eq; c ¼ Ri;eq qo 3 2Ri;eq 2 Re;eq þ 1 2 R3 i;eq R4 e;eq ! " þ qw Re;eq 2 1 2 R3 i;eq R3 e;eq !# ; d ¼ 4 lo Ri;eq þ R2 i;eq R3 e;eq lw lo ð Þ ! ; ¼ row R2 e þ ro R2 i : This equilibrium radius term Ri,eq can in fact be general- ized to be the average bubble radius at any time following the reasoning presented above. The expression is therefore also applicable to the transient thermal state. The calculated resonance frequency given in Eq. (13) also has a strong dependency on the density of the oil and interfacial tensions as one would expect from the mechanical nature of the system as was also shown by Church.21 From the same equation one can obtain the theoretical damping coefﬁcient of the microbubble, that is, If the purely non-linear term 2c €Ri _Ri is neglected, Eq. 2730 J. Acoust. Soc. Am. 141 (4), April 2017 D. Pulsed laser excitation The previous sections describe the case of a modulated continuous wave laser exposure. In practice, pulsed lasers are the preferred excitation tools in photoacoustics. It is therefore worth investigating the case of pulsed light excita- tion. The derivation of the differential equation for the motion of the microbubble in this case also follows from Eq. (5). However, the different timescales are now well sepa- rated. First, the heating of the oil can be treated as instanta- neous as the duration of the pulse is typically a few nanoseconds. Then, the gas heats up by diffusion over a timescale given by sg ¼ R02=Dg where R0 is the bubble radius and Dg the gas thermal diffusivity. sg is typically a few hundreds of nanoseconds, which, following the argu- ment of Eq. (6) is much faster than the bubble motion time- scale. Finally, the heat diffuses away from the bubble over a timescale sw ¼ R02=Dw where Dw is the thermal diffusivity of the water. R0 is also the terminal thickness of the thermal boundary layer in the static case. sw here typically reaches tens of microseconds. An estimate of the impact of heat dif- fusion on a short timescale can be obtained from energy con- servation, such that f0 ¼ 1 2pRi;eq ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2 Ri;eq 2ro þ row 3x x2 ð Þ þ 3Patm x qw qo ð Þ þ qo v u u u t ; (13) where x ¼ Ri;eq=Re;eq is a non-dimensional variable describing the inﬂuence of the oil layer thickness. x also includes a secondary inﬂuence of the laser induced heat- ing via the dilatation of the bubble denoted by the sub- script eq: Ri;eq > Ri;0 implies Ri;eq Re;eq < Ri;0 Re;0 : The undamped natural frequency of the laser-driven micro- bubble [Eq. (13)] has a form similar to that of an acoustically driven bubble and is (mostly) inversely proportional to the equilibrium bubble radius. This corresponds to the The undamped natural frequency of the laser-driven micro- bubble [Eq. (13)] has a form similar to that of an acoustically driven bubble and is (mostly) inversely proportional to the equilibrium bubble radius. This corresponds to the qocpoVoil d Tg T0 ð Þ dt ¼ kw4pR2 i0 Tg T0 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ pDwt p ; (15) (15) 2730 J. Acoust. Soc. Am. 141 (4), April 2017 Lajoinie et al. D. Pulsed laser excitation thickness of the water layer subject to thermal diffusion dur- ing the irradiation cycle. Basically, Eq. (19) is a normaliza- tion function that must be calculated from experimentally measured quantities. The presence of the variables x and y prohibits a simple scaling as far as the laser intensity and oil layer thickness are concerned. On the other hand, varying the frequency alone and using Eq. (13) enables us to reduce the resonant oscillation amplitude description in Eq. (18) to a x5=2 scaling law for a thin oil layer and a x3 scaling law for a thick oil layer. where the small amplitude approximation allows the bubble surface area to be taken as the resting surface area S ¼ 4pR2 i0, where the approximate expression for the devel- opment of the thermal boundary layer for short times, ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ pDwt p , is used and where the inﬂuence of the thickness of the oil layer on the global heat diffusion is neglected. The temperature in the gas then becomes Tg ¼ T0 þ Thot T0 ð ÞH tð Þexp kw8pR2 i0 ﬃﬃt p qocpoVoil ﬃﬃﬃﬃﬃﬃﬃﬃﬃ pDw p ! ; (16) Thus, a thermally driven microbubble experiences a decrease in the maximum response according to a 5/2 power law with increasing excitation frequency for a thin oil layer. As a consequence the parameter space can be divided in two subspaces. The ﬁrst is the modulation frequency and the second captures the inﬂuence of the oil layer thickness and the laser intensity described using Eq. (19). where H represents the Heaviside step function and Thot the temperature of the oil (and gas by extension) just after the laser pulse. Thot can further be approximated to Thot Fa/ qocpo þ T0 where Fa is the thermal energy deposited by the laser per unit volume. Subsequently, the ﬁrst term of Eq. (10) simply becomes Pg ¼ P0R3 i;0 T0R3 i Tg ¼ P0R3 i;0 T0R3 i T0 þ Fa qocpo H tð Þ exp kw8pR2 i0 ﬃﬃt p qocpoVoil ﬃﬃﬃﬃﬃﬃﬃﬃﬃ pDw p !! : (17) J. Acoust. Soc. Am. 141 (4), April 2017 III. FINITE DIFFERENCE MODEL (FDM) In Sec. II, we derived a simple analytical expression that describes, after a number of approximations, the ther- mal and mechanical behavior of the microbubble. The validity of these approximations, using the same initial set of equations, can be veriﬁed by means of a more reﬁned simulation. We therefore design a ﬁnite difference simulation in which the three physical equation describing the problem, i.e., the heat diffusion equation, the momen- tum conservation in the water, and the thermodynamic behavior of the gas core. In short, each equation is simu- lated as such and communicate with the others at each time step. The mass of each grid volume is deﬁned to be constant and the grid is recalculated every time step to hold on to this deﬁnition. Initially, before the laser is turned on and before the bubble starts to oscillate, the grid is deﬁned with a regular size interval of 31.25 nm up to a radius twice the typical bubble size. Beyond this radius the grid gets 15% bigger for each step outward. Without an excessive amount of grid points, the outer point, kept at room temperature, is at a distance well over 3000 times the typical bubble radius making the thermal drift due to a ﬁnite simulation volume negligible. The gas is considered to obey the ideal gas law. The pressure is thus deﬁned by (17) Although simpler, this term is actually similar to that found for the continuous wave (CW) laser and the result- ing equation shows the same characteristics in terms of resonance frequency and damping, without the need for the additional differentiation performed prior to obtaining Eq. (11). 24 October 2024 05:24:01 E. Amplitude response and parameter space The parameter space of the problem has four dimen- sions: the thermal power deposition density, which is propor- tional to the laser intensity and the oil absorption coefﬁcient, the oil layer thickness, the laser modulation frequency and the initial bubble radius. The amplitude of the microbubble response at resonance can be derived from Eq. (12) for x ¼ x0 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 2z2 p x0, ri ¼ Baab 2 þ f ð Þd ; (18) ri a Ri;0 F x ð Þ y 1 y2 qo þ x qw qo ð Þ ð Þ qocpo þ g x ð Þqwcpw 1 lo þ x3 lw lo ð Þ ; (19) (18) P ¼ P0 T0 4 3 pR3 0 1 X Vk=Tk ; (19) with where P kVk=Tk can be written as where P kVk=Tk can be written as F x ð Þ ¼ 3 2 þ x 1 ko kw þ 1 x2 ; 1 2 þ ko kw ; g x ð Þ ¼ 1 þ ew Re;eq 3 1 1 x3 : X Vk Tk ¼ X 4 3 p rk1 þ pk ð Þ3 r3 k1 Tk ; where P is the pressure, V is the volume. The subscript 0 stands for the initial value before the laser is turned on and k is the index on the grid spacing vector p and the radius vec- tor r. The speed of the bubble wall _R is assumed to be much smaller than the speed of sound in air or water so that the Similar to x, the variable y ¼ Ri;eq=Ri;0 is a measure of the thermal dilatation of the microbubble. ew ¼ 0.1 lm is the Lajoinie et al. 2731 2731 Lajoinie et al. 2731 Lajoinie et al. 2731 pressure in the bubble is considered homogeneous. Together with mass conservation, this leads to a condition on the radius of the grid points in the bubble, B. Finite difference model The scheme as describe in Sec. III was coded as such and used to simulate the responses of the microbubbles upon laser irradiation using Fortran and is hereafter referred to as ﬁnite difference model/simulations (FDM). rk1 ¼ Rg ~lP q0Tk kp0 ð Þ3 k 1 ð Þp0 3 h i þ r3 k 1=3 ; A. Undamped natural frequency 24 October 2024 05:24:01 The undamped natural frequency, Eq. (13), was calcu- lated for triacetin oil that has been used in previous studies17 to coat microbubbles; and heptane oil that is a commonly used linear chain organic oil. The results are dis- played in Fig. 3. As expected the natural frequencies for the range of bubble sizes considered lie within the MHz range corresponding to that used in standard ultrasound and photoacoustic imaging systems. There is a noticeable dif- ference in natural frequency between the heptane-coated and triacetin-coated microbubbles owing to the different densities of the two oils. In the same ﬁgure, we also plot the resonance frequency of a free gas bubble driven by ultrasound. The undamped natural frequency of a laser- driven oil-coated microbubble differs from that of the acoustically driven bubble due to both the different proper- ties of the microbubbles (oil density, interfacial tensions, C. Parameter estimate where rk is the radius in meters. To speed up the calculation, a Taylor expansion is carried out to the order 3. The oil and the water are assumed to be incompressible, and therefore, the volume in each grid volume in the oil and water domains remains constant, The four key parameters relevant for biomedical imag- ing applications are the thermal power deposited by the laser, the oil layer thickness, the microbubble initial radius, and the laser modulation frequency. The microbubble size ranges from 1 to 10 lm, corresponding to both the medi- cally relevant bubble size range and to the upper limit of validity of the proposed theory, as demonstrated below. In the present study, the oil layer thickness was varied from 0.1 lm to 3 lm. The power deposition is, to a ﬁrst approxi- mation, equal to Iabs with abs the absorption coefﬁcient of the oil. A value of 2500 m1 was chosen for the absorption that can be reached by dissolution of a dye such as oil red in an organic oil. The laser intensity was varied from 0.25 1010 W/m2, which is necessary for obtaining a sig- niﬁcant bubble response, to 2.5 1010 W/m2 that can still be easily reached in practice. rkþ1 ¼ ð½ððk þ 1Þp0Þ3 ðkp0Þ3 þ r3 kÞ1=3: The heat convection diffusion equation writes The heat convection diffusion equation writes Dr2T þ I qcp ¼ DT Dt ; where I is in units of W/m3. The heat equation can be approximated with a central difference scheme in space and a forward ﬁnite difference scheme in time, which gives a condition on the temporal evolution of the temperature (Fig. 2). The momentum equation deﬁned in Eq. (5) was also discretized in time using ﬁnite differences. Both the velocity and the acceleration are calculated at each time step. More details of the derivation, including the discreti- zation steps, are given in Appendix B. B. Damping coefficient and choice of the oil In order to fully characterize a dynamic system it is essential to determine the damping coefﬁcient. In the present case the damping coefﬁcient is given by Eq. (14) and depends primarily on the oil and water viscosities, as well as the thickness of the oil layer. Common oils have a speciﬁc density of approximately 0.7, but the viscosity can vary over two orders of magnitude, from as low as 386 lPa s for heptane to as high as 17 mPa s for triacetin for example. The simulated response of heptane-coated microbubbles to a square wave modulated laser as a function of the initial bubble radius for different laser intensities, oil layer thick- ness, and modulation frequency is shown in Fig. 5. Figures 5(a)–5(c) show the simulation results of the ﬁnite difference model. Figures 5(d) and 5(e) show the response simulated using the non-linear theory of Eq. (10) and Fig. 5(f)–5(h) show the corresponding phase difference between the laser excitation and the microbubble response. The response simu- lated with both models is very similar both in amplitude and in quality factor, showing that the proposed theory is repre- sentative of the physical problem simulated in the FDM. The only signiﬁcant difference between the ﬁnite difference and the non-linear model is the resonant radius of the initial bub- ble. This discrepancy originates from the fact that an equilib- rium assumption is made in the non-linear theory whereas the FDM is applied over a period of 100 ls. The fully 24 October 2024 05:24:01 Figure 4 shows the variation in the damping coefﬁcient with radius for microbubbles coated with heptane and tria- cetin. The difference in viscosity between these two oils leads to a difference in damping of more than an order of magnitude. A damping coefﬁcient as high as 0.5 for a 3 lm triacetin-coated microbubble drastically limits the beneﬁt of the mechanical resonance of these microbubble as compared to the heptane-coated bubbles. In practice, there will be additional dissipation due to the necessary use of stabilizing agents thereby decreasing even further the oscillation FIG. 4. (Color online) (a) Damping coefﬁcient for heptane (continuous line) and triacetin (dashed line) coated microbubbles with different oil layer thicknesses as a function of the micro- bubble size. (b) Corresponding damp- ing coefﬁcients as a function of the oil layer thickness for a 3 lm oil-coated bubble. J. Acoust. Soc. Am. C. Low viscosity oil in the parameter space We have shown that using a low viscosity oil is crucial for obtaining a strong bubble response. For the remainder of the study, we will therefore consider heptane-coated bubbles only. The shape of the excitation waveform when using ultrasound is limited to quasi-sine waves due to the limita- tions of the relatively narrowband transducer technology. The laser intensity on the other hand can be modulated using any arbitrary waveform with frequency components up to hundreds of MHz, which is the maximum frequency of the currently available acousto-optic modulators. In contrast to ultrasound, light penetration in not further limited by the modulation frequency. Preliminary simulations were there- fore performed using sine and square wave modulated wave- forms. The latter showed a slightly higher efﬁciency as the oscillation amplitude is slightly larger for the same energy deposition. FIG. 3. (Color online) Undamped natural frequencies of laser driven micro- bubbles coated with oil layer of 1 lm. The undamped natural frequencies of heptane coated (short dash red) and triacetin coated (long dash green) micro- bubbles are lower than that of an acoustically driven free gas bubble (solid black curve). etc.) and the different nature of the excitation as discussed in the theory section. A. ODE integration method The ordinary differential equation (ODE) integration for both the non-linear and linear bubble dynamics equations were performed in MATLAB (version R2012a, The Mathworks, Natick, MA). For the non-linear simulations, the integration was performed using the ODE 113 solver as the problem is non-stiff for the considered range of parameters. Simulations were performed to cover the full three- dimensional parameter space using both a sine and a square wave modulation for the laser. FIG. 2. (Color online) Different laser excitation schemes used to predict the bubble response and predicted bubble response near resonance: (a) continuous wave laser modulated in intensity by a square wave, (b) continuous wave laser modulated in intensity by a sine wave and (c) response to a pulsed laser excita- tion. The heat deposited by the laser is displayed in red. FIG. 2. (Color online) Different laser excitation schemes used to predict the bubble response and predicted bubble response near resonance: (a) continuous wave laser modulated in intensity by a square wave, (b) continuous wave laser modulated in intensity by a sine wave and (c) response to a pulsed laser excita- tion. The heat deposited by the laser is displayed in red. 2732 J. Acoust. Soc. Am. 141 (4), April 2017 Lajoinie et al. Lajoinie et al. FIG. 3. (Color online) Undamped natural frequencies of laser driven micro- bubbles coated with oil layer of 1 lm. The undamped natural frequencies of heptane coated (short dash red) and triacetin coated (long dash green) micro- bubbles are lower than that of an acoustically driven free gas bubble (solid black curve). amplitude. Thus, using a low viscosity oil is a necessary condition. Since the damping coefﬁcient is also a function of the oil layer thickness, there is a further difference between high viscosity oils (>1 mPa s) and low viscosity oils (<1 mPa s): for heptane, the damping decreases when increasing the oil thickness whereas for triacetin, the damp- ing increases with increasing oil thickness. This is captured by Eq. (14) and shown in Fig. 4. B. Damping coefficient and choice of the oil 141 (4), April 2017 Lajoinie et al. 2733 FIG. 4. (Color online) (a) Damping coefﬁcient for heptane (continuous line) and triacetin (dashed line) coated microbubbles with different oil layer thicknesses as a function of the micro- bubble size. (b) Corresponding damp- ing coefﬁcients as a function of the oil layer thickness for a 3 lm oil-coated bubble. FIG. 4. (Color online) (a) Damping coefﬁcient for heptane (continuous line) and triacetin (dashed line) coated microbubbles with different oil layer thicknesses as a function of the micro- bubble size. (b) Corresponding damp- ing coefﬁcients as a function of the oil layer thickness for a 3 lm oil-coated bubble. Lajoinie et al. 2733 J. Acoust. Soc. Am. 141 (4), April 2017 FIG. 5. (Color online) Simulated resonance curves and corresponding phase differences for a bubble irradiated by a square wave modulated laser. (a)–(c) The ﬁnite difference simulations of the system for (a) variable oil thickness, a heat deposition rate of 27 TW/m3 and a frequency of 1 MHz; (b) variable heat depo- sition rate, a modulation frequency of 1 MHz and an oil layer thickness of 1 lm and (c) variable frequency, a heat deposition rate of 27 TW/m3 and an oil thickness of 1 lm. (d)–(f) The results from simulations using the non-linear theory for the same parameters and (g)–(i) are the corresponding phase plots the bubble oscillations. 24 October 2024 05:24:01 FIG. 5. (Color online) Simulated resonance curves and corresponding phase differences for a bubble irradiated by a square wave modulated laser. (a)–(c) The ﬁnite difference simulations of the system for (a) variable oil thickness, a heat deposition rate of 27 TW/m3 and a frequency of 1 MHz; (b) variable heat depo- sition rate, a modulation frequency of 1 MHz and an oil layer thickness of 1 lm and (c) variable frequency, a heat deposition rate of 27 TW/m3 and an oil thickness of 1 lm. (d)–(f) The results from simulations using the non-linear theory for the same parameters and (g)–(i) are the corresponding phase plots the bubble oscillations. absorbing oil. Above a critical oil layer thickness, the time required to change the temperature in the oil is no longer negligible compared to the laser modulation period. Thus, the temperature in the oil fails to follow the variation in the heat deposited by the laser. 2734 J. Acoust. Soc. Am. 141 (4), April 2017 B. Damping coefficient and choice of the oil As expected from the linearized equations, the phase varies from p/2 to 3p/2 and crosses p at the for a thick oil layer. Figures 5(b) and 5(e) show the varia- tions in the microbubble response as a function of the heat deposited by the laser. As expected, the microbubble oscilla- tions become stronger with increasing laser intensity. Interestingly, and unlike the temperature variation, the response does not increase linearly with the intensity. The laser intensity in Eq. (19) is represented by the variable y that is a measure of the thermal dilatation of the microbub- ble. Nevertheless, in Eq. (19), the laser intensity is presented as a proportional term that describes the inﬂuence of the density of heat deposition and an inverse term that corre- sponds to the change in bubble size with increasing tempera- ture. Figures 5(c) and 5(f) present the simulation results for different modulation frequencies. The response of the laser- driven microbubbles at resonance increases strongly when decreasing the laser modulation frequency. Physically, the temperature variations in the gas core and therefore the driving pressure will vary with the energy deposited during half the period of the laser excitation and will therefore be larger when the modulation frequency becomes lower. Finally, Fig. 5(g)–5(i) show the variation of the phase differ- ence between the microbubble oscillations and the laser excitation. As expected from the linearized equations, the phase varies from p/2 to 3p/2 and crosses p at the natural frequency. Thus, the bubble oscillates in anti phase with the laser excitation. B. Damping coefficient and choice of the oil (a) Scaled resonance curves at 1 MHz for an oil layer thickness varying from 0.6 lm to 3 lm and a heat deposition ranging from 6.7 TW/m3 to 40.5 TW/ m3. (b) Resonance curves for an oil thickness of 1 lm and a heat deposition of 27 TW/m3 at different frequencies scaled by x5/2. FIG. 6. (Color online) Scaled reso- nance curves for the analytical model. (a) Scaled resonance curves at 1 MHz for an oil layer thickness varying from 0.6 lm to 3 lm and a heat deposition ranging from 6.7 TW/m3 to 40.5 TW/ m3. (b) Resonance curves for an oil thickness of 1 lm and a heat deposition of 27 TW/m3 at different frequencies scaled by x5/2. FIG. 6. (Color online) Scaled reso- nance curves for the analytical model. (a) Scaled resonance curves at 1 MHz for an oil layer thickness varying from 0.6 lm to 3 lm and a heat deposition ranging from 6.7 TW/m3 to 40.5 TW/ m3. (b) Resonance curves for an oil thickness of 1 lm and a heat deposition of 27 TW/m3 at different frequencies scaled by x5/2. for a thick oil layer. Figures 5(b) and 5(e) show the varia- tions in the microbubble response as a function of the heat deposited by the laser. As expected, the microbubble oscilla- tions become stronger with increasing laser intensity. Interestingly, and unlike the temperature variation, the response does not increase linearly with the intensity. The laser intensity in Eq. (19) is represented by the variable y that is a measure of the thermal dilatation of the microbub- ble. Nevertheless, in Eq. (19), the laser intensity is presented as a proportional term that describes the inﬂuence of the density of heat deposition and an inverse term that corre- sponds to the change in bubble size with increasing tempera- ture. Figures 5(c) and 5(f) present the simulation results for different modulation frequencies. The response of the laser- driven microbubbles at resonance increases strongly when decreasing the laser modulation frequency. Physically, the temperature variations in the gas core and therefore the driving pressure will vary with the energy deposited during half the period of the laser excitation and will therefore be larger when the modulation frequency becomes lower. Finally, Fig. 5(g)–5(i) show the variation of the phase differ- ence between the microbubble oscillations and the laser excitation. B. Damping coefficient and choice of the oil This phenomenon of thermal inertia decreases the amplitude of the temperature variation in the gas, and therefore decreases the response amplitude. This effect can be seen mathematically in the term a and its dependency on the oil volume and transiently heated water volume. Asymptotically, the maximum of the resonance curve can be written as developed thermal regime in the FDM is however only reached after several milliseconds of exposure due to the rel- atively slow thermal processes. A laser exposure duration of 100 ls places the system in a regime where the thermal pro- cesses (with the exception of the high-frequency modulation) evolve on a much shorter timescale as compared to the bub- ble oscillations. In this quasi-stable regime, the gas core tem- perature can lag by 20% to 30% from the equilibrium temperature as demonstrated in Appendix C. Figures 5(a) and 5(d) depict the variation of the microbubble response when increasing the oil layer thickness. The oscillation amplitude decreases for a thicker oil layer despite the decreasing damping coefﬁcient. It can also be seen from Figs. 5(a) and 5(d) that the response amplitude passes through a maximum for a heptane oil layer thickness of 1.1 lm. Physically, for a thin oil layer, the energy deposited in the oil is quickly transferred to the gas and the surround- ing water and thus the temperature in the system changes quickly enough to follow the excitation. In this regime, the microbubble response is limited by the energy deposited by the laser that is to ﬁrst order proportional to the volume of ri Ba ¼ e x7=2 T012pcpwlw ﬃﬃﬃﬃﬃﬃ Dw p P0 qw 3=2 (20) (20) in the case of a thin oil layer and in the case of a thin oil layer and ri Ba ¼ x3 12pcpolo T0 þ Bae2 3ko 1 2 þ ko kw ! P0 qo 3=2 (21) Lajoinie et al. Lajoinie et al. 2734 J. Acoust. Soc. Am. 141 (4), April 2017 for a thick oil layer. Figures 5(b) and 5(e) show the varia- tions in the microbubble response as a function of the heat deposited by the laser. As expected, the microbubble oscilla- tions become stronger with increasing laser intensity. Interestingly, and unlike the temperature variation, the response does not increase linearly with the intensity. The laser intensity in Eq. B. Damping coefficient and choice of the oil (19) is represented by the variable y that is a measure of the thermal dilatation of the microbub- ble. Nevertheless, in Eq. (19), the laser intensity is presented as a proportional term that describes the inﬂuence of the density of heat deposition and an inverse term that corre- sponds to the change in bubble size with increasing tempera- ture. Figures 5(c) and 5(f) present the simulation results for different modulation frequencies. The response of the laser- driven microbubbles at resonance increases strongly when decreasing the laser modulation frequency. Physically, the temperature variations in the gas core and therefore the driving pressure will vary with the energy deposited during half the period of the laser excitation and will therefore be larger when the modulation frequency becomes lower. Finally, Fig. 5(g)–5(i) show the variation of the phase differ- ence between the microbubble oscillations and the laser excitation. As expected from the linearized equations, the phase varies from p/2 to 3p/2 and crosses p at the natural frequency. Thus, the bubble oscillates in anti phase with the laser excitation. D. Scaled resonance curves In order to apply these theoretical ﬁndings to an experi- mental case, one must consider the practical difﬁculties involved in producing stable microbubbles with the same oil thickness and to expose them to the same laser intensity. It is therefore desirable to normalize the microbubble resonance curves using parameters that are experimentally accessible. This can be achieved using the normalization functions given in Sec. II E and Eq. (19), derived from the linearized equation Eq. (12). We also know from Eq. (13) that the reso- nant bubble size can be described as a function of the “hot” bubble radius. This hot bubble radius can thus be used instead of the initial bubble radius and then be normalized to the resonant radius using Eq. (13). The resonance curves simulated from the non-linear the- ory for varying intensity and oil thickness and normalized using Eq. (19) are plotted in Fig. 6(a) and those for a varying laser modulation frequency normalized using the power laws derived in Sec. II E are plotted in Fig. 6(b). The normaliza- tion function from Eq. (19) is effective for scaling the FIG. 6. (Color online) Scaled reso- nance curves for the analytical model. D. Scaled resonance curves In order to apply these theoretical ﬁndings to an experi- mental case, one must consider the practical difﬁculties involved in producing stable microbubbles with the same oil thickness and to expose them to the same laser intensity. It is therefore desirable to normalize the microbubble resonance curves using parameters that are experimentally accessible. This can be achieved using the normalization functions given in Sec. II E and Eq. (19), derived from the linearized equation Eq. (12). We also know from Eq. (13) that the reso- nant bubble size can be described as a function of the “hot” bubble radius. This hot bubble radius can thus be used instead of the initial bubble radius and then be normalized to the resonant radius using Eq. (13). 24 October 2024 05:24:01 The resonance curves simulated from the non-linear the- ory for varying intensity and oil thickness and normalized using Eq. (19) are plotted in Fig. 6(a) and those for a varying laser modulation frequency normalized using the power laws derived in Sec. II E are plotted in Fig. 6(b). The normaliza- tion function from Eq. (19) is effective for scaling the FIG. 7. (Color online) Scaled resonance curves for the ﬁnite difference model. (a) Scaled resonance curves at 1 MHz for a heat deposition of 27 TW/m3 with an oil layer thickness varying from 0.6 lm to 3 lm. (b) Scaled resonance curves at 1 MHz for an oil layer thickness of 1 lm and for a heat deposition ranging from 6.7 TW/m3 to 40.5 TW/m3. (b) Scaled resonance curves for a heat deposition of 27 TW/m3 and an oil layer thickness of 1 lm, and for a frequency rang- ing from 0.5 to 1.5 MHz. FIG. 7. (Color online) Scaled resonance curves for the ﬁnite difference model. (a) Scaled resonance curves at 1 MHz for a heat deposition of 27 TW/m3 with an oil layer thickness varying from 0.6 lm to 3 lm. (b) Scaled resonance curves at 1 MHz for an oil layer thickness of 1 lm and for a heat deposition ranging from 6.7 TW/m3 to 40.5 TW/m3. (b) Scaled resonance curves for a heat deposition of 27 TW/m3 and an oil layer thickness of 1 lm, and for a frequency rang- ing from 0.5 to 1.5 MHz. Lajoinie et al. 2735 Lajoinie et al. 2735 J. Acoust. Soc. Am. D. Scaled resonance curves 141 (4), April 2017 microbubble responses simulated using the non-linear the- ory. A scaling according to a 5/2 power law gives a similar result for varying modulation frequencies but with a larger deviation for the lowest frequency (500 kHz). The resonance curves simulated by the FDM and scaled with the same equations are plotted in Fig. 7. For the FDM, the amplitude of the scaled curves for the oil layer thickness and laser intensity [Fig. 7(a) and 7(b)] present a larger amplitude and deviation, which is mostly due to the differences between the thermal equilibrium radius used in the theory and the quasi equilibrium average radius simulated by the FDM. The proposed normalization applied to the results of the FDM reduces a sevenfold variation in the response amplitude to a small error margin. thus depends strongly upon the choice of the laser modula- tion waveform. VI. DISCUSSION As discussed above, the FDM simulations were run over a period of 100 ls, allowing for the model to reach a quasi steady-state that is nonetheless signiﬁcantly different from the perfect equilibrium state used as a reference in the theory. Simulations were also run over a longer timescale with non-modulated laser driving and converged toward the expected thermal equilibrium. A duration of 100 ls or less is, however, more relevant for practical application of these bubbles, as enough time should be allowed for imaging and signal integration/accumulation whilst avoiding excessive heat deposition in the tissue. The theoretical model could be modiﬁed to match this quasi steady state but the timescale to choose would then depend on the experiment or application. Figure 7(c) shows the FDM simulation for different excitation frequencies normalized by the expected 5/2 power law. The observed increase in signal amplitude could potentially increase the tissue depth from which photoa- coustic images can be obtained in two ways. First, the use of a sensitive contrast agent would increase the signal to noise ratio. Second, the use of a CW laser offers tem- poral integration possibility, together with an optimal use of the bubble resonance effect. This last approach is very novel and now needs to be translated into (pre)clinical practice. E. Harmonics and subharmonics Beyond the strength of the fundamental resonance, one feature of microbubble oscillations has become increasingly important and been widely investigated for ultrasound imaging: the harmonic and subharmonic pressure wave generation. Figures 8(a) and 8(b) show the harmonic and subhar- monic microbubble oscillations, respectively, relative to the fundamental response on a dB scale for square wave excitation and for different heat deposition densities. Figures 8(c) and 8(d) depict the same quantities for a sine wave laser modulation. Both the square and sine wave laser modulation generate harmonics from bubbles around the resonant size but the square wave also generates signif- icant harmonics from much smaller bubbles. We attribute this to the harmonic composition of the square wave itself that includes a higher frequency component at three times the fundamental frequency. The generation of harmonics 24 October 2024 05:24:01 The normalization function in Eq. (19) derived from the linear theory fails to satisfactorily scale the thinner oil layers (100 nm) and these are therefore not presented in Fig. 6(a) and Fig. 7(a). We justify this omission by the much lower pre- dicted response for such thin oil layers [Fig. 5(a) and 5(c)] that therefore present a more limited interest in terms of normalization. Both the proposed theory and the FDM were considered in the context of incompressible potential ﬂow. In fact, an oscillating bubble emits an acoustic wave, therefore FIG. 8. (Color online) Harmonic behavior of laser driven microbubbles. (a) harmonic and (b) subharmonic con- tent of the oscillations of the micro- bubbles when irradiated by a 1 MHz square wave modulated laser beam or (c) and (d) a 1 MHz sine wave modu- lated beam. The energy deposited per cycle is identical in both cases. 2736 J. Acoust. Soc. Am. 141 (4), April 2017 Lajoinie et al. FIG. 8. (Color online) Harmonic behavior of laser driven microbubbles. (a) harmonic and (b) subharmonic con- tent of the oscillations of the micro- bubbles when irradiated by a 1 MHz square wave modulated laser beam or (c) and (d) a 1 MHz sine wave modu- lated beam. The energy deposited per cycle is identical in both cases. FIG. 8. (Color online) Harmonic behavior of laser driven microbubbles. (a) harmonic and (b) subharmonic con- tent of the oscillations of the micro- bubbles when irradiated by a 1 MHz square wave modulated laser beam or (c) and (d) a 1 MHz sine wave modu- lated beam. ACKNOWLEDGMENTS where Pg is the gas pressure. Using the state function of the gas and Eq. (9), Eq. (22) becomes This project was made possible by the funding of the Dutch national NanoNextNL program, a micro and nanotechnology consortium of the Government of the Netherlands and 130 partners and the UK Engineering and Physical Sciences Research Council (Grant No. EP/ I021795/1). We also warmly thank Rodolfo Ostilla Monico for his valuable advices concerning the ﬁnite difference simulation. Pac; rad ¼ P0R3 i0 cwT0 1 R2 qocpo þ V01 Voil qwcpw B tð Þ 3 _R R ðt 0 B tð Þdt ! P0R3 i0 _R cwR3 : (23) (23) Here the dominant damping term is on the right side. The addition of this term in the theory and corresponding simula- tions produced only a negligible effect on the total damping. Reradiation is therefore negligible compared to viscous dis- sipation, even in the low viscosity case of heptane oil-coated bubbles. APPENDIX A: MATHEMATICAL DERIVATION OF THE THEORETICAL MODEL The Navier-Stokes equation for an incompressible, Newtonian ﬂuid is as follows q Dv Dt ¼ rP þ qg þ lr2v: E. Harmonics and subharmonics The energy deposited per cycle is identical in both cases. 2736 J. Acoust. Soc. Am. 141 (4), April 2017 Lajoinie et al. selection of the equivalent parameter set (density, thick- ness and viscosity) for the absorbing layer. Finally, we have shown that laser-driven microbubbles are expected to exhibit similar behavior to that of acoustically driven microbubbles in terms of harmonic and subharmonic gen- eration, thereby opening new possibilities for harmonic photoacoustic imaging that could potentially enable a much improved contrast to tissue ratio. producing a weakly compressible ﬂow carrying energy away from the microbubble. This effect has been addressed in ear- lier studies, such as those by Keller et al.22 and Prosperetti et al.23 From these models, an approximate form of the losses by radiation can be included as a supplementary pres- sure term in Eq. (10) for low Mach numbers, Prad ¼ R cw @Pg @t ; (22) Prad ¼ R cw @Pg @t ; (22) VII. CONCLUSIONS 24 October 2024 05:24:01 Body forces will be negligible and a spherical symmetry case is investigated leading to Body forces will be negligible and a spherical symmetry case is investigated leading to On the basis of theoretical considerations, we have demonstrated the possibility of using the mechanical reso- nance of microbubbles in the MHz frequency range, typi- cally used in ultrasound imaging, to increase the signal amplitude in photoacoustic imaging. We have developed a theory supported by ﬁnite difference model simulations that clariﬁes the underlying phenomena and predicts the natural frequencies and resonance characteristics of laser driven microbubbles. The resonance frequency of the laser-driven microbubbles is different from the acousti- cally driven bubble by a factor ﬃﬃﬃj p , j being the poly- tropic exponent of the gas. The natural frequencies of these microbubbles depend on the density of the oil coat- ing, as was already shown for acoustically driven bubbles. The proposed theory also indicates the crucial importance of choosing an oil with a low viscosity, i.e., similar to that of water in order to obtain a sufﬁciently high quality factor for the resonance. It appears that a suitable selec- tion of the oil properties can improve the overall quality of the system, even above that of acoustically driven bub- bles. We have also extracted from the linearized theory normalization functions within the investigated parameter space that will allow for the scaling of datasets using experimentally accessible parameters to reduce signiﬁ- cantly the error due to variations in oil thickness and laser intensity. The proposed theoretical considerations are valid for continuous wave exposure and can also be applied to the more typical case of pulsed excitation pho- toacoustics. Similarly, the response of optically absorbing microbubbles that do not contain an oil layer can be extrapolated from the proposed work by appropriate q @v @t þ v @v @r ¼ @P @r þ lr2v: In the simulation, the bubbles will have an oscillation ampli- tude of the order of lm and the frequency will be in the order of MHz. Speeds will therefore be approximately 1 m/s and thus much lower than the speed of sound. For this reason, incompressibility of the liquid is assumed leading to v ¼ _RR2 r2 ~er; a. Over the oil gas interface Pg P1 ¼ 4lo _Ri Ri þ 2ro Ri þ qo €RiR2 i þ 2 _R 2 i Ri 1 Ri 1 Re 1 2 _R 2 i R4 i 1 R4 i 1 R4 e ! 4 _RiR2 i R3 e lo lw ð Þ þ 2rwo Re þ qw €RiR2 i þ 2 _R 2 i Ri Re 1 2 _R 2 i R4 i R4 e ! : ( ro ~er rg ~er ¼ dP1; where ro is the strain tensor and ~er denotes that it is in the r direction. dP1 is the difference in pressure over the oil-gas interface. 2l0u0 Ri ð Þ P Rþ i þ Pg ¼ 2ro Ri ; where u0 is the velocity derivative to the radius. r is the surface tension. Knowing v ¼ _RiR2 i =r2 we also know v0ðRiÞ ¼ 2 _RiR2 i =R3 i , where u0 is the velocity derivative to the radius. r is the surface tension. Knowing v ¼ _RiR2 i =r2 we also know v0ðRiÞ ¼ 2 _RiR2 i =R3 i , 4lo _RiR2 i R3 i P Rþ i þ Pg ¼ 2ro Ri ; Pg P Rþ i ¼ 4lo _Ri Ri þ 2ro Ri : (A4) b. Over the oil water interface rw ~er ro ~er ¼ dP2; 2lwv0 Re ð Þ P Rþ e 2lov0 Re ð Þ P R e ð Þ ¼ 2rwo Re ; knowing v ¼ _RiR2 i =r2 we also know v0ðReÞ ¼ 2 _RiR2 i =R3 e. 2. Combining We know that Pg P1 ¼ PðR i Þ P1 because the pressure at the inside of the inner radius of the bubble is by deﬁnition in the gas and therefore Pg ¼ PðR i Þ. We can rewrite by adding and subtracting similar terms, rewritten as Pg P1 ¼ PgðR i Þ PðRþ i Þ \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} þ PðRþ i Þ PðR e Þ \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} þ PðR e Þ PðRþ e Þ \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} þ PðRþ e Þ P0 \|ﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄ} : (A6) P Rþ i P R e ð Þ ¼ qo €RiR2 i þ 2 _R 2 i Ri 1 Ri 1 Re 1 2 _R 2 i R4 i 1 R4 i 1 R4 e : (A3 Part 1 of (A6) is deﬁned in (A4), part 2 is deﬁned in (A3), part 3 is deﬁned in (A5) and part 4 is deﬁned in (A1). Thus, the complete equation is where _R is dR=dt. With this we ﬁnd : (A1) grating over the oil domain, 4 _RiR2 i R3 e lw P Rþ e " # 4 _RiR2 i R3 e lo P R e ð Þ " # ¼ 2rwo Re 4 _RiR2 i R3 e lw lo ð Þ 2rwo Re ¼ P Rþ e P R e ð Þ: Resulting in P Rþ e P R e ð Þ ¼ 4 _RiR2 i R3 e lo lw ð Þ 2rwo Re : (A5) P B ð Þ P A ð Þ q ¼ €RR2 r þ 2 _R 2R r 1 2 _R 2R4 r4 " #B A : Taking the inner bubble radius Ri for R and integrating over the water domain, P Rþ e P1 ¼ qw €RiR2 i þ 2 _R 2 i Ri Re 1 2 _R 2 i R4 i R4 e ! : (A1) Resulting in P Rþ P R ð Þ 4 _RiR2 i l ð P Rþ e P1 ¼ qw €RiR2 i þ 2 _R 2 i Ri Re 1 2 _R 2 i R4 i R4 e ! : (A1) ntegrating over the oil domain, Resulting in P Rþ e P R e ð Þ ¼ 4 _RiR2 i R3 e lo lw ð Þ 2rwo Re : (A5) P Rþ e P1 ¼ qw €RiR2 i þ 2 _R 2 i Ri Re 1 2 _R 2 i R4 i R4 e ! : (A1) ntegrating over the oil domain, Resulting in P Rþ e P R e ð Þ ¼ 4 _RiR2 i R3 e lo lw ð Þ 2rwo Re : (A5) Resulting in Integrating over the oil domain, P R e ð Þ P Rþ i ¼ qo €RiR2 i þ 2 _R 2 i Ri Re 1 2 _R 2 i R4 i R4 e €RiR2 i þ 2 _R 2 i Ri Ri 1 2 _R 2 i R4 i R4 i !! ; (A2) a. Over the oil gas interface Rewriting the equation above then gives Rewriting gives Pg P1 ¼ €Ri qoR2 i 1 Ri 1 Re þ qw R2 i Re " # þqo 2R2 i Ri 1 Ri 1 Re 1 2 _R 2 i R4 i 1 R4 i 1 R4 e " # þqw 2 _R 2 i Ri Re 1 2 _R 2 i R4 i R4 e " # þ 4lo _Ri Ri _RiR2 i R3 e " # þ 4 _RiR2 i R3 e lw þ 2row Re þ 2ro Ri ; (A8) and rewriting further Rewriting gives Rewriting gives 4lo _RiR2 i R3 i P Rþ i þ Pg ¼ 2ro Ri ; Pg P Rþ i ¼ 4lo _Ri Ri þ 2ro Ri : (A4) Rewriting gives Pg P1 ¼ €Ri qoR2 i 1 Ri 1 Re þ qw R2 i Re " # 1 1 1 1 1 " # 4lo _RiR2 i R3 i P Rþ i þ Pg ¼ 2ro Ri ; Pg P Rþ i ¼ 4lo _Ri Ri þ 2ro Ri : Pg P1 ¼ €Ri qoR2 i 1 Ri 1 Re þ qw R2 i Re " # þqo 2R2 i Ri 1 Ri 1 Re 1 2 _R 2 i R4 i 1 R4 i 1 R4 e " # þqw 2 _R 2 i Ri Re 1 2 _R 2 i R4 i R4 e " # þ 4lo _Ri Ri _RiR2 i R3 e " # þ 4 _RiR2 i R3 e lw þ 2row Re þ 2ro Ri ; (A8) 1. Normal component stress tensor 24 October 2024 05:24:01 24 October 2024 05:24:01 where _R is dR=dt. With this we ﬁnd where _R is dR=dt. With this we ﬁnd q 1 r2 @ @t _RR2 ð Þ 2 _RR2 ð Þ2 r5 ! ¼ @P @r þ lr2v; 1 q rð Þ @P @r þ lr2v ¼ 1 r2 d dt _RR2 ð Þ 2 _RR2 ð Þ2 r5 : This equation can be written for both the oil layer and the water. When integrating from r ¼ A to r ¼ B the term lr2v drops out and this gives P B ð Þ P A ð Þ q ¼ 1 r d dt _RR2 ð Þ 1 2 _RR2 ð Þ2 r4 " #B A ; J. Acoust. Soc. Am. 141 (4), April 2017 Lajoinie et al. 2737 Lajoinie et al. 2737 P B ð Þ P A ð Þ q ¼ €RR2 r þ 2 _R 2R r 1 2 _R 2R4 r4 " #B A : 4 _RiR2 i R3 e lw P Rþ e " # 4 _RiR2 i R3 e lo P R e ð Þ " # 2 4 _R R2 2 P B ð Þ P A ð Þ q ¼ €RR2 r þ 2 _R 2R r 1 2 _R 2R4 r4 " #B A : ing the inner bubble radius Ri for R and integrating over water domain, P Rþ e P1 ¼ qw €RiR2 i þ 2 _R 2 i Ri Re 1 2 _R 2 i R4 i R4 e ! b. Over the oil water interface rw ~er ro ~er ¼ dP2; 2lwv0 Re ð Þ P Rþ e 2lov0 Re ð Þ P R e ð Þ ¼ 2rwo Re ; knowing v ¼ _RiR2 i =r2 we also know v0ðReÞ ¼ 2 _RiR2 i =R3 e. Rewriting the equation above then gives and rewriting further knowing v ¼ _RiR2 i =r2 we also know v0ðReÞ ¼ 2 _RiR2 i =R3 e. Rewriting the equation above then gives Re Re Ri and rewriting further and rewriting further 2738 J. Acoust. Soc. Am. 141 (4), April 2017 Lajoinie et al. 2738 J. Acoust. Soc. Am. 141 (4), April 2017 Lajoinie et al. 2738 J. Acoust. Soc. Am. 141 (4), April 2017 Lajoinie et al. Pg P1 ¼ €Ri R2 i Re qw qo ð Þ þ qoRi þqo _R 2 i Ri 2 Ri 2 Re 1 2 1 Ri þ 1 2 R3 i R4 e ! " # þ qw _R 2 i Ri Re 2 1 2 R3 i R3 e ! 2 4 3 5 þ 4lo _Ri Ri _RiR2 i R3 e " # þ 4 _RiR2 i R3 e lw þ 2row Re þ 2ro Ri : (A9) ðqoVoilcpo þ qwVw0:1cpwÞdT ¼ BaðtÞVoildt; with qo the density of the oil, Vw0.1 the volume of the ﬁrst 0.1 lm of water, cpo the heat capacity at constant pressure of the oil and Ba(t) the absorbed laser power (W/m3): with qo the density of the oil, Vw0.1 the volume of the ﬁrst 0.1 lm of water, cpo the heat capacity at constant pressure of the oil and Ba(t) the absorbed laser power (W/m3): dT dt ¼ Ba tð ÞVoil qoVoilcpo þ qwVw0:1cpw ; (A11) ! Tg ¼ ðt 0 Ba tð ÞdtVoil qoVoilcpo þ qwVw0:1cpw ð Þ þ constant: (A12) (A11) To reach the modiﬁed Rayleigh-Plesset equation, Using the initial equilibrium solution, Pg P1 ¼ €Ri R2 i Re qw qo ð Þ þ qoRi þ _R 2 i Ri qo 3 2Ri 2 Re þ 1 2 R3 i R4 e ! þ qw Re 2 1 2 R3 i R3 e ! b. Over the oil water interface " # þ4lo _Ri Ri _RiR2 i R3 e " # þ 4 _RiR2 i R3 e lw þ 2row Re þ 2ro Ri ; (A10) Tg ¼ ðt 0 Ba tð ÞdtVoil qoVoilcpo þ qwVw0:1cpw ð Þ þ Ba;avR2 i;eq 6ko C1o Ri;eq þ C2o þ Troom; Ba,av is the average laser power deposited (W/m3). Filling in C1o and C2o and rewriting gives (A10) Tg ¼ ðt 0 Ba tð ÞdtVoil qoVoilcpo þ qwVw0:1cpw ð Þ þ Ba;avR2 i;eq 6ko Ba;avR2 i;eq 3ko þ Ba;avR3 i;eq 3koRe;eq 1 ko kw þ Ba;avR2 e;eq 3ko 0:5 þ ko kw þ Troom; where the viscosity of water lw is temperature dependent following the following relation:18 where the viscosity of water lw is temperature dependent following the following relation:18 lw ¼ 2:414 105 10247:8=ðT140Þ; 24 October 2024 05:24:01 where T is the temperature of the water at the water-oil inter- face. This new RP equation reduces to the classic RP equa- tion for a bubble with only one liquid around it when the properties of water and oil are chosen to be identical. ! Tg ¼ ðt 0 Ba tð ÞdtVoil qoVoilcpo þ qwVw0:1cpw ð Þ þ Ba;av 3ko 1:5R2 i;eq þ R3 i;eq Re;eq 1 ko kw þ R2 e;eq 0:5 þ ko kw þ Troom: J. Acoust. Soc. Am. 141 (4), April 2017 3. Small variations around equilibrium In this part small variations are added to the static solu- tion in order to obtain a simple model describing the simula- tion result. The static solution assumes the temperature in the gas to be homogeneous. For this to be true for a modu- lated laser signal, the diffusion time of the heat in the gas should be smaller than the half period of the laser modula- tion. This can be contained in the following equations: We know that We know that P ¼ P0V0Tg T0 4 3 pR3 i ; (A15) P ¼ P0V0Tg T0 4 3 pR3 i ; t ¼ R2 i pDg ¼ Period laser 2 ¼ 1 2f ; t ¼ R2 i pDg ¼ Period laser 2 ¼ 1 2f ; (A15) where Tg can be substituted and P is the total pressure as used in the modiﬁed Rayleigh-Plesset equation [Eq. (A10)]: we make the assumption a priori (veriﬁed a posteriori) that the microbubble resonance frequency is in the same range as its acoustic resonance frequency and using a Minnaert approximation, the temperature in the gas can be considered constant for bubbles smaller than Pg P1 ¼ €Ri R2 i Re qw qo ð Þ þ qoRi þ _R 2 i Ri qo 3 2Ri 2 Re þ 1 2 R3 i R4 e ! þ qw Re " 2 1 2 R3 i R3 e !# þ 4lo _Ri Ri _RiR2 i R3 e " # þ 4 _RiR2 i R3 e lw þ 2row Re þ 2ro Ri ; (A16) R ¼ pDg 6:6 11lm: A diffusion distance estimation can be made for the water to ﬁnd app. 0.1 lm for 1 MHz frequency. The change in temperature over time can be described as follows: (A16) Lajoinie et al. 2739 Lajoinie et al. 2739 where Pg is the found P and P1 is the pressure at inﬁnity. Expressing this in the new variables P0 as the pressure at the begin- ning of the static solution and Pg as the total pressure in the gas, and ﬁlling in Tg and the found pressure, this gives P0R3 i;0 T0R3 i ðt 0 Ba tð ÞdtVoil qoVoilcpo þ qwVw0:1cpw þ Ba;av 3ko 1:5R2 i;eq þ R3 i;eq Re;eq 1 ko kw þ R2 e;eq 0:5 þ ko kw ! 3. Small variations around equilibrium þ Troom 2 64 3 75 P1 ¼ €Ri R2 i Re qw qo ð Þ þ qoRi þ _R 2 i Ri qo 3 2Ri 2 Re þ 1 2 R3 i R4 e ! þ qw Re 2 1 2 R3 i R3 e ! " # þ 4lo _Ri Ri _RiR2 i R3 e " # þ 4 _RiR2 i R3 e lw þ 2row Re þ 2ro Ri : (A1 (A17) Organizing for _R; _R 2, and €R and, as an approximation, taking all Ri and Re to be Ri,eq and Re,eq in case they are multi- plied by _R; €R 2, or €R, Organizing for _R; _R 2, and €R and, as an approximation, taking all Ri and Re to be Ri,eq and Re,eq in case they are multi- plied by _R; €R 2, or €R, ð Badt P0V0 T0 4 3 pR3 i;eq qocpo þ qw Vw0:1 Voil cpw 2 64 3 75 \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} a þ P0V0 T0 4 3 pR3 i Tgas;eq þ Troom ½ P1 ¼ €Ri R2 i;eq Re;eq qw qo ð Þ þ qoRi;eq " # \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} b þ _R 2 i Ri;eq qo 3 2Ri;eq 2 Re;eq þ 1 2 R3 i;eq R4 e;eq ! þ qw Re;eq 2 1 2 R3 i;eq R3 e;eq ! " # \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} c _Ri 4 lo Ri;eq þ R2 i;eq R3 e;eq lw lo ð Þ ! " # \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} d þ 2 row Re þ ro Ri : (A18) ð Badt P0V0 T0 4 3 pR3 i;eq qocpo þ qw Vw0:1 Voil cpw 2 64 3 75 \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} a þ P0V0 T0 4 3 pR3 i Tgas;eq þ Troom ½ P1 ¼ €Ri R2 i;eq Re;eq qw qo ð Þ þ qoRi;eq " # \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} b þ _R 2 i Ri;eq qo 3 2Ri;eq 2 Re;eq þ 1 2 R3 i;eq R4 e;eq ! þ qw Re;eq 2 1 2 R3 i;eq R3 e;eq ! 3. Small variations around equilibrium " # \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} c 24 October 2024 05:24:01 24 October 2024 05:24:01 In order to ﬁnd an equation that does not contain an inte- gral, everything is derived to time: aBa 3P0Tg2 T0 Ri;0 R4 i;eq \|ﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄ} f _Ri ¼ Ri ::: b þ 2c €Ri _Ri þ d €Ri þ 2 _Ri row R2 e þ ro R2 i \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} ; (A19) aB f _Ri ¼ Ri ::: b þ 2c €Ri _Ri þ d €Ri þ 2 _Ri: (A20) which has the shape of a transfer function which has the shape of a transfer function which has the shape of a transfer function O I ¼ G 1 þ jx 2z x0 x2 x2 o 1 jx ; (A27) þ 2 _Ri row R2 e þ ro R2 i \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} ; (A19) with G being the gain, O the output, I the input, z the damping and x0 the angular eigen frequency. One thing that can be noted here is that this transfer function is of third order where a standard RP equation would be of sec- ond order. The expected phase difference in our case is therefore p at resonance instead of p/2 such as in the nor- mal RP equation: In which case _Re is assumed to be approximately _Ri and Tgas,eq þ Troom is now called Tg2. The term 2c €Ri _Ri is of higher order and is therefore neglected. Ri is expected to act as an harmonic oscillator and will therefore have the shape of Ri ¼ Ri;eq þ riejxt þ u ¼ Ri;eq þ ri; (A21) _Ri ¼ jxriejxt þ u ¼ ðjxÞri; (A22) €Ri ¼ x2riejxt þ u ¼ ðjxÞ2ri; (A23) Ri ::: ¼ jx3riejxt þ u ¼ ðjxÞ3ri; (A24) aB ¼ jxri 2 þ f þ jwd x2b ; (A25) ! x0 ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2 þ f b s ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2 row R2 e þ ro R2 i þ 3 P0Tg2 T0 Ri;0 R4 i;eq R2 i;eq Re;eq qw qo ð Þ þ qoRi;eq v u u u u u u t : (A28) From Eq. (A15), we can ﬁnd From Eq. (A15), we can ﬁnd 2740 J. Acoust. Soc. Am. 141 (4), April 2017 Lajoinie et al. 2740 J. Acoust. Soc. Am. 141 (4), April 2017 Lajoinie et al 2740 J. Acoust. Soc. Am. 3. Small variations around equilibrium 141 (4), April 2017 x0 ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2 þ f b s ; (A37) ! z ¼ x0 2 d 2 þ f ¼ 1 2 d ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ b 2 þ f ð Þ p ; (A38) z ¼ 1 2 4 lo Ri;eq þ R2 i;eq R3 e;eq lw lo ð Þ ! ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ b 2 þ f ð Þ p : (A39) Tg2R3 i;0 R4 i;eq ¼ T0Pg;eq P0Ri;eq : (A29) x0 ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2 þ f b s ; (A37) Tg2R3 i;0 R4 i;eq ¼ T0Pg;eq P0Ri;eq : (A29) x0 ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2 þ f b s ; Therefore, f can be simpliﬁed f ¼ 3 P0 T0 Tg2Ri;0 R4 i;eq ¼ 3 Pg;eq Ri;eq ; (A30) where the equilibrium pressure Pg,eq is the atmospheric pres- sure plus the Laplace pressure jump over both interfaces, f ¼ 3 Patm Ri;eq þ 2ro R2 i;eq þ 2row Ri;eqRe;eq ! ; (A31) !x0¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2 row R2 e;eq þ ro R2 i;eq þ3 Patm Ri;eq þ 2ro R2 i;eq þ 2row Ri;eqRe;eq ! R2 i;eq Re;eq qwqo ð ÞþqoRi;eq v u u u u u u u t ; (A32) (A31) (A33) (A33) 1. The mesh The simulation is based on a time dependent mesh. The mass of each grid volume is deﬁned to be constant and the grid is recalculated every time step to hold on to this deﬁni- tion. Initially, before the laser is turned on and before the bubble starts to oscillate, the grid is deﬁned with a regular size interval of 31.25 nm up to a radius of 6 lm which is twice the typical bubble radius. Beyond this radius the grid gets 15% bigger each step outward. This way, without hav- ing an excessive amount of grid points, the most outer grid point, kept at room temperature is more than 3000 times the typical bubble radius making negligible the thermal drift due to a ﬁnite simulation volume. 2. The ideal gas law In order to ﬁnd an expression for the pressure, we look at the ideal gas law, PV ¼ m ~l RgT; PV ¼ m ~l RgT; where P is pressure, V is volume, m is mass, ~l is the molar mass in kilograms per mole, Rg is the ideal gas constant. The simulation is deﬁned such that the mass in each grid-volume remains constant in time: where P is pressure, V is volume, m is mass, ~l is the molar mass in kilograms per mole, Rg is the ideal gas constant. The simulation is deﬁned such that the mass in each grid-volume remains constant in time: PV T ¼ constant tð Þ: (B1) PV T ¼ constant tð Þ: (B1) (B1) (A32) x0 is not a function of time so all Ri and Re are now Ri,eq and Re,eq: ! x0 ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 4ro R2 i;eq þ 2row 1 R2 e;eq þ 3 Ri;eqRe;eq ! þ 3Patm Ri;eq R2 i;eq Re;eq qw qo ð Þ þ qoRi;eq v u u u u u u u t ; (A33) ! x0 ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 Ri;eq 4ro Ri;eq þ2row Ri;eq R2 e;eq þ 3 Re;eq ! þ3Patm ! Ri;eq Ri;eq Re;eq qw qo ð Þþqo v u u u u u u t ; (A34) ! x0 ¼ 1 Ri;eq ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 4ro Ri;eq þ 2row Ri;eq R2 e;eq þ 3 Re;eq ! þ 3Patm Ri;eq Re;eq qw qo ð Þ þ qo v u u u u u u t ; (A35) ! x0 ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 4ro R2 i;eq þ 2row 1 R2 e;eq þ 3 Ri;eqRe;eq ! þ 3Patm Ri;eq R2 i;eq Re;eq qw qo ð Þ þ qoRi;eq v u u u u u u u t ; (A33) 24 October 2024 05:24:01 (A35) The speed of the bubble wall _R is much smaller than the speed of sound in air or water. Therefore the pressure in the bubble is considered homogeneous. With this information it can be shown that the pressure is deﬁned by which altogether is an expression for the angular eigenfre- quency as a function of Ri,eq and Re,eq. This shows the eigen- frequency is inversely related to the bubble size but also shows that the oil layer thickness plays a role. The denomi- nator under the square root shows an inertial shift of the res- onance curve: because oil and water have different densities, the thickness of the oil layer inﬂuences the mass to be dis- placed and therefore the resonance frequency. PVk Tk ¼ constant tð Þ ! P X Ri 0 Vk Tk ¼ constant tð Þ ! P X Vk Tk ¼ P0 V0 T0 ¼ P0 T0 4 3 pR3 0; P ¼ P0 T0 4 3 pR3 0 1 X Vk=Tk ; Now to ﬁnd an expression for the damping. According to Eq. (A27), 2z x0 ¼ d 2 þ f ; (A36) (A36) where P Vk=Tk can be written as where P Vk=Tk can be written as where P Vk=Tk can be written as Lajoinie et al. 2741 Lajoinie et al. 2741 J. Acoust. Soc. Am. 141 (4), April 2017 J. Acoust. Soc. Am. 141 (4), April 2017 Lajoinie et al. 2741 X Vk Tk ¼ X 4 3 p rk1 þ pk ð Þ3 r3 k1 Tk ; 3. Water and oil grids The mass in each grid volume is deﬁned to be constant over time, the oil and the water are assumed incompressible and therefore the volume in each grid volume in the oil and water domains is constant in time, where subscript 0 stands for the initial value before the laser is turned on. The constant pressure condition within the bubble writes rkþ1 ¼ ð½ððk þ 1Þp0Þ3 ðkp0Þ3 þ r3 kÞ1=3; Vk mkTk ¼ constant rð Þ: Vk mkTk ¼ constant rð Þ: Vk mkTk ¼ constant rð Þ: which can be Taylor expanded to give rkþ1 ¼ rk 1 þ 1 3 H 1 9 H2 þ 10 162 H3 ; Further rewriting and using that mk is constant in time, gives Vk Tk 1 q0V0k ¼ 1 q0Tk Vk V0k ¼ 1 q0Tk r3 k r3 k1 kp0 ð Þ3 k 1 ð Þp0 3 ¼ Rg ~lP; r3 k r3 k1 ¼ Rg ~lPq0Tk kp0 ð Þ3 k 1 ð Þp0 3 h i ; rk1 ¼ Rg ~lPq0Tk kp0 ð Þ3 k 1 ð Þp0 3 h i þ r3 k 1=3 ; with H ¼ k þ 1 ð Þp0 ð Þ3 kp0 ð Þ3 h i1 rk 3 : (B3) (B3) The heat convection diffusion equation writes The heat convection diffusion equation writes where rk is the radius in meters that belongs to grid point k. To speed up the calculation in MATLAB or Fortran, an approximation based on a Taylor expansion is made, Dr2T þ I qcp ¼ DT Dt ; !1=3 rk1 ¼ Rg ~lPq0Tk rk3 rk3 kp0 ð Þ3 k1 ð Þp0 3 h i þr3 k !1=3 ; rk1 ¼rk Rg ~lPq0Tk 1 rk3 kp0 ð Þ3 k1 ð Þp0 3 h i þ1 1=3 : where I is in units of W/m3. Since the simulation calculates the temperature for each grid point, and the grid points fol- low the movement of the ﬂuid particles, the simulation is performed in the lagrangian referential which allows for treating the total derivative as a local one. The heat equation can be approximated with a central difference scheme in space and a forward ﬁnite difference scheme in time, 24 October 2024 05:24:01 We now assume 1 to be the dominant term, which is true for small spatial steps, We now assume 1 to be the dominant term, which is true for small spatial steps, 1 r2 @ @r r2 @T @r ¼ 1 r2 @ @r r2 Tkþ1 Tk pkþ1 ¼ 1 r2 k @ @r rk þ 1 2 pkþ1 2 Tkþ1 Tk pkþ1 ! : rk1 ¼ rk 1 þ 1 3 H 1 9 H2 þ 10 162 H3 with H ¼ Rg ~lP q0Tk 1 rk3 kp0 ð Þ3 k 1 ð Þp0 3 h i : (B2) Approximating even further gives ference scheme: H ¼ Rg ~lP q0Tk 1 rk3 kp0 ð Þ3 k 1 ð Þp0 3 h i : (B2) Approximating even further gives a second order central dif- ference scheme: Approximating even further gives a second order central dif- ference scheme: Approximating even further gives a second order central dif- ference scheme: @2T @r2 ¼ 1 r2 k rk þ 1 2 pkþ1 2 Tkþ1 Tk pkþ1 ! rk1 þ 1 2 pk 2 Tk Tk1 pk ! 5. Second order precision over the gas-oil interface and the oil-water interface 5. Second order precision over the gas-oil interface and the oil-water interface ko @To @r Re ¼ kw @Tw @r Re : The heat equation that was deﬁned above can be used as long as the temperature is known at grid points k þ 1, k and k 1. When crossing the interface between gas and oil, or between oil and water the heat equation must be deﬁned separately. These equations will be based on a second order Taylor expansion and are therefore more precise than the heat equation for the bulk. This is required for a satisfactory stability of the simulation. We therefore need to know the derivative of the temperature with respect to the radius. To ﬁnd this we look for T0 k ¼ ATk þ BTkþ1 þ CTkþ2. A, B, and C should therefore be such that the coefﬁcient of Tk is zero, that of T0 k is one and that of T00 k is zero. This results in A þ B þ C ¼ 0; A þ B þ C ¼ 0; pkþ1B þ pkþ1 þ pkþ2 ð ÞC ¼ 1; p2 kþ1 2 B þ pkþ1 þ pkþ2 ð Þ2 2 C ¼ 0; The heat convection diffusion equation writes Second order precision over the gas-oil interface and the oil-water interface ko @To @r Re ¼ kw @Tw @r Re : In the simulation it can now be used that the new temperature at grid point k in terms of the temperature at t dt in the grid points k 1, k, k þ 1 is as follows: In the simulation it can now be used that the new temperature at grid point k in terms of the temperature at t dt in the grid points k 1, k, k þ 1 is as follows: Tnþ1 k ¼ Tn k1 dtD r2 k rk1 þ 1 2 pk 2 pk 1 2 pk þ 1 2 pkþ1 2 6664 3 7775 þ Tn k 1 dtD r2 k 1 2 pk þ 1 2 pkþ1 rk þ 1 2 pkþ1 2 pkþ1 þ rk1 þ 1 2 pk 2 pk 0 B @ 1 C A 2 66664 3 77775 þTn kþ1 dtD r2 k 1 2 pk þ 1 2 pkþ1 rk þ 1 2 pkþ1 2 pkþ1 2 6664 3 7775 þ dtI qcp : (B6) Tnþ1 k ¼ Tn k1 dtD r2 k rk1 þ 1 2 pk 2 pk 1 2 pk þ 1 2 pkþ1 2 6664 3 7775 þ Tn k 1 dtD r2 k 1 2 pk þ 1 2 pkþ1 rk þ 1 2 pkþ1 2 pkþ1 þ rk1 þ 1 2 pk 2 pk 0 B @ 1 C A 2 66664 3 77775 þTn kþ1 dtD r2 k 1 2 pk þ 1 2 pkþ1 rk þ 1 2 pkþ1 2 pkþ1 2 6664 3 7775 þ dtI qcp : (B6) (B6) 24 October 2024 05:24:01 24 October 2024 05:24:01 J. Acoust. Soc. Am. 141 (4), April 2017 The heat convection diffusion equation writes 1 2 pk þ 1 2 pkþ1 2 6664 3 7775: The derivative of the temperature with respect to time can be given by The derivative of the temperature with respect to time can be given by The derivative of the temperature with respect to time can be given by @T @t ¼ Tnþ1 k Tn k dt : @T @t ¼ Tnþ1 k Tn k dt : The full heat equation now becomes 2742 J. Acoust. Soc. Am. 141 (4), April 2017 Lajoinie et al. D r2 k rk þ 1 2 pkþ1 2 Tkþ1 Tk pkþ1 ! rk1 þ 1 2 pk 2 Tk Tk1 pk ! 1 2 pk þ 1 2 pkþ1 2 6664 3 7775 þ I qcp ¼ Tnþ1 k Tn k dt : (B4) This can be rearranged D r2 k rk þ 1 2 pkþ1 2 Tkþ1 Tk pkþ1 ! rk1 þ 1 2 pk 2 Tk Tk1 pk ! 1 2 pk þ 1 2 pkþ1 2 6664 3 7775 þ I qcp ¼ Tnþ1 k Tn k dt : (B4) hi b d (B4) This can be rearranged Tnþ1 k ¼ dtD r2 k rk þ 1 2 pkþ1 2 Tkþ1 Tk pkþ1 ! rk1 þ 1 2 pk 2 Tk Tk1 pk ! 1 2 pk þ 1 2 pkþ1 2 6664 3 7775 þ dtI qcp þ Tn k: (B5) (B5) Tnþ1 k ¼ dtD r2 k pkþ1 ! pk ! The heat convection diffusion equation writes 1 2 pk þ 1 2 pkþ1 664 775 þ dtI qcp þ Tk: (B5) In the simulation it can now be used that the new temperature at grid point k in terms of the temperature at t dt in the grid points k 1, k, k þ 1 is as follows: Tnþ1 k ¼ Tn k1 dtD r2 k rk1 þ 1 2 pk 2 pk 1 2 pk þ 1 2 pkþ1 2 6664 3 7775 þ Tn k 1 dtD r2 k 1 2 pk þ 1 2 pkþ1 rk þ 1 2 pkþ1 2 pkþ1 þ rk1 þ 1 2 pk 2 pk 0 B @ 1 C A 2 66664 3 77775 þTn kþ1 dtD r2 k 1 2 pk þ 1 2 pkþ1 rk þ 1 2 pkþ1 2 pkþ1 2 6664 3 7775 þ dtI qcp : (B6) 5. a. Outer side of an interface F ¼ 1 pk þ pk1 ð Þ2 pk pk þ pk1 ð Þ ; B ¼ pkþ1 þ pkþ2 ð Þ p2 kþ1 pkþ1 pkþ1 þ pkþ2 ð Þ ; B ¼ pkþ1 þ pkþ2 ð Þ p2 kþ1 p2 kþ1 pkþ1pkþ2 ; B ¼ pkþ1 þ pkþ2 ð Þ pkþ1pkþ2 : and thus, and thus, Now ﬁlling in B and C to ﬁnd A, Now ﬁlling in B and C to ﬁnd A, , E ¼ pk þ pk1 ð Þ2 p2 k 1 pk þ pk1 ð Þ2 pk pk þ pk1 ð Þ ; E ¼ pk þ pk1 ð Þ p2 k 1 pk þ pk1 ð Þ pk 1 ; E ¼ pk þ pk1 ð Þ pk pk þ pk1 ð Þ p2 k ; E ¼ pk þ pk1 ð Þ pkpk1 : A ¼ B C; A ¼ pkþ1 þ pkþ2 ð Þ pkþ1pkþ2 1 pkþ1 þ pkþ2 ð Þ pkþ1 þ pkþ2 ð Þ2 pkþ1 ; A ¼ pkþ1 þ pkþ2 ð Þ pkþ1pkþ2 1 pkþ1 þ pkþ2 ð Þ pkþ1 þ pkþ2 ð Þ2 pkþ1 ; B ¼ pkþ1 þ pkþ2 ð Þ pkþ1pkþ2 ; C ¼ 1 pkþ1 þ pkþ2 ð Þ pkþ1 þ pkþ2 ð Þ2 pkþ1 : A ¼ B C; A ¼ pkþ1 þ pkþ2 ð Þ pkþ1pkþ2 1 pkþ1 þ pkþ2 ð Þ pkþ1 þ pkþ2 ð Þ2 pkþ1 ; A ¼ pkþ1 þ pkþ2 ð Þ pkþ1pkþ2 1 pkþ1 þ pkþ2 ð Þ pkþ1 þ pkþ2 ð Þ2 pkþ1 ; B ¼ pkþ1 þ pkþ2 ð Þ pkþ1pkþ2 ; C ¼ 1 pkþ1 þ pkþ2 ð Þ pkþ1 þ pkþ2 ð Þ2 pkþ1 : A ¼ B C; A ¼ pkþ1 þ pkþ2 ð Þ pkþ1pkþ2 1 pkþ1 þ pkþ2 ð Þ pkþ1 þ pkþ2 ð Þ2 pkþ1 ; A ¼ pkþ1 þ pkþ2 ð Þ pkþ1pkþ2 1 pkþ1 þ pkþ2 ð Þ pkþ1 þ pkþ2 ð Þ2 pkþ1 ; 24 October 2024 05:24:01 24 October 2024 05:24:01 Using D ¼ E F and ﬁlling in E and F, D ¼ pk þ pk1 ð Þ pkpk1 1 pk þ pk1 ð Þ2 pk pk þ pk1 ð Þ : Finally, D ¼ pk þ pk1 ð Þ pkpk1 1 pk þ pk1 ð Þ2 pk pk þ pk1 ð Þ ; E ¼ pk þ pk1 ð Þ pkpk1 ; F ¼ 1 pk þ pk1 ð Þ2 pk pk þ pk1 ð Þ : D ¼ pk þ pk1 ð Þ pkpk1 1 pk þ pk1 ð Þ2 pk pk þ pk1 ð Þ : Finally, D ¼ pk þ pk1 ð Þ pkpk1 1 pk þ pk1 ð Þ2 pk pk þ pk1 ð Þ ; E ¼ pk þ pk1 ð Þ pkpk1 ; F ¼ 1 pk þ pk1 ð Þ2 pk pk þ pk1 ð Þ : a. Outer side of an interface Just like in the heat equation for the bulk, we express the new temperature Tnþ1 k (just beyond the interface) in terms of the temperature at t dt on three grid points Tn k; Tn k 1, and Tn kþ2: solving this solving this p2 kþ1 2 B ¼ pkþ1 þ pkþ2 ð Þ2 2 C; B ¼ pkþ1 þ pkþ2 ð Þ2 p2 kþ1 C Tk ¼ Tk: Using a Taylor expansion up to second order to get enough precision Tkþ1 ¼ Tk þ pkþ1T0 k þ p2 kþ1 2 T00 k ; Tkþ2 ¼ Tk þ pkþ1 þ pkþ2 ð ÞT0 k þ pkþ1 þ pkþ2 ð Þ2 2 T00 k : Tkþ1 ¼ Tk þ pkþ1T0 k þ p2 kþ1 2 T00 k ; and Tkþ2 ¼ Tk þ pkþ1 þ pkþ2 ð ÞT0 k þ pkþ1 þ pkþ2 ð Þ2 2 T00 k : pkþ1B þ pkþ1 þ pkþ2 ð ÞC ¼ 1; pkþ1 pkþ1 þ pkþ2 ð Þ2 p2 kþ1 C þ pkþ1 þ pkþ2 ð ÞC ¼ 1; The heat ﬂux across the oil water interface must be con- served leading to the following condition: Lajoinie et al. 2743 Lajoinie et al. 2743 J. Acoust. Soc. Am. 141 (4), April 2017 C pkþ1 þ pkþ2 ð Þ pkþ1 þ pkþ2 ð Þ2 pkþ1 ! ¼ 1 ! C ¼ 1 pkþ1 þ pkþ2 ð Þ pkþ1 þ pkþ2 ð Þ2 pkþ1 D þ E þ F ¼ 0; pkE pk þ pk1 ð ÞF ¼ 1; p2 k 2 E þ pk þ pk1 ð Þ2 2 F ¼ 0; solving this p2 k 2 E ¼ pk þ pk1 ð Þ2 2 F; E ¼ pk þ pk1 ð Þ2 p2 k F and thus, and thus, and pkE pk þ pk1 ð ÞF ¼ 1; pk pk þ pk1 ð Þ2 p2 k F pk þ pk1 ð ÞF ¼ 1; F pk þ pk1 ð Þ2 pk pk þ pk1 ð Þ ! ¼ 1; ! b. Inner side of an interface Finally, Finally, Until now we considered the temperature to be known for grid points k or higher. This is the case when looking at the outer side of a boundary. For looking at the inner side of a boundary grid points bigger than k are not known and the same analysis can be done for this side. The results are an expression like T0 k ¼ ATk þ BTk1 þ CTk2 Where D, E, and F are D ¼ pk þ pk1 ð Þ pkpk1 1 pk þ pk1 ð Þ2 pk pk þ pk1 ð Þ ; E ¼ pk þ pk1 ð Þ pkpk1 ; F ¼ 1 pk þ pk1 ð Þ2 pk pk þ pk1 ð Þ : Tk ¼ Tk; Tk1 ¼ Tk pkT0 k þ p2 k 2 T00 k ; Tk2 ¼ Tk pk þ pk1 ð ÞT0 k þ pk þ pk1 ð Þ2 2 T00 k ; c. Resulting interface conditions in the simulation with k being the gridpoint on the boundary between water and oil. Similarly, the boundary condition between the oil and the gas is py p p 10R. Qin, J. Xu, R. Xu, C. Kim, and L. V. Wang, “Fabricating multifunc- tional microbubbles and nanobubbles for concurrent ultrasound and photo- acoustic imaging,” in Proceedings SPIE (2010). 24 October 2024 05:24:01 g g g 11J. D. Dove, M. A. Borden, and T. W. Murray, “Optically induced reso- nance of nanoparticle-loaded microbubbles,” Opt. Lett. 39, 3732–3735 (2014). kg @Tg @r Ri ¼ ko @To @r Ri ; kg @Tg @r Ri ¼ ko @To @r Ri ; ( ) 12T. Faez, M. Emmer, K. Kooiman, M. Versluis, A. F. W. van der Steen, and N. de Jong, “20 years of ultrasound contrast agent modeling,” IEEE T. Ultrason. Ferr. 60, 7–20 (2013). with Ri being the radius of the bubble at the gas–oil inter- face. Rearranging this gives with Ri being the radius of the bubble at the gas–oil inter- face. Rearranging this gives 13N. de Jong, M. Emmer, A. van Wamel, and M. Versluis, “Ultrasonic char- acterization of ultrasound contrast agents,” Med. Biol. Eng. Comput. 47, 861–873 (2009). Tk ¼ kg ETk1 þ FTk2 ð Þ þ ko BTkþ1 þ CTkþ2 ð Þ kgD koA : 14D. Cosgrove and C. Harvey, “Clinical uses of microbubbles in diagnosis and treatment,” Med. Biol. Eng. Comput. 47, 813–826 (2009). 15 15D. Cosgrove, “Ultrasound contrast agents: An overview,” Eur. J. Radiol. 60, 324–330 (2006). 16K. Firouzi, E. Stride, and N. Saffari, “A theoretical investigation of photoacoustic contrast agents,” J. Acoust. Soc. Am. 133, 3853–3862 (2013). c. Resulting interface conditions in the simulation This simulation is performed for a 3 lm radius bubble with a 1 lm thick triacetin oil layer and a continuous laser exposure and (b) simulation result of the same bubble irradiated with a laser intensity modu- lated at 1 MHz, showing the quasi con- vergence of the gas temperature after 100 ls. 5Y. Wang, X. Xie, X. Wang, G. Ku, K. L. Gill, D. P. O’Neal, G. Stoica, and L. V. Wang, “Photoacoustic tomography of a nanoshell contrast agent in the in vivo rat brain,” Nano Lett. 4, 1689–1692 (2004). 6C. Kim, E. C. Cho, J. Chen, K. H. Song, L. Au, C. Favazza, Q. Zhang, C. M. Cobley, F. Gao, Y. Xia, and L. V. Wang, “In vivo molecular photo- acoustic tomography of melanomas targeted by bio-conjugated gold nano- cages,” ACS Nano. 4, 4559–4564 (2010). 5Y. Wang, X. Xie, X. Wang, G. Ku, K. L. Gill, D. P. O’Neal, G. Stoica, and L. V. Wang, “Photoacoustic tomography of a nanoshell contrast agent in the in vivo rat brain,” Nano Lett. 4, 1689–1692 (2004). 6C. Kim, E. C. Cho, J. Chen, K. H. Song, L. Au, C. Favazza, Q. Zhang, C. M. Cobley, F. Gao, Y. Xia, and L. V. Wang, “In vivo molecular photo- acoustic tomography of melanomas targeted by bio-conjugated gold nano- cages,” ACS Nano. 4, 4559–4564 (2010). with Re being the radius of the bubble at the oil water inter- face. Filling in for what was found in Appendix B 5, with Re being the radius of the bubble at the oil water inter- face. Filling in for what was found in Appendix B 5, 7H. Huang, C. He, Y. Zeng, X. Xia, X. Yu, P. Yi, and Z. Chen, “Preparation and optical properties of worm-like gold nanorods,” J. Colloid Interf. Sci. 322, 136–142 (2008). koðDTk þ ETk1 þ FTk2Þ ¼ kwðATk þ BTkþ1 þ CTkþ2Þ: , ( ) 8E. M. Strohm, M. Rui, M. C. Kolios, I. Gorelikov, and N. Matsuura, “Optical droplet vaporization (ODV): Photoacoustic characterization of perﬂuorocarbon droplets,” in Proceedings IEEE Ultrasonics Symposium (2010), pp. 495–498. Rearranging gives Rearranging gives Tk ¼ ko ETk1 þ FTk2 ð Þ þ kw BTkþ1 þ CTkþ2 ð Þ koD kwA ; 9J. D. Dove, P. A. Mountford, T. W. Murray, and M. A. Borden, “Engineering optically triggered droplets for photoacoustic imaging and therapy,” Biomed. Opt. Express 5, 4417–4427 (2014). c. Resulting interface conditions in the simulation The boundary condition between the oil and the water is the following: 2744 J. Acoust. Soc. Am. 141 (4), April 2017 2744 J. Acoust. Soc. Am. 141 (4), April 2017 2744 J. Acoust. Soc. Am. 141 (4), April 2017 2744 J. Acoust. Soc. Am. 141 (4), April 2017 ko @To @r Re ¼ kw @Tw @r Re ; with Re being the radius of the bubble at the oil water inter- face. Filling in for what was found in Appendix B 5, koðDTk þ ETk1 þ FTk2Þ ¼ kwðATk þ BTkþ1 þ CTkþ2Þ: Rearranging gives Tk ¼ ko ETk1 þ FTk2 ð Þ þ kw BTkþ1 þ CTkþ2 ð Þ koD kwA ; 5Y. Wang, X. Xie, X. Wang, G. Ku, K. L. Gill, D. P. O’Neal, G. Stoica, and L. V. Wang, “Photoacoustic tomography of a nanoshell contrast agent in the in vivo rat brain,” Nano Lett. 4, 1689–1692 (2004). 6C. Kim, E. C. Cho, J. Chen, K. H. Song, L. Au, C. Favazza, Q. Zhang, C. M. Cobley, F. Gao, Y. Xia, and L. V. Wang, “In vivo molecular photo- acoustic tomography of melanomas targeted by bio-conjugated gold nano- cages,” ACS Nano. 4, 4559–4564 (2010). 7H. Huang, C. He, Y. Zeng, X. Xia, X. Yu, P. Yi, and Z. Chen, “Preparation and optical properties of worm-like gold nanorods,” J. Colloid Interf. Sci. 322, 136–142 (2008). 8E. M. Strohm, M. Rui, M. C. Kolios, I. Gorelikov, and N. Matsuura, “Optical droplet vaporization (ODV): Photoacoustic characterization of perﬂuorocarbon droplets,” in Proceedings IEEE Ultrasonics Symposium (2010), pp. 495–498. 9J. D. Dove, P. A. Mountford, T. W. Murray, and M. A. Borden, FIG. 9. (Color online) (a) Convergence of the FDM simulation result towards the static solution. This simulation is performed for a 3 lm radius bubble with a 1 lm thick triacetin oil layer and a continuous laser exposure and (b) simulation result of the same bubble irradiated with a laser intensity modu- lated at 1 MHz, showing the quasi con- vergence of the gas temperature after 100 ls. FIG. 9. (Color online) (a) Convergence of the FDM simulation result towards the static solution. APPENDIX C: FDM MODEL CONVERGENCE TOWARD THE STATIC SOLUTION ( ) 17R. Shih, D. Bardin, T. D. Martz, P. S. Sheeran, P. A. Dayton, and A. P. Lee, “Flow-focusing regimes for accelerated production of monodisperse drug-loadable microbubbles toward clinical-scale applications,” Lab. Chip 13 4816 4826 (2013) Please see Fig. 9. drug-loadable microbubbles toward clinical-scale applications,” Lab. Chip 13, 4816–4826 (2013). 13, 4816–4826 (2013). 18T. T. Al. Shemmeri, Engineering Fluid Dynamics (Ventus Publishing, Telluride, CO, 2012), Chap. 1. 1P. Beard, “Biomedical photoacoustic imaging,” Interface Focus 1, 602–631 (2011). 2 19A. Prosperetti, “A generalization of the Rayleigh–Plesset equation of bub- ble dynamics,” Phys. Fluids 25, 409–410 (1982). 2K. Wilson, K. Homan, and S. Emelianov, “Biomedical photoacoustics beyond thermal expansion using triggered nanodroplet vaporization for contrast-enhanced imaging,” Nat. Commun. 3, 618 (2012). 20M. Minnaert, “On musical air-bubbles and the sound of running water,” Philos. Mag. 16, 235–248 (1933). 3W. Lu, Q. Huang, G. Ku, X. Wen, M. Zhou, D. Guzatov, P. Brecht, R. Su, A. Oraevsky, L. V. Wang, and C. Li, “Photoacoustic imaging of living mouse brain vasculature using hollow gold nanospheres,” Biomaterials 31, 2617–2626 (2010). 21C. C. Church, “The effects of an elastic solid surface layer on the radial pulsations of gas bubbles,” J. Acoust. Soc. Am. 97, 1510–1521 (1995). 22J. B. Keller and M. Miksis, “Bubble oscillations of large amplitude,” J. Acoust. Soc. Am. 68, 628–633 (1980). 4S. Mallidi, S. Kim, A. Karpiouk, P. P. Joshi, K. Sokolov, and S. Emelianov, “Visualization of molecular composition and functionality of cancer cells using nanoparticle-augmented ultrasound-guided photo- acoustics,” Photoacoustics 3, 26–34 (2015). 23A. Prosperetti and A. Lezzi, “Bubble dynamics in a compressible liquid. Part 1. First-order theory,” J. Fluid Mech. 168, 457–478 (1986). Lajoinie et al. 2745 J. Acoust. Soc. Am. 141 (4), April 2017
https://openalex.org/W3116990551	https://zenodo.org/record/61302/files/22_IJRG16_C08_105.pdf	English	null	THE IMPACTS OF CLIMATE CHANGE ON LIVESTOCK PRODUCTION AND PRODUCTIVITIES IN DEVELOPING COUNTRIES: A REVIEW	International journal of research - granthaalayah	2,016	cc-by	3,840	THE IMPACTS OF CLIMATE CHANGE ON LIVESTOCK PRODUCTION AND PRODUCTIVITIES IN DEVELOPING COUNTRIES: A REVIEW Getachew Bekele Fereja 1 1 Department of Animal Science, College of Agriculture and Natural Resource, Gambella i i 6 b ll Getachew Bekele Fereja 1 1 Department of Animal Science, College of Agriculture and Natural Resource, Gambella University, PO Box 126, Gambella, ETHIOPIA DOI: 10.5281/zenodo.61302 ABSTRACT It is true that most proportions of Africans livestock production and productivities are declined when the climate condition is not comfortable. Therefore this review was conducted to show the impacts of climate change on livestock production and productivities. The climate change especially global warming may highly influence production performance of farm animals throughout the world. While there has been occasional mention, in the global discussion on climate change there has generally been a dearth of attention paid to the animal production and productivities impacts of the proposed abatement options and some of the suggested livestock management approaches would have severe and wide-ranging impacts on the animal’s products. This is on the background that the impact of climate change on crops is well known, much less is known about the impact of climate change on livestock. Unfortunately, livestock production and productivities assumes an overriding emphasis as one of the core sectors to solve the current food challenges and bringing future food sustainability in developing countries. Out of all the factors influencing livestock production, climate, and location are undoubtedly the most significant. Climate change will have far-reaching consequences for animal production, especially in vulnerable parts of the world where it is vital for nutrition and livelihoods. The impact of climate change can heighten the vulnerability of livestock systems and exacerbate existing stresses upon them, such as drought. Parasites and diseases are among the most severe factors that impact livestock production and productivity. Animal diseases have great impact on food supplies, trade and commerce, and human health globally. Animal genetic diversity is critical for food security and rural development. It allows farmers to select stocks or develop new breeds in response to changing conditions. ISSN- 2350-0530(O) ISSN- 2394-3629(P) IF: 4.321 (CosmosImpactFactor), 2.532 (I2OR) ISSN- 2350-0530(O) ISSN- 2394-3629(P) IF: 4.321 (CosmosImpactFactor), 2.532 (I2OR) [Fereja , Vol.4 (Iss.8): August, 2016] Science 1. INTRODUCTION Agriculture is the back bone of most African countries economy. The agricultural sector is the largest domestic producer across the continent and employs about 70% to 90% of the total labor force. Moreover this sector supplies up to 50% of household food demand and up to 50% of their income. In addition most of the income generated by livestock rearing such as dairy cattle, beef cattle, sheep, goat and chickens. In most rural communities livestock is the only asset of the poor. But it is highly susceptible to climate variability extremes. The effect of climate change is anticipated to heighten the susceptibility of livestock system and reinforce the existing factors that are affecting livestock production systems. Therefore the objective of this review is to review the impact of climate change on livestock production and productivities. Climate change is actually the most important environmental issue of any time. With severe and widespread destructive effects, warming of the planet threatens ecological systems, peoples’ livelihoods, and species survival. Animal production under agriculture is an important source of greenhouse gas (GHG) emissions and has been implicated as a serious contributor to climate change. The climate impacts anticipated for developing country are similar to those being experienced around the world: general warming (day and night temperatures all year round); changes in rainfall timing and quantities; changes in seasons (longer summers); increased climate variability (e.g. floods, droughts and heat waves); higher sea-levels; and increasing frequency and intensity of extreme weather events (IPPC, 2007). While there has been occasional mention, in the global discussion on climate change there has generally been a dearth of attention paid to the animal production and productivities impacts of the proposed abatement options, and some of the suggested livestock management approaches would have severe and wide-ranging impacts on the animal’s products. This is on the background that the impact of climate change on crops is well known, much less is known about the impact of climate change on livestock. Unfortunately, livestock production and productivities assumes an overriding emphasis as one of the core sectors to solve the current food challenges and bringing future food sustainability in developing countries. The intergovernmental Panel on Climate Change (IPCC, 2007) is now overwhelmingly convinced that climate change is real, that it will become worse, and that the poorest regions and small scale famers will be the worst affected. Keywords: y impact, livestock, climate, production. y impact, livestock, climate, production. Cite This Article: Getachew Bekele Fereja, “THE IMPACTS OF CLIMATE CHANGE ON LIVESTOCK PRODUCTION AND PRODUCTIVITIES IN DEVELOPING COUNTRIES: A REVIEW” International Journal of Research - Granthaalayah, Vol. 4, No. 8 (2016): 181-187. Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [181-1 [181-187] [181-187] ISSN- 2350-0530(O) ISSN- 2394-3629(P) IF: 4.321 (CosmosImpactFactor), 2.532 (I2OR) [Fereja , Vol.4 (Iss.8): August, 2016] 1. INTRODUCTION The impact of climate change will call for a balanced type of animal which can produce in stressful environment as a result the utilization of adaptive livestock genotypes which may sustain production and productivities in this regard. Climate change taking the centre stage the merit of indigenous livestock apart from their ability to adapt to stressful environment are a valuable asset to the smallholder farmers as they provide their basic needs. On the other hand, it is believed that livestock production and productivities will be one of the most susceptible sectors to climate change due to changes in hydrological cycle, temperature balance and rainfall patterns which have a negative impact on livestock production and productivity (Mwiturubani, 2010). The direct impacts of air temperature, humidity and wind speed capable of influencing growth rate, milk production, wool production and reproduction have been reported by (Houghton, 2001) and (Rust and Rust (2013). Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [181-187] [Fereja , Vol.4 (Iss.8): August, 2016] ISSN- 2350-0530(O) ISSN- 2394-3629(P) IF: 4.321 (CosmosImpactFactor), 2.532 (I2OR) 2. MEASURING THE IMPACTS OF CLIMATE CHANGE ON ANIMAL PRODUCTION AND PRODUCTIVIES All stocks however, showed varying degrees of vulnerability to diseases associated with climate variability. Cattle, Sheep Goats Equine and Camels recorded the maximum vulnerability for diseases. For floods, owners noted that large animals had some advantage, due to their ability to float/swim in water channels, as it is by their high adaptability. All other stocks are extremely vulnerable. During review time which was noted that during at the time of floods, camels and small ruminants are almost totally immobile and thus, cannot access pasture areas for their feeds. In general this contributed highly to animal losses. In the face of climate challenges, adaptation of different livestock species to tropical conditions becomes highly imperative. A report by the FAO’s Committee on Genetic Resources for Food and Agriculture (CGRFA, 2009) noted that the management of animals under natural selection by pastoralists in marginal areas plays an essential role in their adaptation and fitness in such environments. 3. THE IMPACTS OF CLIMATE CHANGE ON LIVESTOCK PRODUCTION AND PRODUCTIVITIES The direct effects of air temperature, humidity and wind speed capable of influencing growth rate, milk production, wool production and reproduction have been reported by (Houghton, 2001, Rust, 2013). The quantity and quality of livestock feed stuffs such as pasture and forage can be indirectly affected by climate change (McCarthy et al., 2001) significantly influencing farmers` livestock selection choices. Also, the severity and distribution of livestock diseases and parasites is conditioned by climate change (Thornton and Herrero, 2008). The challenges of climate change will call for a balanced type of animal which can produce in stressful environment as a result the utilization of adaptive livestock genotypes which may sustain production in this regard. Climate change taking the centre stage the merit of indigenous livestock apart from their ability to adapt to stressful environment are a valuable asset to the smallholder farmers as they provide their basic needs. 5. IMPACT OF CLIMATE CHANGE ON ANIMAL FEED RESOURCES Out of all the factors influencing livestock production, climate, and location are undoubtedly the most significant. In fact, climatology characteristics such as ambient temperature and rainfall patterns have great influence on pasture and food resources availability cycle throughout the year among animal populations. As discussed before, the rain pattern during the year strongly conditions livestock production systems through pasture development and disease and parasites outbreaks, therefore influencing animal production systems, productivity. Tropical and Mediterranean climates are characterized by the existence of a season of varied duration, when rainfall is scanty or non-prevalent. Such season is termed dry season in the tropics and summer in Mediterranean climates. During rainy season pastures are available in higher quantities and show good nutritional quality whereas dry season’s pastures have poor nutritional quality with high fiber and low protein contents (Butterworth 1984), which often results in declining the animal production. 4. IMPACTS OF CLIMATE CHANGE ON ANIMAL PRODUCTION Climate change will have far-reaching consequences for animal production, especially in vulnerable parts of the world where it is vital for nutrition and livelihoods. The impact of climate change can heighten the vulnerability of livestock systems and exacerbate existing stresses upon them, such as drought. Ensuring good animal welfare will be paramount to addressing these challenges; breeds suited to the local environment are often more robust and resilient than industrially farmed breeds (www.fao.org/docrep/meeting/022/mb386e.pdf (accessed 28 November 2011)).There is a range of thermal conditions within which animals are able to maintain a relatively stable body temperature by means of behavioral and physiological means (Champaign, IL 61820, pp: 609-618). Heat stress results from the animal’s inability to dissipate sufficient heat to maintain homoeothermic. Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [181-187] [Fereja , Vol.4 (Iss.8): August, 2016] ISSN- 2350-0530(O) ISSN- 2394-3629(P) IF: 4.321 (CosmosImpactFactor), 2.532 (I2OR) 6. IMPACT OF DISEASES AND PARASITES ON LIVESTOCK PRODUCTIVITY DUE TO CLIMATE CHANGE Parasites and diseases are among the most severe factors that impact livestock production and productivity. Animal diseases have great impact on food supplies, trade and commerce, and human health globally. The last few decades have seen a general reduction in the burden of livestock diseases. Such reduction is the direct result of the availability and effectiveness of drugs and vaccines, as well as improvements in diagnostic technologies (Pearson 2006; Thornton 2010). Future disease trends are likely to be effectively managed by disease surveillance and control technologies. At the same time, new diseases have emerged and will continue to spread by the international movement of animals and animal products, such as avian influenza. This disease has caused considerable global concern about the potential for a change in host species from poultry to man and an emerging global pandemic of human influenza (Murray 2006; Thornton 2010). Livestock diseases can cause direct losses (deaths, stunting, reduced fertility, and changes in herd structure) and indirect losses (additional costs for drugs and vaccines, added labor costs and profit losses due to denied access to better markets and use of suboptimal production technology) in revenue (Rushton 2009). Large ruminants are generally regarded as the most important domestic livestock species in the world. The importance is demonstrated by the list of products they provide. In developed countries, their contributions are mainly restricted to commercial products such as meat and milk. In developing countries they are a source of food, particularly protein for human diets, and they provide income, employment, transport, can serve as a store of wealth, and provide draft power and organic fertilizer for crop production (Perry et al. 2005; Rushton 2009). 7. IMPACT OF CLIMATE CHANGE ON ANIMAL GENETICS RESOURCE Animal genetic diversity is critical for food security and rural development. It allows farmers to select stocks or develop new breeds in response to changing conditions, including climate change, new or resurgent disease threats, new knowledge of human nutritional requirements, and [181-187] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH ISSN- 2350-0530(O) ISSN- 2394-3629(P) IF: 4.321 (CosmosImpactFactor), 2.532 (I2OR) [Fereja , Vol.4 (Iss.8): August, 2016] changing market conditions or changing societal needs – all of which are largely unpredictable. What is predictable is increased future human demand for food. The effects will be most acute in developing countries, where the increase in demand is expected to be greatest, and occur at a rate faster than increases in production (FAO, 2003; 2006a), and where climate change is projected to have its greatest impact. changing market conditions or changing societal needs – all of which are largely unpredictable. What is predictable is increased future human demand for food. The effects will be most acute in developing countries, where the increase in demand is expected to be greatest, and occur at a rate faster than increases in production (FAO, 2003; 2006a), and where climate change is projected to have its greatest impact. Most flows of genetic material occur among developed countries, most of which are without zoosanitary restrictions, and involve animals suited to high-input production systems (Valle- Zarate et al., 2006; Hiemstra et al., 2007). More than 90% of exports originate from developed countries, and the share of trade in genetic material from developed to developing countries increased from 20% in 1995 to 30% in 2005 (Gollin et al., 2008). In many cases, the improved components of the high-input management systems needed to express the genetic potential of the high-output breeds have been transferred to developing countries. Industrial systems utilizing sophisticated technology and based on internationally sourced feed and animal genetics already produce 55% of pork, 68% of eggs and 74% of poultry meat globally (FAO, 2003; Steinfeld et al., 2006). Climate change will affect the products and services provided by agricultural biodiversity. But this biodiversity has not yet been properly integrated into strategies for adaptation to and mitigation of climate change. Its role in the resilience of food systems still needs to be addressed. 8. CONCLUSION The review reported here addressed the very complex issue of impacts of climate change livestock production and productivities. Climate change could affect animal production and well-being, especially because of increases in air temperature. However, the knowledge of animal responses to heat stress during the hot months in several areas of the world, as well as during extreme heat events, may be used to evaluate the impacts of global change. However, farmers are not quite aware about the impacts global warming can produce in their operation. At some point the people have experienced drought, and all this points to a changing climate. Livestock production is under threat from the changing climate. This is because the natural pastures which a majority of the livestock owners rely on for feeding their animals are deteriorating in quality and the amount of fodder available. In addition to that, water sources available are not reliable as they sometimes dry up due to high temperatures and shortage of rainfall. Livestock has been lost due to among other factors excessive heat, shortage of water and feed and unknown diseases. Therefore, the following recommendations are forwarded for future action.  Training about impact of climate change must be given for the livestock ow  The choice of representative climate stations for livestock enterprises, particularly in the arid and semi-arid regions must be considered.  The choice of representative climate stations for livestock enterprises, particularly in the arid and semi-arid regions must be considered.  The recordation of additional inputs to pasture and livestock production, especially in climatically favorable zones must be applied.  The recordation of additional inputs to pasture and livestock production, especially in climatically favorable zones must be applied. 7. IMPACT OF CLIMATE CHANGE ON ANIMAL GENETICS RESOURCE Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [181-187] ISSN- 2350-0530(O) ISSN- 2394-3629(P) IF: 4.321 (CosmosImpactFactor), 2.532 (I2OR) [Fereja , Vol.4 (Iss.8): August, 2016] 7. IMPACT OF CLIMATE CHANGE ON ANIMAL GENETICS RESOURCE The Intergovernmental Panel on Climate Change (IPCC) report on biological diversity (Gitay et al., 2002) and likewise the report of the Convention on Biological Diversity on climate change (CBD, 2009) contain little mention of agricultural biodiversity, and a recent literature review (Campbell et al., 2009) largely ignores livestock diversity. In a survey on threats to livestock diversity (FAO, 2009c), climate change was only mentioned as a minor factor in the context of extensive land-based production systems. The findings show that many stakeholders do not yet perceive climate change as a problem for the management and conservation of livestock biodiversity. While substantial differences in thermal tolerance lie between species, there are also differences between breeds of a species. Ruminants generally have a higher degree of thermal tolerance than monogastric species, but species and breed environmental envelopes overlap. The ability to thermoregulate depends on complex interactions among anatomical and physiological factors. Factors such as properties of the skin and hair, sweating and respiration capacity, tissue insulation, the relationship between surface area per unit body weight or relative lung size, endocrinological profiles and metabolic heat production are known to influence heat loads, but the underlying physiological, behavioural or genetic mechanisms are largely unknown (Hall, 2004; McManus et al., 2008). While substantial differences in thermal tolerance lie between species, there are also differences between breeds of a species. Ruminants generally have a higher degree of thermal tolerance than monogastric species, but species and breed environmental envelopes overlap. The ability to thermoregulate depends on complex interactions among anatomical and physiological factors. Factors such as properties of the skin and hair, sweating and respiration capacity, tissue Factors such as properties of the skin and hair, sweating and respiration capacity, tissue insulation, the relationship between surface area per unit body weight or relative lung size, endocrinological profiles and metabolic heat production are known to influence heat loads, but the underlying physiological, behavioural or genetic mechanisms are largely unknown (Hall, 2004; McManus et al., 2008). With increasing milk yield in dairy cattle, growth rates and leanness in pigs or poultry, metabolic heat production has increased and the capacity to tolerate elevated temperatures has declined (Zumbach et al., 2008; Dikmen & Hansen, 2009). In the long term, single-trait selection for yields will therefore result in animals with lower heat tolerance. [9] FAO (2009a). Status and trends report on animal genetic resources – 2008. CGRFA/WG- AnGR-5/09/Inf. 7. [10] Food Agricultural Organisation (FAO), 2007. Climate Change: Climate Change Impacts, Adaptation and Vulnerability. IPCC WG II Forht Assessment Report. [11] Framework Convention on Climate Change, Technical Paper. 2008. Available online: Genetic components of heat stress in finishing pigs: Genetic resources for the farming of the future. Blackwell, 264pp [12] Harper, R.; House, J.; Jafari, M.; et al. Agriculture, forestry, and other land use (AFOLU). In Hiemstra, S.J., A.G. Drucker, M.W. Tvedt, N. Louwaars, J.K. Oldenbroek K. Awgichew, S. Abegazhttp://unfccc.int/resource/docs/2008/tp/08.pdf (accessed on 14 May 2015). IFAD, Rome. [13] International Fund for Agricultural Development (IFAD)., 2011. Rural Poverty Report, final report,Gerber, P.J.; Steinfeld, H.; Henderson, B.; Mottet, A.; Opio, C.; Dijkman, J.; Falcucci, A.; Tempio, G. Tackling Climate Change through Livestock—A Global Assessment of Emissions and Mitigation Opportunities; Food and Agriculture Organization of the United Nations: Rome. [14] IPCC, Impacts, adaptation and vulnerability: Contributions of Working Group II to the Fourth Assessment Report, Cambridge University Press, 2007, 444. Italiana 42:319–336 Italy, 2013. [15] J.M. Rust, T. Rust, Climate change and livestock production: A review with emphasis on Africa. S. Afri. J. Anim. Sci. 43(3), 2013. [16] J.T. Houghton, L.G. Meira Filho, B. Lim, K. Treanton, I. Mamaty, Y. Bonduki, D.J. Griggs, and B.A. Callender, (Eds). Revised 1996 IPCC Guidelines for National Greenhouse Gas Inventories, IPCC/OECD/IEA, 1997. available from http://www.ipcc- nggip.iges.or.jp/public /gl/ invs1 .htm, IPCC NGGIP, Japan. [17] Journal of Animal Science, 86 (9), 2082 - 2088. Kebede, P.N. Bhat, A. da Silva Mariante, 2007. What’s on the menu? Options for strengthening the lactating dairy cows in a subtropical environment? Journal of Dairy Science. 92, 109-116. [18] linkages between biodiversity and climate change adaptation. A review of the recent scientific literature. UNEP World Conservation Monitoring Centre. livestock products with fair market access for the poor (Pro-Poor Livestock Policy Initiative London. management. Am. Dairy Sci. Assoc., Champaign, IL 61820, pp: 609-618. [19] McManus C., Prescott E., Paludo G.R., Bianchini E., Louvandini H. & Mariante A.S. (2008). [20] Murray N (2006) International trade and the spread of animal diseases: assessing the risks. Vet Italiana 42:319–336 [21] Pearson JE (2006) Worldwide risks of animal diseases: introduction. Vet Ita [21] Pearson JE (2006) Worldwide risks of animal diseases: introduction. Vet Italiana 42:293 [22] Place, S.E.; Mitloehner, F.M. The nexus of environmental quality and livestock welfare. Annu. Rev. Anim. Biosci. 2014, 2, 555–569. ( ) f [22] Place, S.E.; Mitloehner, F.M. 9. REFERENCES [1] Animal Genetic Resources Information 41, 65-74. assessment. Livestock Science 120 (3), 248-255. biodiversity. IPCC Technical Paper V. IPCC, Geneva, Switzerland. pp 85. Biol Sci 365:2853–2867. [1] Animal Genetic Resources Information 41, 65-74. assessment. Livestock Science 120 (3), 248-255. biodiversity. IPCC Technical Paper V. IPCC, Geneva, Switzerland. pp 85. Biol Sci 365:2853–2867. [2] Brunner, S., Eickemeier, P., et al., Eds.; Cambridge University Press: New York, NY, USA, 2014; pp. 811–922. [3] Bucklin, R.A., Hahn, G.L., Beede, D.K., Bray, D.R. 1992. Physical facilities for Butterworth MH (1984) Beef cattle nutrition and tropical pastures. Longman, London (England) [4] Campbell A., Kapos V., Chenery A., Kahn, S.I., Rashid M., Scharlemann J.P.W. & Dickson B. (2009). The. Carr DH (1984) The regulation of parotid and submandibular salivary secretion in sheep. 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Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [181-187] [Fereja *, Vol.4 (Iss.8): August, 2016] ISSN- 2350-0530(O) ISSN- 2394-3629(P) IF: 4.321 (CosmosImpactFactor), 2.532 (I2OR) [9] FAO (2009a). Status and trends report on animal genetic resources – 2008. CGRFA/WG- AnGR-5/09/Inf. 7. [9] FAO (2009a). Status and trends report on animal genetic resources – 2008. CGRFA/WG- AnGR-5/09/Inf. 7. The nexus of environmental quality and livestock welfare. Annu. Rev. Anim. Biosci. 2014, 2, 555–569. [23] Summary for policymakers. In Climate Change 2014: Impacts, Adaptation, and Vulnerability. Part A: Global and Sectoral Aspects. [24] Thornton PK (2010) Livestock production: recent trends, future prospects. Philos Trans R Soc B Biol Sci 365:2853–2867.warm climates. In: Van Horn, H.H.; Wilcox, C.J. (eds.). R Soc B Biol Sci 365:2853–2867.warm climates. In: Van Horn, H.H.; Wilcox, C.J. (eds.). [25] Zumbach, B., Misztal I. Tsuruta S., Sanchez J. 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https://openalex.org/W3085413090	https://ieeexplore.ieee.org/ielx7/6287639/8948470/09189856.pdf	English	null	Automation of Dynamic Power Management in FPGA-Based Energy-Constrained Systems	IEEE access	2,020	cc-by	7,912	Received August 27, 2020, accepted September 6, 2020, date of publication September 9, 2020, date of current version September 22, 2020. Received August 27, 2020, accepted September 6, 2020, date of publication September 9, 2020 date of current version September 22, 2020. Digital Object Identifier 10.1109/ACCESS.2020.3022955 Digital Object Identifier 10.1109/ACCESS.2020.3022955 INDEX TERMS Design automation, energy efﬁciency, FPGA design, Internet of Things, power management. INDEX TERMS Design automation, energy efﬁciency, FPGA design, Internet of Things, power management. FPGA technology as a prototyping platform, for functional veriﬁcation and characterization of hardware designs that will be later fabricated as ASIC chips. To ‘‘try-out’’ some new circuit is just not feasible in ASIC technology, mainly due to time and cost resources to manufacture single or a few chips. Modern FPGAs can be very complex circuits, which often integrate various special-purpose components (e.g. multipli- ers, memories, specialized processors) to support as many applications as possible (to widen market of their own). These additional components are not always used (in each conﬁgu- ration); however, they increase power requirements of FPGA devices. under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ VOLU Received August 27, 2020, accepted September 6, 2020, date of publication September 9, 2020, date of current version September 22, 2020. MICHAL ŠKUTA, DOMINIK MACKO , (Member, IEEE), AND KATARÍNA JELEMENSKÁ, (Member, IEEE) , ( , ) Faculty of Informatics and Information Technologies, Slovak University of Technology in Bratislava, 842 16 Bratislava, Slovakia Corresponding author: Dominik Macko (dominik.macko@stuba.sk) This work was support in part by the Ministry of Education, Science, Research and Sport of the Slovak Republic (Incentives for Research and Development, under Grant 2018/14427:1-26C0, and in part by the Eset Research Centre. ABSTRACT The era of the Internet of Things comes with a huge number of interconnected communicating devices, which are often rather limited on the energy supply (e.g. battery powered or energy harvesting). Therefore, the pressure on energy efﬁciency of their operation (inﬂuencing lifetime or amount of functions) is especially crucial. In spite of a growing number of IoT devices, there still are many applications that are very speciﬁc and their market is quite limited. This is where the FPGAs offer a good alternative to dedicated application-speciﬁc chips, which would be too costly for such a purpose. Therefore, we target the power-management automation that simpliﬁes energy-efﬁcient design for FPGA platforms. A designer is then able to specify just an abstract power management in the commonly used SystemC model and it is automatically transformed in the more-complex form acceptable by a speciﬁc FPGA device. The proposed simpliﬁcation and automation shortens the time-to-market of energy-efﬁcient IoT products and prevents possible human-errors that could be otherwise introduced to the design. The alleviated veriﬁcation and debugging spare even more time in the development process. The experiments have proved the beneﬁts of the proposed automation method. I. INTRODUCTION Thus, a high amount of manual effort was still required to successfully ﬁnish power-management integration into the design. efﬁciency – if not for low-power application market, then to reduce costs, or at least for sustainability reasons. The usage of well-known power-reduction techniques, such as power gating of voltage scaling, is limited by the hardware architec- ture of the FPGA device itself. For example, a multivoltage design technique can be used only if the FPGA platform supports multiple voltages and the power-gating technique cannot be used in the application design if not enabled by the FPGA device. Nowadays, the most efﬁcient power-reduction techniques are commonly applied by some sort of dynamic power management, enabling to control power in various parts of the system depending on current tasks executed by the application (i.e. during runtime). However, it complicates the design and is often just too difﬁcult to be applied by inexperienced designers. Therefore, various power-reduction techniques are not used, even if they could be, wasting the valuable energy. p g g g In this work, we extend our previous results [1] into a new method, which enables to analyze the abstract ESL speciﬁcation of the power-managed system described in Sys- temC/PMS [26] and automatically synthesizes Verilog model of the PMU that enables to scale the frequency of the system, as well as other required logic, such as synchronization ele- ments between clock domains of the system. In comparison to [1], which proposed the method and provided early experi- mental veriﬁcation of manual power-management techniques application to an FPGA-based design, this work is accom- panied by the fully functional automation tool that has automatically synthesized the power management compara- ble to the previously used manual one (a golden model). The key contribution of the work is that the synthesized power management is application-speciﬁc (i.e. optimized, to accomplish just the intent speciﬁed in SystemC/PMS), instead of the use of general-purpose power controllers. Just to be clear, this article does not introduce a new power-reduction technique, it focuses on simpliﬁed applica- tion of the existing popular techniques that are rather com- plicated for introduction into an FPGA-based system design. Usage of abstract power-management speciﬁcation signiﬁ- cantly simpliﬁes the ESL modeling, and thus shortens the design time. Moreover, the automated transition from ESL to RTL (Register-Transfer Level) abstraction levels prevents possible human errors that could be introduced to the design, and thus reduces debugging effort. I. INTRODUCTION The proposed method simpliﬁes and speeds-up the design process of low-power and energy-efﬁcient FPGA-based systems. This way, it enables even designers unfamiliar with power-reduction techniques to target energy efﬁciency, making their products to run longer on batteries, integrate more functions into the products while not exceeding energy limits, and contribute to sustainability. gy There are various research works focused on increasing the energy efﬁciency of FPGA-based systems. For example, a modiﬁcation of the place-and-route process can reduce power requirements of the FPGA interconnect, as proposed in [9]. This method is useful when synthesizing the applica- tion design for the selected FPGA platform; however, it can- not optimize the power consumption during runtime. Another method [10] utilizes the autonomous power-gating technique in a ﬁne-grained manner, targeting LUTs (Look-Up Tables) in asynchronous FPGAs. It thus focuses on the FPGA-chip architecture rather than on reducing power in the application design. A similar focus was preferred by [11] that uses power gating in so-called mega cells to optimize power require- ments. However, it also cannot be utilized for dynamic power management by the application design. Another modiﬁcation of the FPGA architecture was proposed in [12]. Although it enables a sort of power management, it cannot be used on commercial FPGA devices. Dynamic power management, applying software-driven power gating, was used in [13], which targeted the Xilinx ZYNQ platform. However, it can- not be used on devices that do not contain any embedded processor in addition to the FPGA chip. A method utiliz- ing clock domains and algorithm partitioning was applied in [14]. Although such architectural modiﬁcations can indeed help to reduce power requirements, they do not enable man- aging power dynamically during runtime. There also exist FPGA-based methods that enable to estimate power con- sumption of system prototypes [15], or methods that demon- strate beneﬁts of various technologies in low-power system design [16]. Neither one although enables dynamic power management. Incorporating dynamic power management is deﬁnitely a challenge for design teams, since it substantially increases complexity of the system. To improve design productivity of complex systems, the International technology roadmap for semiconductors [17] suggested the adoption of higher abstraction in the design process. The ESL (Electronic System Level) progressively becomes the industrial design starting point, especially in FPGA-based systems [18]–[22]. The remaining part of the article is structured as follows. The next section includes an overview of the back- ground, regarding the SystemC/PMS speciﬁcation. I. INTRODUCTION The market forces regarding cost reduction and time- to-market of the products increase popularity of the FPGA (Field-Programmable Gate Array) devices. These devices can be programmed to perform some function in hardware (i.e. at hardware speed); however, they are premanufactured, thus the hardware designers do not need to wait for their designs to be manufactured as a chip to integrate them in their products. Therefore, FPGAs are often used for hard- ware acceleration of some speciﬁc tasks. In contrast to ASIC (Application-Speciﬁc Integrated Circuit) or PLD (Pro- grammable Logic Device) chips, the hardware function of the FPGA device can be changed by its reconﬁguration. This is very useful for space applications (e.g. in satellites or in space-exploration missions devices), since it provides a way to update the device function remotely [2]–[4]. The recon- ﬁguration possibility also widens a high interest of using the Energy costs, as well as the green-computing ideology, put the energy efﬁciency of the hardware designs to the forefront [5], [6]. Also, the rapidly growing market of the Internet of Things (IoT) strictly requires low-power operation of the devices limited by the energy source (e.g. battery powered or energy harvesting) [7], [8]. Therefore, the energy demands of FPGA-based systems have to be reduced as much as possible and each hardware design should focus on energy The associate editor coordinating the review of this manuscript and approving it for publication was Kim-Kwang Raymond Choo . 165894 VOLUME 8, 2020 VOLUME 8, 2020 M. Škuta et al.: Automation of Dynamic Power Management in FPGA-Based Energy-Constrained Systems Although the ESL-based design methods deﬁnitely increase the productivity in programming FPGAs, they tend to omit the energy point in the high-level synthesis, focusing on the functional aspect of the design. There were some methods already developed for adoption of power manage- ment into ESL speciﬁcation [23]–[26]. However, all exist- ing methods are focused on automatically generating power intent at lower levels in a standard format, suitable only for the ASIC technology. In our previous work, we have already outlined the idea of power-management automation in FPGA-based systems [27]. It was mainly focused on the power-management unit (PMU) automated generation, based on an ESL speciﬁcation. However, important parts of the power management, such as clock gating, isolation cells, or other support logic, have not been generated. FIGURE 1. A partial example of power-management specification in SystemC/PMS. FIGURE 1. A partial example of power-management specification in SystemC/PMS. its constructor. The special variable POWER_MODE holds information about the current power mode of the system, and it can be switched in some other SystemC process of the functional model. It must be noted that SystemC/PMS represents just an abstract speciﬁcation of power management, it does not model the effects of power management. It means that it spec- iﬁes ‘‘what’’ should be achieved, but ‘‘how’’ it is achieved must be modeled and implemented at lower abstraction levels. TABLE 1. The predefined abstract power states in SystemC/PMS. TABLE 1. The predefined abstract power states in SystemC/PMS. TABLE 1. The predefined abstract power states in SystemC/PMS. As previously mentioned, the SystemC/PMS speciﬁcation is based on UPF concepts. The original goal was to simplify power-intent speciﬁcation by abstracting from unnecessary details at the system level, and then automatically synthe- size a more-complex equivalent speciﬁcation in UPF. How- ever, the UPF standard is intended for ASIC-based chips (Application Speciﬁc Integrated Circuits) and its concepts are not easily applicable to FPGA-based systems. The whole power-management synthesis algorithm must be adjusted to this kind of devices. II. SystemC/PMS SPECIFICATION y The abstract power-management speciﬁcation in SystemC/ PMS was introduced in [26]. It is based on the standard power concepts deﬁned by the UPF (Uniﬁed Power Format) standard [28], which are introduced into a system-level model described in SystemC [29] in an easy-to-use way. The key utilized concepts include power states, power domains, power modes, and power policy. There are ﬁve abstract states pre- deﬁned by SystemC/PMS, which are summarized in Table 1 along with the corresponding power-reduction techniques that are expected to be applied by the power states. A power domain groups multiple components that are always operat- ing in the same power states (the state changes simultaneously in all components). Power modes represent allowed combina- tions of power states in all power domains (one power state for each power domain) – i.e. like a system-wide power state. The power policy speciﬁes when and how the system switches between power modes. Unlike in UPF, the SystemC/PMS abstract power state is not limited to the speciﬁcation of the supply-voltage level only, but also the frequency level. Such a frequency-voltage pair is called a performance level, which must be speciﬁed for each active power state (i.e. either NORMAL or DIFF_LEVEL). I. INTRODUCTION Section III describes the proposed power-management automation method for FPGA-based systems. In Section IV, the experi- mental results supporting the beneﬁts of the proposed method 165895 165895 VOLUME 8, 2020 M. Škuta et al.: Automation of Dynamic Power Management in FPGA-Based Energy-Constrained Systems are reported and discussed. And in the last section, the work is concluded. are reported and discussed. And in the last section, the work is concluded. FIGURE 1. A partial example of power-management specification in SystemC/PMS. II. SystemC/PMS SPECIFICATION III. THE PROPOSED POWER-MANAGEMENT AUTOMATION An initial idea of adopting power-management simpliﬁcation beneﬁts offered by SystemC/PMS to FPGA-based system was presented in [27]. This work extends the previous idea by automated synthesis of FPGA-supported power-management execution logic, such as clock gating, isolation or synchro- nization elements, in addition to the power-management unit. The proposed low-power FPGA-based application design ﬂow is illustrated in Fig. 2. The red dashed line marks the process in the design ﬂow, targeted by the proposed automa- tion method (the ﬂow step 4b in the ﬁgure). Similarly to the ASIC-based ﬂow [26], we expect the design process to start at the highly abstract ESL, as a crude model speciﬁ- cation (step 1). The abstraction-reﬁnement process (step 2) To illustrate how such SystemC/PMS speciﬁcation of architectural power management looks like, we provide an example in Fig. 1. The abstract power management is spec- iﬁed in the top module called example_top. The declara- tion part of the module contains a declaration of available power domains and power modes. However, a speciﬁcation of power states for power domains and power modes, along with the assignment of components to the power domains, is contained in the functional part of the module, such as 165896 VOLUME 8, 2020 VOLUME 8, 2020 M. Škuta et al.: Automation of Dynamic Power Management in FPGA-Based Energy-Constrained Systems FIGURE 2. The proposed low-power FPGA-based application design flow. FIGURE 3. Components of the automation tool. to obtain user-deﬁned inputs via a user interface (either FIGURE 2. The proposed low-power FPGA-based application design flow. FIGURE 3. Components of the automation tool. FIGURE 3. Components of the automation tool. FIGURE 2. The proposed low-power FPGA-based application design flow. to obtain user-deﬁned inputs via a user interface (either GUI - Graphical User Interface or CLI - Command Line Interface), redirects them to the other components, and pro- vides the synthesized model to the user again via a user interface. is then used to specify abstract power management directly into the functional model using SystemC/PMS (step 3). Since the abstract power-management speciﬁcation does not affect the system function, the designer does not need to worry that it will corrupt the simulation results (the optional step A). The designer can rely on the previously developed abstract power-management static analysis [30] to validate the speciﬁcation. III. THE PROPOSED POWER-MANAGEMENT AUTOMATION Just before the functional high-level synthe- sis (HLS) takes place (step 4b), the power-management HLS extracts the power-related information from the ESL model (step 4a), in order to be synthesizable by commonly used HLS tools (e.g. Vivado HLS). After the functional RTL model is synthesized, the power-management HLS automatically generates the appropriate power-management components and integrates them into the functional model (step 5). The Analyzer component is used to load the input ESL speciﬁcation of the system model in SystemC/PMS, extract the power-related data from the speciﬁcation, and ﬁll them into an intermediate structure. For this structure to be kept in a simple and easy-to-use form (for a human as well as for a computer), we have proposed a JSON-based format of the internal structure, as illustrated in Fig. 4. The synthesized power-managed RTL model can then be veriﬁed during a functional simulation (the optional step B). After logical synthesis and place and route process (step 6), the design can be analyzed for power consumption and resource utilization (the optional step B). The analyzed infor- mation (the optional step C) can be then used (while taking into account the trade-off between power, performance, and area) to adjust the abstract power-management speciﬁcation (step 3) and resynthesize the model (steps 4 and 5). If the functionality was not modiﬁed, the functional HLS process (step 4b) does not need to be run again. This speeds-up power-management exploration. The proposed method was implemented into a tool, called pmuToFPGA, which automates the power-management syn- thesis process. It was implemented in the Python program- ming language, version 3.7, using IDE editor PyCharm CE at the Mac OS X platform. Since Python is a multiplatform language, the tool is also usable at different platforms, such as Linux or Windows. The modular architecture of the tool is illustrated in Fig. 3. It consists of ﬁve separate components with dedicated functions. Such a modular design makes the tool ﬂexible for extension and future modiﬁcations. VOLUME 8, 2020 FIGURE 4. An example of the internal JSON structure. The Controller component interconnects all the other components and enables to exchange data among them in an efﬁcient and meaningful way. For example, it enables Firstly, Analyzer has to ﬁnd all the speciﬁed performance levels in SystemC/PMS model. The information about the VOLUME 8, 2020 VOLUME 8, 2020 165897 165897 M. Škuta et al.: Automation of Dynamic Power Management in FPGA-Based Energy-Constrained Systems 5. The Mako-based template for PMU synthesis. power state name, the supply voltage, and the frequency value, is stored. Since the performance levels of inactive power states (i.e. HOLD, OFF, OFF_RET) are not speciﬁed explicitly, the Analyzer must deduce them. Then, the Ana- lyzer component ﬁnds all the speciﬁed power domains, along with the assigned components and the allowed power states for each domain. Also, all the signals interconnecting the components of the modeled system must be indexed, and the information about which components are connected and the direction of the communication are stored. It is inevitable for synchronization and isolation purposes. Last but not least, Analyzer must ﬁnd all the speciﬁed power modes and store the information about the selected power states for each domain in each mode. The analyzed data in the internal JSON structure are then forwarded to the Generator component. Using the internal structure, Generator synthesizes the power-management unit, driving all the clock signals according to the speciﬁcation, as well as the control signals for other power-management elements. Generator also synthesizes the power-management support logic, such as synchronizers. For the code synthesis, the Mako-based templates [31] are used. To show an example, a portion of the template for the PMU synthesis is provided in Fig. 5. It is used to create the main body of the synthesized PMU; however, individual parts of the PMU are synthesized by utilization of another more-complex template (the whole synthesis code is available on GitHub [32]). The Generator component is conﬁgured by a conﬁguration ﬁle, which is used by the designer to set some synthesis parameters for a speciﬁc FPGA device (e.g. whether the PLL module should be used or not, or what are the acceptable boundaries of the generated clock frequencies). An example of the conﬁguration ﬁle is provided in Fig. 6. The synthe- sized PMU uses the PLL unit, if use_pll attribute is acti- vated. Input parameters for the PLL to generate a speciﬁc frequency are computed using Algoritm 1. FIGURE 4. An example of the internal JSON structure. If the PLL cannot generate the clock frequency in the speciﬁed boundaries, the divider is synthesized to adjust the frequency value of the main clock signal (the divide_clock attribute must be activated). The divide_pll attribute enables dividing the PLL output signal. The main clock signal has precedence before the PLL-generated signal. Using the strict_freq and all_freq attributes, it is possible to specify whether the power domains are limited only to the frequencies deduced from the speci- ﬁed abstract power states, or they can use all the generated frequencies. FIGURE 5. The Mako-based template for PMU synthesis. must be created by the designer – their creation is not yet automated. After the synthesis of PMU and synchronizers, the Generator component must ﬁnd a suitable place in the top module of the modeled system for their automated inte- gration. According to the direction of a speciﬁc signal of some component, it is renamed to the signal generated by the synchronization element (e.g. signal_synced). The conﬁguration ﬁle also enables to activate the reconﬁg- uration support. The synthesized PMU is then able to not only switch between power modes, but also switch between mul- tiple conﬁgurations. It is usable, for example, as a replace- ment for unsupported power gating by some FPGA devices (like the selected FPGA device iCE40 for our prototype). Instead of powering down some component, it is possible to switch to the alternative design, not including that com- ponent. Although this functionality is fully supported by the synthesized PMU, the alternative designs (i.e. conﬁgurations) After the power-managed model is automatically synthe- sized, a few manual modiﬁcations are required. The designer needs to ﬁnd the place in the code, where the synthesized power-management modules have been inserted. To simplify the code search, we have identiﬁed such a place by a com- mentary of ‘‘Start of auto-generated components PMU + synchronizers’’. Since the main clock signal can have various 165898 165898 VOLUME 8, 2020 M. Škuta et al.: Automation of Dynamic Power Management in FPGA-Based Energy-Constrained Systems to connect a suitable signal to the FlagIn_clkA port. There is an optional FlagOut_clkB port that informs about new value at the BusOut output, which can also be connected. Another optional output port is Busy_clkA, which can be connected to inform the input power domain (generating the signal) that the signal has not been processed yet by the output domain. FIGURE 6. An example of configuration file contents. FIGURE 6. An example of configuration file contents. Algorithm 1 An Algorithm to Calculate PLL Parameters to Generate a Given Frequency 1~best_fout = 0 2~for divr in range(16): 3 f_pfd = device.clk_freq / (divr + 1) 4 for divf in range(128): 5 f_vco = f_pfd * (divf + 1) 6 for divq in range(1, 7): 7 fout = f_vco * math.pow(2, -divq) 8 if math.fabs(fout - freq_setting.frequency) < math.fabs(best_fout - freq_setting.frequency) or not found_something: 9 best_fout = fout 10 best_divr = divr 11 best_divf = divf 12 best_divq = divq Algorithm 1 An Algorithm to Calculate PLL Parameters to Generate a Given Frequency IV. RESULTS AND DISCUSSION FIGURE 4. An example of the internal JSON structure. All modules have a reset signal, preset to the static zero value. It is recommended to replace it so that the signal is driven dynamically. In the prototype, we have activated reset signal upon the start for the period of one clock cycle and the system worked correctly. The last required modiﬁcation is to connect clock signals of the functional modules to the clock signals generated for their power domains. To assist with this, the commentaries are generated in the code, e.g. ‘‘Change clock to pd_clk_0’’. All of the speciﬁed functional and non-functional require- ments have been met, which can be summarized as follows. The developed tool is able to load SystemC/PMS speciﬁca- tion and extract the information about power management. It is able to synthesize PMU that applies power-reduction techniques supported by the target FPGA device. The tool is multi-platform and is able to run by various operating systems. The tool is available as an easy to use tool offering both, command line and graphical, user interfaces. Besides, the created source code can be used as a module for other Python projects. The tool supports simple user information about the current state (i.e. progress) and it is also able to generate a detailed SystemC/PMS analysis report. Conﬁg- urability of the tool is achieved mainly by the JSON-based conﬁguration ﬁle. IV. RESULTS AND DISCUSSION There were three performance levels speciﬁed. Other models described small variations of the basic model. For example, an additional power state was used or there was another power domain with an extra component speciﬁed. The intermediate JSON ﬁles with extracted power-management information have then been manually checked whether they correspond to the speciﬁcations. The result of this part of the experiments is that the analysis and extraction features of the tool worked correctly. represents a signiﬁcant simpliﬁcation for the power-managed FPGA-applications design process. To evaluate the proposed method in real hardware, we have selected the Lattice iCEstick Evaluation Kit [33] (the iCE40 device, speciﬁcally suitable for low-power designs), mainly due to good support by open-source tools and small purchase costs. A drawback of the selected FPGA is its sup- port of just a single voltage level for the application logic. Due to this fact, we were limited to clock gating and frequency scaling techniques to increase energy efﬁciency. However, it was sufﬁcient to show the beneﬁts and applicability of the proposed method. In the second part, six intermediate JSON ﬁles specifying various power management have been used to verify the synthesis function of the developed tool. In these ﬁles, the dif- ferences were, for example, in the speciﬁed frequencies or in the number of power domains. These six power-information JSON ﬁles were accompanied by another 13 conﬁgurations for FPGA, testing various possible parameters. All possible combinations of these ﬁles and conﬁgurations have been used to synthesize the PMUs and other code required by the FPGA to successfully use the power-reduction techniques (e.g. syn- chronizers). Simple testbenches were also generated to run simple Verilog simulations to verify the syntactical correct- ness. The logical correctness of the automatically generated code has been manually checked whether it corresponds to the original PMS speciﬁcations. For a single randomly selected combination, the manually created more complex testbench was used to verify all possible states during the simulation. The results conﬁrmed that the automatically synthesized Ver- ilog code is generated correctly. FIGURE 7. The implemented case-study system overview. FIGURE 7. The implemented case-study system overview. As a demonstration application to be running on the FPGA, a simple open-source 8-bit microprocessor (CPU) was interconnected with the UART (Universal Asynchronous Receiver-Transmitter) interface. We have created the abstract model of the top module of this system using SystemC/PMS and speciﬁed power management for such a system. IV. RESULTS AND DISCUSSION To evaluate the proposed method, the experiments have consisted of four parts. Firstly, the ability of the devel- oped tool (implementing the proposed method) to analyze a SystemC/PMS model and to extract the power-management information has been veriﬁed. The second part has veri- ﬁed the ability of the tool to create a PMU, including the power-reduction techniques available on the selected FPGA device, based on the extracted power-management informa- tion. The third part evaluated the simpliﬁcation of the design process offered by the proposed automation method. And last but not least, it has been veriﬁed that the generated code is accepted by the development environment for the selected FPGA and that the energy requirements of the ﬁnal hardware device (i.e. the FPGA with the running application including automated power management) can be reduced using the proposed method. identiﬁers, the designer must connect it with the clk port of the PMU module. If the sync_control attribute has been activated in the Generator conﬁguration ﬁle, the synchro- nizers for PMU inputs are also synthesized. It is necessary to connect two input clock signals, one for the controlling component (requesting power-mode switching, e.g. CPU) and one for the PMU. Also, two control signals driven by the controlling component have to be connected manually. The power-mode switching request signal must be connected to the FlagIn_clkA port, and the switching vector must be con- nected to the BusIn port. Also, in case of other synchronizers (between other communicating components), it is necessary Although the pmuToFPGA tool has been developed at the Mac OS X platform, which was also used in the following experiments, the tool is multiplatform thanks to the Python nature. The environment requirements are Python version 3.7 or higher with the following libraries: pyparsing, Mako, and PyQt5. Besides the Mac platform, we have successfully tested the tool using the Windows 10 Home 64-bit operating 165899 165899 VOLUME 8, 2020 M. Škuta et al.: Automation of Dynamic Power Management in FPGA-Based Energy-Constrained Systems TABLE 2. Abstract and synthesized power management comparison. TABLE 2. Abstract and synthesized power management comparison. system running at the machine with the Intel Core i5 proces- sor and 8 GB of RAM. In the ﬁrst part of the experiments, we have used various SystemC/PMS speciﬁcations described manually. The basic SystemC model described a simple FPGA application with one Intel 8080-compatible processor and the RS232 interface. IV. RESULTS AND DISCUSSION Škuta et al.: Automation of Dynamic Power Management in FPGA-Based Energy-Constrained Systems required for signals and buses interconnecting the two power domains (clock domains) and to control the PMU by the CPU. applied to reduce idling of the components). Since the pro- posed method was not targeted to any new power-reduction technique, but to easier application of existing ones, this evaluation was focused on showing that the automatically applied technique works. This experiment has proved that the automatically synthesized power management using the proposed method can be used to reduce power, and thus it contributes to energy-efﬁcient systems design. The result of this experiment was the correctly functioning FPGA application – i.e. the speed of text-strings appearing while reading the UART interface by the laptop was notice- able when the special key was pressed. Although the chosen application was pretty simple, it was enough to illustrate the beneﬁts of the proposed method. The proposed automa- tion method helped to scale the frequency of multiple clock domains, while the automatically generated synchronizers successfully avoided the metastability issues. Although there is still a small amount of manual effort needed to ﬁnish the integration of the automatically synthe- sized power-management code into the Verilog model, it is insigniﬁcant. In the used case-study system, the pure-manual introduction of power management has taken several hours and subsequent adding/modiﬁcation of the frequency has taken another hour of work. Using the developed automation tool, it has taken up to an hour of complete time (including the mentioned post-synthesis manual modiﬁcations), while the synthesis itself has taken few seconds. We expect that the designer can save hours of design time using the pro- posed automation method. Even more, if we take into account possible introduction of human errors into the design and subsequent debugging effort. In order to ensure that the developed tool and the imple- mented method are not limited to speciﬁcally selected iCE40 FPGA device, we have used the tool to implement the same application on commonly known Xilinx Spar- tan 3 FPGA [35]. Both the Vivado synthesis tools and the Spartan 3 device accepted the pmuToFPGA-generated RTL model without any problems. Thus, the tool is not suited just to the speciﬁcally selected prototype device – since the pro- posed automation method is general enough, the tool is easily extendible for other target FPGA devices. IV. RESULTS AND DISCUSSION The system was split into two clock domains, one for CPU (PD_CPU) and the other for UART (PD_UART). Four power states were speciﬁed for the CPU domain: the ﬁrst one to stop its operation, the second to scale its operating frequency to 1.2 kHz, the third to scale the frequency to 12 MHz, and the last to scale the frequency to 48 MHz. Based on this speciﬁcation, a new top-module Verilog code along with the PMU have been synthesized automatically. We have made a few small manual modiﬁcations (to identify clock signals of the components) and used the freely available synthesis tools of the IceStorm project [34] to program the FPGA device with the designed system. In order to visually see in which power state the CPU is operating, we have created a simple assembly program writing speciﬁc text strings via the UART interface. Special keys were preconﬁgured to switch the power states. An abstract overview of such a setup is illustrated in Fig. 7. The synchronizers are automatically introduced into the design by the proposed method. These are g g y The main goal of the proposed method was to simplify and speed-up the design process for FPGA-application designers, who need to develop energy-efﬁcient systems. To evaluate these beneﬁts of the method, we have summarized the statis- tics (concerning the code size) from the previous experiments. Six samples of different power-management speciﬁcations were used and the average values have been compared. For the abstract power management, only the SystemC/PMS information about power management is counted. For the synthesized power management, the Verilog code describing the PMU and generated synchronization and isolation nodes are counted, as well as the modiﬁcations in the top-level Verilog module. Since various code-style discipline could inﬂuence a single-parameter comparison, a number of lines, a number of words, and a number of characters have been compared. The results reported in Table 2 show that the synthesized power management in Verilog is approximately 6.6 times more complex than the abstract power manage- ment in SystemC/PMS. It means that the manual effort of designers regarding power management is 6.6 times smaller using SystemC/PMS, and thus the design is faster (since the automated-synthesis time is negligible). Such a result 165900 VOLUME 8, 2020 VOLUME 8, 2020 M. IV. RESULTS AND DISCUSSION The device-speciﬁc components (such as PLL) must be predeﬁned in the tool to correctly generate the enriched RTL model for a given platform. If not, a general RTL code is synthesized, accept- able by any FPGA device – however, it might not be optimal (e.g. a common counter is used to generate the required clock frequencies instead of dedicated clock-management circuitry, such as PLL). The whole project implementing the proposed automation method along with the created/modiﬁed source code and the used test samples are available on GitHub [32], [37] to increase the reproducibility of our results. To summa- rize, the beneﬁts and limitations of the proposed method are brieﬂy stated in Table 4. Using the Kkmoon RD Tester UM24C [36] device, the cur- rent consumption in the four speciﬁed power states have been measured (see Table 3). Since the current consumption directly relates to the power consumption (P = I × V), the energy requirements of the device can be thus deduced. The calculated power in the HOLD state can be approxi- mated to the static power of the device (no operation). Thus, the dynamic power can be assumed as the remaining amount of the calculated power. TABLE 4. Advantages and disadvantages of the proposed automation method. TABLE 4. Advantages and disadvantages of the proposed automation method. TABLE 4. Advantages and disadvantages of the proposed automation method. TABLE 3. Current measurements in individual power states. TABLE 3. Current measurements in individual power states. It must be noted that the amount of actually reduced power consumption and increased energy efﬁciency depends on the used techniques and their actual implementation on the FPGA device done by logic synthesis and place & route tools (not targeted in our work). The proposed automation method helps to create an RTL model of the power-managed system based on the abstract speciﬁcation and it relies on existing tools (provided by the FPGA vendor) to implement the model into the device. Thus, different tools can provide different results. To derive the energy, we would need to measure also the time, which the device has spent in each power state. The dynamic power calculations (provided in the last column of the table) however are enough to show that the applied frequency scaling can indeed reduce power consumption, and thus the energy can be saved (e.g. when frequency scaling is V. CONCLUSION This work was focused on the simpliﬁcation of application of power-reduction techniques in FPGA-based designs using power management. Especially, it was focused on the auto- mated insertion of power-management elements (the control 165901 VOLUME 8, 2020 M. Škuta et al.: Automation of Dynamic Power Management in FPGA-Based Energy-Constrained Systems unit as well as the supporting logic) required by the FPGA device based on the abstract speciﬁcation in SystemC/PMS. The power-intent speciﬁcation in SystemC/PMS is simple and intuitive, thus usable even by inexperienced design- ers. 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His research interests are in the area of low-power digital-systems design and energy-efﬁcient communications within the Internet of Things. [28] IEEE. IEEE Standard for Design and Veriﬁcation of Low-Power, Energy- Aware Electronic Systems, IEEE Standard 1801-2018, 2019. DOMINIK MACKO (Member, IEEE) received the master’s degree in computer engineering and the Ph.D. degree in applied informatics from the Fac- ulty of Informatics and Information Technologies, Slovak University of Technology in Bratislava, in 2011 and 2015, respectively. [29] IEEE. IEEE Standard for Standard SystemC Language Reference Manual, IEEE Standard 1666-2011, 2012. [30] D. Macko, K. Jelemenská, and P. Čičák, ‘‘Veriﬁcation of power- management speciﬁcation at early stages of power-constrained sys- tems design,’’ J. Circuits, Syst. Comput., vol. 26, no. 8, Aug. 2017, Art. no. 1740002. He is currently with the Institute of Com- puter Engineering and Applied Informatics of his Alma Mater. 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Available: https://www.kkmoon.com/p-e3358-2.html Š [37] M. Škuta. (2019). iCE40HX1K Demos. [Online]. Available: https://github. com/mintos5/iCE40HX1K-demos [38] D. Macko, ‘‘Contribution to automated generating of system power- management speciﬁcation,’’ in Proc. IEEE 21st Int. Symp. Design Diag. Electron. Circuits Syst. (DDECS), Apr. 2018, pp. 27–32. KATARÍNA JELEMENSKÁ (Member, IEEE) received the Ph.D. REFERENCES degree in computer science from the Slovak University of Technology in Bratislava, in 1995. She is currently the Director of the Faculty of Informatics and Information Technologies, Institute of Computer Engineering and Applied Informatics, Slovak University of Technology in Bratislava. She has been working with the Slo- vak University of Technology in Bratislava, since Her research interests include digital systems design, modeling, and cation, means of hardware speciﬁcation, as well as efﬁcient use of mation and communication technologies in education. KATARÍNA JELEMENSKÁ (Member, IEEE) received the Ph.D. degree in computer science from the Slovak University of Technology in Bratislava, in 1995. She is currently the Director of the Faculty of Informatics and Information Technologies, Institute of Computer Engineering and Applied Informatics, Slovak University of Technology in Bratislava. She has been working with the Slo- vak University of Technology in Bratislava, since 1986. Her research interests include digital systems design, modeling, and veriﬁcation, means of hardware speciﬁcation, as well as efﬁcient use of information and communication technologies in education. MICHAL ŠKUTA received the bachelor’s and master’s degrees in computer engineering from the Faculty of Informatics and Information Tech- nologies, Slovak University of Technology in Bratislava, in 2017 and 2019, respectively. MICHAL ŠKUTA received the bachelor’s and master’s degrees in computer engineering from the Faculty of Informatics and Information Tech- nologies, Slovak University of Technology in Bratislava, in 2017 and 2019, respectively. In his work, he focused on low power IoT devices, speciﬁcally the development of an access point for the LoRa technology and automation of power management on FPGA platforms. In his work, he focused on low power IoT devices, speciﬁcally the development of an access point for the LoRa technology and automation of power management on FPGA platforms. 165903 VOLUME 8, 2020
https://openalex.org/W2090607834	https://eprints.whiterose.ac.uk/84079/11/Bacterial%20community%20dynamics%20during%20the%20early%20stages%20of%20biofilm%20formation%20in%20a%20chlorinated%20experimental%20drinking%20water%20distribution%20system%3A%20implications%20for%20drinking%20water%20discolouration.pdf	English	null	Bacterial community dynamics during the early stages of biofilm formation in a chlorinated experimental drinking water distribution system: implications for drinking water discolouration	Journal of applied microbiology	2,014	cc-by	12,255	Journal of Applied Microbiology 117, 286--301 © 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Correspondence Isabel Douterelo, Pennine Water Group, Department of Civil and Structural Engineering, The University of Shefﬁeld, Mappin Street, Shefﬁeld S1 3JD, UK. E-mail: i.douterelo@shefﬁeld.ac.uk 2013/2496: received 12 December 2013, revised 19 March 2014 and accepted 1 April 2014 2013/2496: received 12 December 2013, revised 19 March 2014 and accepted 1 April 2014 doi:10.1111/jam.12516 Conclusions: Shifts in the bacterial community structure were observed along with an increase in cell coverage, bacterial richness and diversity. Species related to Pseudomonas spp. and Janthinobacterium spp. dominated the process of initial attachment. Based on ﬁngerprinting results, the hydraulic regimes did not affect the bacteriological composition of bioﬁlms, but they did inﬂuence their mechanical stability. doi:10.1111/jam.12516 Signiﬁcance and Importance of the Study: This study gives a better insight into the early stages of bioﬁlm formation in DWDS and will contribute to the improvement of management strategies to control the formation of bioﬁlms and the risk of discolouration. are able to survive by forming bioﬁlms (Flemming et al. 2002; Batte et al. 2003). The process of bioﬁlm formation on surfaces can be relatively fast even in chlorinated net- works. Morvay et al. (2011) reported that bioﬁlm forma- tion on different plumbing material in chlorinated drinking water systems reaches values of 107 cells cm2 after only 30 days. Journal of Applied Microbiology ISSN 1364-5072 Journal of Applied Microbiology ISSN 1364-5072 Journal of Applied Microbiology ISSN 1364-5072 Abstract 16s rRNA sequencing, bacterial community structure, bioﬁlm development, discolouration, drinking water distribution systems, terminal restriction fragment length polymorphism. Aims: To characterize bacterial communities during the early stages of bioﬁlm formation and their role in water discolouration in a fully representative, chlorinated, experimental drinking water distribution systems (DWDS). Methods and Results: Bioﬁlm development was monitored in an experimental DWDS over 28 days; subsequently the system was disturbed by raising hydraulic conditions to simulate pipe burst, cleaning or other system conditions. Bioﬁlm cell cover was monitored by ﬂuorescent microscopy and a ﬁngerprinting technique used to assess changes in bacterial community. Selected samples were analysed by cloning and sequencing of the 16S rRNA gene. Fingerprinting analysis revealed signiﬁcant changes in the bacterial community structure over time (P < 005). Cell coverage increased over time accompanied by an increase in bacterial richness and diversity. Bacterial community dynamics during the early stages of bioﬁlm formation in a chlorinated experimental drinking water distribution system: implications for drinking water discolouration I. Douterelo1, R. Sharpe2 and J. Boxall1 1 Pennine Water Group, Department of Civil and Structural Engineering, University of Shefﬁeld, Shefﬁeld, UK 2 School of Civil and Building Engineering, Loughborough University, Loughborough, UK 1 Pennine Water Group, Department of Civil and Structural Engineering, University of Shefﬁeld, Shefﬁeld, UK 2 School of Civil and Building Engineering, Loughborough University, Loughborough, UK Introduction It is commonly accepted that the use of a disinfectant residual such as chlorine in drinking water distribution systems (DWDS) does not completely prevent bacterial occurrence. Free-living bacteria can enter the distribution system through, for example, the treatment works, cross- connections or contamination ingress and can adhere to the pipe inner surfaces and form bioﬁlms (Szewzyk et al. 2000). Bioﬁlms are an advantageous way of living in hos- tile environments (Costerton et al. 1987; Simoes et al. 2010), and most of micro-organisms present in DWDS Bioﬁlms are structurally complex and consist of micro- organisms attached to a surface and to each other and embedded in an extracellular polymeric matrix (EPS) made of polysaccharides, proteins, extracellular DNA, etc. (Lopes et al. 2009). The EPS of bioﬁlms offers protection 286 Bacteria in drinking water bioﬁlms I. Douterelo et al. to the direct action of disinfectants and also provides physical stability against the inﬂuence of shear forces (Flemming and Wingender 2010). Even in highly oligo- trophic environments, such as DWDS, bioﬁlms are diverse microbial ecosystems where different micro- organisms can coexist interacting with each other, acting as a unique entity and contributing with their different metabolic capabilities to the acquisition of nutrients (Stoodley et al. 2002). Microbial bioﬁlms can modify the quality of drinking water both due to their presence and through their metabolic activities. The growth and accu- mulation of bioﬁlms can modify hydraulics within the pipes, including pipe clogging, enhanced corrosion in metallic pipes and changes in water taste and odour (Zacheus et al. 2000). If the hydraulic conditions change in a way that overcomes bioﬁlm adhesive forces, bioﬁlms can detach from the pipe surfaces and contribute to the deterioration of water quality including discolouration (Ginige et al. 2011) and even have the potential to release pathogens into the bulk water. ment of this test loop facility offers a signiﬁcant advance in overcoming the difﬁculties of bench-scale experimentation. The facility was operated to allow a material accumulation phase, to represent material development in the network, as well as a ﬂushing/mobilisation phase, to represent a network disturbance with the potential to cause bioﬁlm mobilisation and associated discolouration. In general, the formation of a bioﬁlm is a successional process that can take years (Martiny et al. 2003) but starts when free-living bacteria (i.e. planktonic) attach to surfaces under certain conditions. Introduction These primary colonizing micro- organisms, mainly bacteria, start growing and are able to modify the substratum, providing more adhesion sites, and allowing for the colonization by other micro-organisms (Lee et al. 2008; Andrews et al. 2010). Different factors can affect the initial adhesion of planktonic cells to surfaces. In DWDS, these are likely to include pipe characteristics (material, diameter and roughness), source water parame- ters (e.g. pH, temperature and organic matter content), hydrodynamic conditions (ﬂow, shear stress, etc.) and the characteristics of the bacterial cells themselves such as ability to produce extracellular polymeric substance, cell hydro- phobicity and motility (Liu et al. 2004; Simoes et al. 2007a). It is generally accepted that DWDS are inhabited by dif- ferent bacterial species that can form bioﬁlms (Simoes et al. 2007b; Li et al. 2010). Using a model water distribution sys- tem with stainless steel plugs and operated under turbulent ﬂow, Martiny et al. (2003) demonstrated that bioﬁlm devel- opment is a dynamic process where bacterial succession occurs. However, most bioﬁlm studies in aquatic environ- ments are carried out under idealized laboratory conditions, using bench-top reactors and/or inoculating a limited num- ber of micro-organisms (Moritz et al. 2010; Yu et al. 2010). As a consequence, little is known about which bacteria are involved in the initial development of bioﬁlms under realis- tic conditions in water distribution networks. In this study, we use a full-scale experimental DWDS (Fig. 1) which fully recreates hydraulic and physico-chemical conditions of real distribution systems to obtain more accurate information about the initial process of bioﬁlm formation. The develop- In this study, we explore who are the primary colonizers of a chlorinated distribution system, which bacteria are able to leave the bulk water and adhere to the pipe sur- faces, how they are changing in their ﬁrst month of bioﬁlm formation and whether different hydraulic regimes affected their ability for initial adherence and the physical structure of the bioﬁlm itself. Figure 1 (a) full-scale laboratory pipe loop experimental facility at the University of Shefﬁeld, (b) section of pipe designed to ﬁt PWG coupons and (c) PWG coupon showing the insert and the outer part of the coupon. Experimental drinking water distribution system To achieve the aims of this article, experiments were conducted in a purpose-built, temperature-controlled re-circulating test (a) oop f o ﬁt owing upon (a) (b) (c) (a) (b) (c) Figure 1 (a) full-scale laboratory pipe loop experimental facility at the University of Shefﬁeld, (b) section of pipe designed to ﬁt PWG coupons and (c) PWG coupon showing the insert and the outer part of the coupon. 287 Journal of Applied Microbiology 117, 286--301 © 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology. (c) Figure 1 (a) full-scale laboratory pipe loop experimental facility at the University of Shefﬁeld, (b) section of pipe designed to ﬁt PWG coupons and (c) PWG coupon showing the insert and the outer part of the coupon. Figure 1 (a) full-scale laboratory pipe loop experimental facility at the University of Shefﬁeld, (b) section of pipe designed to ﬁt PWG coupons and (c) PWG coupon showing the insert and the outer part of the coupon. 287 Bacteria in drinking water bioﬁlms I. Douterelo et al. I. Douterelo et al. Sample collection for DNA extraction from bulk water and bioﬁlms loop facility (Fig. 1). The temperature-controlled test facil- ity has been previously described (Douterelo et al. 2013). Brieﬂy, the system has a total volume of 45 m3 and con- sists of three 200-m-long high-density polyethylene (HDPE) recirculating loops, fed by a common pump and returning to a common closed reservoir. The system was ﬁtted with a trickle feed from the local water network and is set to obtain a system residence time of 24 h. The tem- perature for the experiment reported here was set at 16°C to reproduce average UK temperatures in DWDS during warmer months. The ﬂow in each loop was individually controlled to generate different hydraulic regimes. The hydraulic regimes were: steady-state conditions (04 l s1) and two variable ﬂows with different daily hydraulic pat- terns based on costumer’s drinking water demand in the UK; low varied ﬂow, ranging from 02 to 05 l s1 and highly varied ﬂow ranging from 02 to 08 l s1 (Husband et al. 2008). To study changes in the bacterial community structure over time, PWG coupons were collected in triplicate from each loop during the material build-up phase at speciﬁc sampling days (days 0, 3, 7, 14, 21 and 28). Experimental drinking water distribution system Coupons were collected on day 0 after the disinfection process when chlorine concentrations matched that of the inlet water. Coupons collected on day 0 were used as controls. In total, 54 coupons were collected during the 28-day growth phase. To assess the mechanical stability of the material devel- oped on the pipe and the inﬂuence of ﬂushing on bacte- rial community structure, coupons were also collected in triplicate from each loop before and after ﬂushing the system (total of 18 coupons). To examine the impact that ﬂushing had on the bulk water, triplicate 1l pre- and post ﬂushing samples were taken directly from the outlet of each of the three loops. In total, 18 water samples were collected for this experiment and ﬁltered through 022- lm nitrocellulose membrane ﬁlters (Millipore UK Ltd., Watford, UK). Before starting the experiment, the test facility was disinfected with 20 mg l1 of RODOLITE H (RODOL Ltd, Liverpool, UK) by ﬂushing the system for three turnovers at the maximum ﬂow rate (42 l s1) and then being left stagnant with RODOLITE for 24 h. After that period, the system was ﬂushed again at the maxi- mum ﬂow rate with fresh water until the levels of chlorine were similar to those of the local tap water. After disinfection of the facility, sterile PWG coupons of the same material (Deines et al. 2010) were ﬁtted along and around the pipe length of each loop. Coupons were removed for bioﬁlm analysis at days 0, 3, 7, 14, 21 and 28. After the 28-day growth phase, each loop was ﬂushed from an initial ﬂow of 04 l s1 to a maximum ﬂow of 45 l s1 to assess the mechanical stability of bioﬁlms. Bioﬁlms were removed from PWG coupons, and cells were concentrated in nitrocellulose membrane ﬁlters as described in (Deines et al. 2010). Filters containing water and bioﬁlms samples were kept in the dark and at 80°C for subsequent DNA analysis. DNA extraction, including samples from day 0, was car- ried out by a method based on proteinase K digestion fol- lowed by a standard phenol/chloroform–isoamyl alcohol extraction (Neufeld et al. 2007). The quantity and purity of the extracted DNA were assessed using Nanodrop ND- 1000 spectrophotometer (NanoDrop, Wilmington, DE). Statistical analysis of T-RFLPs proﬁles T-RFLPs were aligned on the basis of fragments length and peak areas using the T-ALIGN Software (Smith et al. 2005). The aligned T-RFLPs were square root transformed, and Bray–Curtis similarity matrixes were calculated using the software PRIMER v6 (PRIMER-E, Plymouth, UK). Bray– Curtis similarity matrixes were visualized using multiple dimensional scaling (MDS) diagrams. Analysis of similarity statistics (ANOSIM) was calculated using the same Bray– Curtis distance matrix to test the signiﬁcance of differences among samples based on hydraulic regimes and ﬂushing. Sequencing and phylogenetic analysis DNA was sequenced in both directions and consensus sequences obtained using the Phred-Phrap programs (Phil Green, Brent Ewing, University of Washington) by utilizing Perl scripts written by NBAF-Edinburgh and modiﬁed by Dr Gavin Horsburgh at NBAF-Shefﬁeld, UK. Sequences were analysed for chimeric artefacts using DECIPHER (Wright et al. 2012), and any chimeric sequences identiﬁed were removed from the clone libraries for further analysis. The MultiClassiﬁer tool based on naive Bayesian classiﬁer in the Ribosomal Data- base Project (RDP) II (Cole et al. 2009) was used to clas- sify query sequences at a conﬁdence threshold of 80% (Wang et al. 2007). The online analysis function from the RDP was used to align sequences by the Infernal aligner (Nawrocki et al. 2009) and to cluster the aligned sequences by the complete-linkage clustering method; the cluster ﬁles were used to calculate Shannon diversity index (Shannon and Weaver 1963) and Chao richness estimator (Chao 1984). Column formatted distance matrices were generated using the RDP online analysis tool and used in MOTHUR (Schloss et al. 2009) to calculate a Venn diagram with the number of shared OTUs between samples at 97% sequence similarity cut-off. The Basic Logic Alignment Search Tool (BLAST) (Altschul et al. 1990) was used to evaluate similarities with sequences deposited in the GenBank and to create a table with the summarized BLAST outputs at species level. Water physico-chemical analysis Differences in abundance and length of T-RFLPs were determined by comparison with the known size internal standard, and the actual fragment sizes were estimated by interpolation using a Local Southern algorithm with the software GENEMAPPER 3.7 (Applied Biosystems). Terminal fragments smaller than 50 bp and with a peak height of <50 were excluded, considered background noise and eliminated from the analysis. Kit (Qiagen Inc., Valencia, CA) and then digested at 37°C for 2 h with restriction enzyme Alu I (Roche Diag- nostics Ltd., Burgess Hill, UK). Desalted restriction digests were mixed with 1 ll of deionized formamide and 05 ll of a ROX-labelled Genescan 500-bp internal size standard (Applied Biosystems), denatured at 94°C for 3 min and immediately transferred to ice. T-RFLPs were separated by capillary electrophoresis using an auto- mated genetic analyser ABI3730 (Applied Biosystems). Differences in abundance and length of T-RFLPs were determined by comparison with the known size internal standard, and the actual fragment sizes were estimated by interpolation using a Local Southern algorithm with the software GENEMAPPER 3.7 (Applied Biosystems). Terminal fragments smaller than 50 bp and with a peak height of <50 were excluded, considered background noise and eliminated from the analysis. et al. 1989). Inserts were sequenced from both directions using plasmid-vector-speciﬁc primers M13F (50- CGCCAGGGTTTTCCCAGTCACGAC-30) and M13R (50- TAACAATTTCACACAGGA-30) primers. Samples were puriﬁed with EXOSAP and sequenced at The University of Shefﬁeld, Medical School with an Applied Biosystems 3730 automated DNA analyser. et al. 1989). Inserts were sequenced from both directions using plasmid-vector-speciﬁc primers M13F (50- CGCCAGGGTTTTCCCAGTCACGAC-30) and M13R (50- TAACAATTTCACACAGGA-30) primers. Samples were puriﬁed with EXOSAP and sequenced at The University of Shefﬁeld, Medical School with an Applied Biosystems 3730 automated DNA analyser. Cloning and sequencing Based on T-RFLPs results and to detect main shifts in bioﬁlm bacterial composition, selected DNA samples obtained from bioﬁlms grown under steady state condi- tions on days 7, 14 and 28 were PCR-ampliﬁed with bacterial primers 27F (50-AGAGTTTGATCCTTGGCT- CAG-30) and 1492R (50-GCYTACCTTGTTACGACTT-30) (Lane 1991). PCR conditions were 5 min at 94°C, 30 cycles of 30 s at 94°C, 30 s at 54°C, and 90 s at 72°C and a ﬁnal extension for 10 min at 72°C. The PCR mas- ter mix was prepared as explained in the previous section. PCR products from three biological replicates were pooled and puriﬁed using the QIAquick PCR Puriﬁcation Kit (Qiagen Inc.). Puriﬁed PCR products were cloned using the pGEM-T Easy Vector Systems (Promega UK Ltd, Southampton, UK), and ligations were performed overnight at 4°C. Transformations were carried out using competent cells of Escherichia coli JM109, following manu- facturer’s instructions (Promega). Transformants were selected by ampicillin resistance, and blue-white screening was performed to identify clones with inserts (Sambrook In this study, GenBank and the RDP provided similar taxonomic assignments from domain to genus. However, at species level, GenBank provides a measurement of the percent identity of a sequence and RDP measures related- ness values which is close to but inferior than the GenBank percent identity (Clarridge 2004). As a consequence, we have included the results from BLAST in Table 2 to support the information obtained by the RDP. Sequences included in the analysis were submitted to the GenBank and are available under the accession num- bers KF611923-KF611976. Water physico-chemical analysis Water samples were collected on the day of coupon sampling during the growth phase and before and after ﬂushing. The water samples were analysed for several physico-chemical factors (i.e. pH, temperature, chlorine, iron and manganese). Every analysis was performed in triplicate, and the average of the three replicates was cal- culated. Free chlorine was measured using a HACH DR/ 2010 spectrophotometer. Measurements of temperature and pH were made using a Hanna H1991003 meter and probes. Water samples for total iron and manganese were sent to an independent accredited laboratory AlCon- trol Laboratories (Deeside, UK) for analysis. During ﬂushing, turbidity was constantly measured by a Chem- trac TM2200 turbidity meter installed in the experimental facility to measure the particles mobilized as described in Sharpe et al. (2010). A fragment of approximately 490 bp of the bacterial 16S rRNA gene, targeting the region V1-V3, was ampliﬁed using primer pair 63F (50-CAGGCCTAACACATGCA AGTC-30) and 518R (50-CGTATTACCGCGGCTGCTCG-30) (Girvan et al. 2003). The oligonucleotide 63F was labelled at the 50 end with the phosphoramidite dye FAM (Applied Biosystems, Life Technologies Ltd, Paisley, UK). PCR mixtures were carried out using 125 ll of Taq- ReadyMix with MgCl2 (Sigma-Aldrich Company Ltd., Dorset, UK) and 5 lmol l1 of each primer, using 1 ll of template DNA, and sterile nuclease-free water to a ﬁnal volume of 25 ll. PCR conditions were 5 min at 95°C; 30 cycles of 30 s at 94°C, 30 s at 55°C and 1 min at 72°C; and a ﬁnal extension for 10 min at 72°C. PCR products were puriﬁed using QIAquick PCR Puriﬁcation 288 f Applied Microbiology 117, 286--301 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology. I. Douterelo et al. Bacteria in drinking water bioﬁlms Kit (Qiagen Inc., Valencia, CA) and then digested at 37°C for 2 h with restriction enzyme Alu I (Roche Diag- nostics Ltd., Burgess Hill, UK). Desalted restriction digests were mixed with 1 ll of deionized formamide and 05 ll of a ROX-labelled Genescan 500-bp internal size standard (Applied Biosystems), denatured at 94°C for 3 min and immediately transferred to ice. T-RFLPs were separated by capillary electrophoresis using an auto- mated genetic analyser ABI3730 (Applied Biosystems). Confocal laser scanning microscopy Three replicate coupons with bioﬁlm grown under steady state conditions were removed on days 7, 14 and 289 Applied Microbiology 117, 286--301 © 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology Bacteria in drinking water bioﬁlms I. Douterelo et al. 28. These particular coupons were selected to match the data obtained from cloning and sequencing the 16S rRNA gene. The ﬂat insert section was separated from the coupon and ﬁxed in 5% formaldehyde for 24 h, then transferred to phosphate buffer solution (PBS) and stored at 4°C until analysed. After ﬁxing, the inserts were stained with 20 lmol l1 Syto 63 for 30 min. Syto 63 is a cell-permeative nucleic acid stain which is used to visualize cells (McSwain et al. 2005). Once stained, the samples were washed three times in sterile water and air-dried for 10 min before being stored in the dark at 4°C (<1 month). Imaging was performed at the Kroto Research Institute Confocal Imaging Facility, using a Zeiss LSM 510 Meta Confocal Florescent Micro- scope. Images were taken using an 920 EC Plan Neof- laur Objective (05 NA) at 3154 uz per pixel speed, pin hole set to an optical slice of 47 lm, resolution 832 9 832 pixels and a frame size of 420 9 420 lm. LSM510 Image Examiner Software was used to visualize the images (Zeiss, UK). Each insert was imaged for ﬁve random ﬁelds of view to provide an accurate represen- tation of cell coverage. The images were then processed to extract a relative quantiﬁcation of the bioﬁlm at each layer. The images were ﬁrstly un-mixed, based on the spectral response of the Syto 63 at different laser wave- lengths, to remove any inﬂuence of the plastic coupon substrate. The images were then median ﬁltered to reduce noise, whereby the value of any pixel is calcu- lated as the median of itself and the 8 pixels that sur- round it. Finally, the area of the bioﬁlm per slice was calculated by the count of the pixels above a threshold, chosen to further reduce noise from the microscope and stain. higher value of 828. The temperature of the facility was set to operate at 16°C, and during the experiment, water temperature ﬂuctuated from 159 to 1627°C. Confocal laser scanning microscopy Average iron concentration of the water samples obtained at dif- ferent sampling times during the 28-day accumulation phase test was 207 lg l1 227 reaching a maximum on day 21 of 24 lg l1. Manganese concentrations were also highly consistent ranging from 21 to 29 lg l1. Free chlorine was high (044 mg l1) on day 0 when the experiment started due to the ﬁlling of the system with fresh drinking water; the subsequent water samples once the system residence time of 24 h was established gave values ranging from 01 to 017 mg l1. During the ﬂush- ing experiment, free chlorine levels ranged between 019 and 028 mg l1 (data not shown), showing higher values than those measured during the growth phase due to the reﬁlling of the reservoir with fresh water between ﬂushing successive loops. Figure 2 shows the changes in levels of turbidity, iron and manganese concentration before and after ﬂushing the system. Before ﬂushing, the pipe loop which had been operated at the higher varied ﬂow had higher turbidity levels (0114 NTUs) than the other loops. After ﬂushing the system, water turbidity levels and simultaneously iron and manganese concentrations increased. This was greatest in steady state and least in high varied ﬂow, supporting previous laboratory investi- gations (Sharpe et al. 2010) and ﬁeldwork observations (Husband et al. 2008) that daily hydraulics, characterized by daily peak, inﬂuences discolouration response. Succession over time h b ﬁl From the bioﬁlm samples obtained on coupons collected on day 0 and used as controls, no DNA was obtained as conﬁrmed both by Nanodrop quantiﬁcations and the lack of PCR ampliﬁcations from these samples. Similarly, not all the bioﬁlm samples from day 3 and 7 yielded enough DNA to obtain good-quality amplicons for ﬁngerprinting analysis and these were excluded from the analysis. The nonmetric multidimensional scaling analysis (MDS) of the T-RFLPs from bioﬁlm samples (n = 36) obtained Below detection. 290 Journal of Applied Microbiology 117, 286--301 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology. Water physico-chemical analysis Table 1 shows the characteristics of the water at speciﬁc sampling days during the 28 days of growth phase. pH values were near neutral (714–777) for most of the sam- pling days with the exception of day 21 with a slightly Table 1 Average value (n = 3) and standard deviation of the physico-chemical parameters analysed in the bulk water samples during the 28 days of growth phase Sampling day pH T (°C) Fe (lg l1) Mn (lg l1) Free Chlorine (mg l1) 0 777 005 1593 011 220 000 21 000 044 000 7 714 000 1620 000 187 058 21 000 010 002 14 743 002 1607 005 197 115 23 011 21 828 000 1627 011 240 265 29 070 011 001 28 730 010 1603 005 190 000 29 006 017 003 *B l d t ti = 3) and standard deviation of the physico-chemical parameters analysed in the bulk water samples during the Table 1 Average value (n = 3) and standard deviation of the physico-chemical parameters analysed in the bulk water sam 28 days of growth phase 290 Journal of Applied Microbiology 117, 286--301 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology. Journal of Applied Microbiology 117, 286--301 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied 290 I. Douterelo et al. Bacteria in drinking water bioﬁlms 0·00 B A LVF B A SS HVF B A B A LVF B A SS HVF B A 0 Fe and Mn (µg.µl–1) 10 20 30 40 50 60 70 80 90 100 Turbidity (NTU) 0·10 0·20 0·30 0·40 0·50 0·60 (a) (b) Figure 2 (a) Differences in bulk water turbidity (NTU) and (b) iron and manganese (lgll1) concentrations among the three hydraulic regimes before and after ﬂushing the system. B (before ﬂushing) and A (after ﬂushing). SS (steady state); LVF (low varied ﬂow); HVF (high varied ﬂow). Please note that for (a), the standard deviations are conventionally plotted positively to the bars. However, for (b), this is not possible due to the composite iron and manganese data being displayed. Figure (b) hence has standard deviation bars plotted negatively. ( ) Fe; ( ) MN. Water physico-chemical analysis I. Douterelo et al. Bacteria in drinking water bioﬁlms B A LVF B A SS HVF B A 0 Fe and Mn (µg.µl–1) 10 20 30 40 50 60 70 80 90 100 (b) Figure 2 (a) Differences in bulk water turbidity (NTU) and (b) iron and manganese (lgll1) concentrations among the three hydraulic regimes before and after ﬂushing the system. B (before ﬂushing) and A (after ﬂushing). SS (steady state); LVF (low varied ﬂow); HVF (high varied ﬂow). Please note that for (a), the standard deviations are conventionally plotted positively to the bars. However, for (b), this is not possible due to the composite iron and manganese data being displayed. Figure (b) hence has standard deviation bars plotted negatively. ( ) Fe; ( ) MN. 2D Stress: 0·19 Figure 3 Nonmetric Multidimensional Scaling (MDS) ordination based on a Bray–Curtis resemblance matrix calculated from T-RFLPs proﬁles during bioﬁlm growth phase. The plot shows distribution of samples according to hydraulic regime. SS (steady state); LVF (low varied ﬂow); HVF (high varied ﬂow). ( ) SS; ( ) LVF; ( ) HVF 2D Stress: 0·19 Figure 4 Two-dimensional plot of the nonmetric multidimensional scaling (MDS) ordination based on a Bray–Curtis resemblance matrix calculated from T-RFLPs proﬁles during bioﬁlm growth phase. The plot shows differences in bacterial community structure at different days of bioﬁlm development.( ) 3 days; ( ) 7 days; ( ) 14 days; ( ) 21 days; ( ) 28 days 2D Stress: 0·19 Figure 4 Two-dimensional plot of the nonmetric multidimensional scaling (MDS) ordination based on a Bray–Curtis resemblance matrix calculated from T-RFLPs proﬁles during bioﬁlm growth phase. The plot shows differences in bacterial community structure at different days of bioﬁlm development.( ) 3 days; ( ) 7 days; ( ) 14 days; ( ) 21 days; ( ) 28 days. 2D Stress: 0·19 Figure 3 Nonmetric Multidimensional Scaling (MDS) ordination based on a Bray–Curtis resemblance matrix calculated from T-RFLPs proﬁles during bioﬁlm growth phase. The plot shows distribution of samples according to hydraulic regime. SS (steady state); LVF (low varied ﬂow); HVF (high varied ﬂow). ( ) SS; ( ) LVF; ( ) HVF 2D Stress: 0·19 2D Stress: 0·19 Figure 4 Two-dimensional plot of the nonmetric multidimensional scaling (MDS) ordination based on a Bray–Curtis resemblance matrix calculated from T-RFLPs proﬁles during bioﬁlm growth phase. Water physico-chemical analysis The plot shows differences in bacterial community structure at different days of bioﬁlm development.( ) 3 days; ( ) 7 days; ( ) 14 days; ( ) 21 days; ( ) 28 days. Figure 3 Nonmetric Multidimensional Scaling (MDS) ordination based on a Bray–Curtis resemblance matrix calculated from T-RFLPs proﬁles during bioﬁlm growth phase. The plot shows distribution of samples according to hydraulic regime. SS (steady state); LVF (low varied ﬂow); HVF (high varied ﬂow). ( ) SS; ( ) LVF; ( ) HVF during the growth phase did not show a clear separation of the bacterial communities developed under the three different hydraulic regimes (Fig. 3). However, when the factor analysed is time, changes in the bacterial commu- nity ﬁngerprints of bioﬁlm samples were detected (Fig. 4). The analysis of similarity (ANOSIM) revealed that these temporal differences were particularly signiﬁ- cant between days 7 and 21 (R = 0607, P = 002). structure of the bacterial communities before and after ﬂushing were analysed. Eighteen bioﬁlm samples were collected during the ﬂushing experiment; however, two samples (one before ﬂushing at LVF and one after ﬂush- ing at HVF) did not yield good ampliﬁcations for T-RFLPs analysis, and consequently, only 16 samples were used. The nonmetric MDS of the T-RFLPs proﬁles (Fig. 5a) showed a clear separation between bacterial communities from the bulk water and those inhabiting the bioﬁlms, ANOSIM (R = 0852, statistic = 0001). When considering only pre- and postﬂushing bioﬁlm samples (n = 16) (Fig. 5b), differences were detected between different hydraulic regimes (LVF vs HVF = 0479, P = 006, SS vs HVF = 0375, P = 013 and SS vs LVF = 0143, P = 026) between pre- and Flushing Flushing of the pipes was used to gain a practically rele- vant measure of bioﬁlm stability and to assess the poten- tial for detachment of material from the pipe walls into the bulk water. Terminal restriction fragments were obtained from 18 water samples and 16 bioﬁlm samples during the ﬂushing experiment and differences in the 291 Applied Microbiology 117, 286--301 © 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology Bacteria in drinking water bioﬁlms I. Douterelo et al. 2D Stress : 0·08 2D Stress : 0·11 (a) (b) Figure 5 Two dimensional plot of the nonmetric multidimensional 2D Stress : 0·11 (a) With this aim, three clone libraries were constructed from bioﬁlm samples developed on coupons under steady state conditions on days 7, 14 and 28. These days were selected because not enough amplicons were obtained from 3-day-old bioﬁlms and changes were detected in the T-RFLPs proﬁles from these 3 days. After the removal of chimeric artefacts, a total of 237 sequences were obtained from the three libraries. The analysis of 16S rRNA gene sequences using the RDP II classiﬁer showed that all three libraries were dominated by sequences related to the phylum Proteobacteria (Fig. 6). The Betaproteobacteria subdivision was the predominant group in all the bioﬁlm samples followed by Gammaproteobacteria and Alphapro- teobacteria over the studied period of time. Over time, the percentage of Betaproteobacteria decreased and Alpha- proteobacteria increased. Figure 7 shows the results obtained at genera level for the sequences analysed using the RDP Na€ıve Bayesian classiﬁer at a sequence threshold of 80%. Most of the clones selected on day 7 were closely related to the genus Pseudomonas (32%) followed by Janthinobacterium (23%), Methylophilus (18%) and Steno- trophomonas (11%). In the clone library obtained from day 14, Methylophilus was the most represented genus (305%) followed by Pseudomonas (236%), Dechloromon- as (21%) and Curvibacter (7%). The percentage of clones related to Pseudomonas spp. decreased over time, and on day 28, they only represented 10% of the total number of clones in the library; other genera were more abundant in this particular clone library such as Undibacteria (15%) and Porphyrobacter (125%). However, on day 28, a high proportion of clones (21%) were not classiﬁed at genus level. Flushing 2D Stress : 0·08 (b) (b) Figure 5 Two-dimensional plot of the nonmetric multidimensional scaling (MDS) ordination based on Bray–Curtis resemblance matrix calculated from T-RFLPs proﬁles during ﬂushing (a) water vs bioﬁlm and (b) pre- and post ﬂushing. (a) ( ) Water _B; ( ) Water_A; ( ) Bioﬁlm _B; ( ) Bioﬁlm_A (b) ( ) SS_B; ( ) LVF_B; ( ) HVF_B; ( ) SS_A; ( ) LVF_A; ( ) HVF_A Figure 5 Two-dimensional plot of the nonmetric multidimensional scaling (MDS) ordination based on Bray–Curtis resemblance matrix calculated from T-RFLPs proﬁles during ﬂushing (a) water vs bioﬁlm and (b) pre- and post ﬂushing. (a) ( ) Water _B; ( ) Water_A; ( ) Bioﬁlm _B; ( ) Bioﬁlm_A (b) ( ) SS_B; ( ) LVF_B; ( ) HVF_B; ( ) SS_A; ( ) LVF_A; ( ) HVF_A Table 2 shows the results obtained from the GenBank database search at species level using the BLAST algorithm. The majority of Gammaproteobacteria in the clone library of day 7 were afﬁliated to the genus Pseudomonas. The remaining clones in this phylum were related to Stenotrophomonas maltophilia. Within the group of Betaproteobacteria, clones related to Janthinobacterium sp., Methylophilus methylotrophus and Duganella sp. were highly represented; other clones were related to species of Acidovorax sp., Curvibacter gracilis and Dechloromonas sp. With low sequence similarity (<95%), we detected one postﬂushing samples (LVF = 077, P = 01, SS = 0917, P = 01, HVF = 1, P = 01). Analysis of sequencing data from days 7, 14 and 28 clone libraries As the T-RFLPs analysis did not show signiﬁcant differ- ences over time between different hydraulic regimes (Fig. 3), we selected steady-state bioﬁlm samples to assess changes in the bacterial community structure over time. Day 7 Day 14 Day 28 Figure 6 Pie charts showing the relative abundance of bacterial class in the three clone libraries from days 7, 14 and 28 at steady-state conditions. ( ) Betaproteobacteria; ( ) Gammaproteobacteria; ( ) Flavobacteria; ( ) Alphaproteobacteria. 292 Journal of Applied Microbiology 117, 286--301 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology. Day 28 Day 14 ( ) Flavobacteria; ( ) Alphaproteobacteria. 292 Journal of Applied Microbiology 117, 286--301 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Bacteria in drinking water bioﬁlms I. Douterelo et al. Microscopy analysis On day 14, the presence of Betaprotebacteria representatives increased and species related to other genera such as Methylomonas clara, Sphingopyxis sp. and Methylovorus glucosotrophus were abundant; however, most of the other clones in these bacterial phyla were related to unknown uncultured bacteria. Gammaproteobacteria was mainly represented by species of Pseudomonas (21%) in particular Pseudomonas ﬂuorescens. Alphaproteobacteria is represented in this clone library by only two clones related to Sphingopyxis sp. Finally, within the clone library of day 28, the Betaproteobacteria phylum was represented mainly by clones related to Comamonadaceae bacterium (97% similarity), Undibacterium sp. (similarity > 95%), Methylo- phylus methilotrophus and Oxalobacteriaceae bacterium. Other Betaproteobacteria were represented by only one clone. Within Gammaproteobacteria, the majority of the clones were highly similar to Stenotrophomonas maltophilia (74% of clones) and different species of Pseudomonas spp. and Nevskia spp. In this clone library, the Alphaproteobac- teria phylum was mainly represented by clones related to species such as Porphyrobacter sanguineus (134%), Sphingomonas sp. and several species of Methylobacterium sp. and Novosphingobium sp. Microscopy analysis Microscopy analysis Given that no signiﬁcant changes in the bacterial com- munity structure were detected using ﬁngerprinting tech- niques between the three different hydraulic regimes, only bioﬁlms developed under steady state conditions were imaged to assess the rate of bioﬁlm build-up in our sys- tem. Graphs in Fig. 10 represent changes in bioﬁlm area fraction, calculated to quantify changes in cell coverage (i.e. the relative area of each 2D image covered by stained cells) on pipe surfaces over time under steady state con- ditions. On day 7, most of the analysed ﬁeld of views from the three different coupons were not above the ﬂu- orescence threshold established to distinguish microbial cells from ﬂuorescence emitted by the plastic coupon and considered as background or noise. Throughout time, cell coverage increased from a maximum area fraction on day 14 of 0003 to a maximum fraction on day 28 of 0009, showing that cell coverage can be triplicated after 2 weeks of bioﬁlm development under steady state conditions. clone related to Herbapirillum sp., another clone related to Methylotenera mobilis and one clone showed only 93% sim- ilarity to Flavobacterium sp. which belongs to the Flavobac- teria phylum. On day 14, the presence of Betaprotebacteria representatives increased and species related to other genera such as Methylomonas clara, Sphingopyxis sp. and Methylovorus glucosotrophus were abundant; however, most of the other clones in these bacterial phyla were related to unknown uncultured bacteria. Gammaproteobacteria was mainly represented by species of Pseudomonas (21%) in particular Pseudomonas ﬂuorescens. Alphaproteobacteria is represented in this clone library by only two clones related to Sphingopyxis sp. Finally, within the clone library of day 28, the Betaproteobacteria phylum was represented mainly by clones related to Comamonadaceae bacterium (97% similarity), Undibacterium sp. (similarity > 95%), Methylo- phylus methilotrophus and Oxalobacteriaceae bacterium. Other Betaproteobacteria were represented by only one clone. Within Gammaproteobacteria, the majority of the clones were highly similar to Stenotrophomonas maltophilia (74% of clones) and different species of Pseudomonas spp. and Nevskia spp. In this clone library, the Alphaproteobac- teria phylum was mainly represented by clones related to species such as Porphyrobacter sanguineus (134%), Sphingomonas sp. and several species of Methylobacterium sp. and Novosphingobium sp. clone related to Herbapirillum sp., another clone related to Methylotenera mobilis and one clone showed only 93% sim- ilarity to Flavobacterium sp. which belongs to the Flavobac- teria phylum. Flushing Aligned sequences were clustered into OTUs at 95% (used as genus level) and 97% (used as species level) sequence similarities thresholds, and rarefaction curves were plotted for the observed OTUs at 97% cut-off (Fig. 8). As it was expected, the rarefaction curves show- ing the number of OTUs per clone library did not show a plateau due to the limited number of clones in the sequenced libraries. Species richness (Chao I) and diver- sity (Shannon index) of the clone libraries calculated at 95 and 97% cut-offs increased over time from day 7 to 28 with the exception of Chao I at 95% cut-off for day 14 (Fig. 8). The Venn diagram generated to calculate the shared OTUs between clone libraries showed that the total estimated richness of all groups at 97% sequence similarity threshold (i.e. species level) was 32 OTUs and that the estimated richness shared by the three clone libraries was 5 OTUs (Fig. 9). The data indicate that after the initial ﬁrst week of material development within the pipes, the bacterial community changes towards a more diverse community. % Relative genera abundance Day 7 Day 14 Day 28 20 0 60 80 100 40 Figure 7 Relative abundance of bacterial genera found in the clone libraries according to the RDP classiﬁer. ( ) Pseudomonas; ( ) Janthi- nobacterium; ( ) Methylophilus; ( ) Stenotrophomonas; ( ) Undi- bacterium; ( ) Dechloromonas; ( ) Acidovorax; ( ) Bacteroidetes; ( ) Flavobacterium; ( ) Curvibacter; ( ) Porphyrobacter; ( ) Sphingo- monas; ( ) Nevskia; ( ) Sphingopyxis; ( ) Novosphingobium; ( ) Methylobacterium; ( ) Acinetobacter; ( ) Unclassiﬁed_Methylophila- ceae; ( ) Unclassiﬁed_Rhizobiales; ( ) Unclassiﬁed_Erythrobactera- ceae; ( ) Unclassiﬁed_Pseudomonadaceae; ( ) Unclassiﬁed. % Relative genera abundance Day 7 Day 14 Day 28 20 0 60 80 100 40 Day 14 Day 28 Figure 7 Relative abundance of bacterial genera found in the clone libraries according to the RDP classiﬁer. ( ) Pseudomonas; ( ) Janthi- nobacterium; ( ) Methylophilus; ( ) Stenotrophomonas; ( ) Undi- bacterium; ( ) Dechloromonas; ( ) Acidovorax; ( ) Bacteroidetes; ( ) Flavobacterium; ( ) Curvibacter; ( ) Porphyrobacter; ( ) Sphingo- monas; ( ) Nevskia; ( ) Sphingopyxis; ( ) Novosphingobium; ( ) Methylobacterium; ( ) Acinetobacter; ( ) Unclassiﬁed_Methylophila- ceae; ( ) Unclassiﬁed_Rhizobiales; ( ) Unclassiﬁed_Erythrobactera- ceae; ( ) Unclassiﬁed_Pseudomonadaceae; ( ) Unclassiﬁed. Bacterial community composition and succession over time The T-RFLPs proﬁles (Fig. 4) and the sequencing analysis of the clone libraries (Figs 6–9) showed clear changes in the bacterial community composition over time. The dominant phylum in the clone libraries was Proteobacteria, and within this, Gammaproteobacteria and Betaproteobacte- ria were predominant (Fig. 6). However, the percent- age of these two bacterial classes varied over time and 293 Applied Microbiology 117, 286--301 © 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology Bacteria in drinking water bioﬁlms I. Douterelo et al. Table 2 Phylogenetic afﬁliations and percentage of similarity between the cloned 16S rRNA gene and its closest relative in the NCBI database using the BLAST algorithm Closest relative in GenBank No. of clones Similarity % Accession number Bacterial group Clone Library Day 7 Janthinobacterium sp. HC3-3 19 97 JF312973.1 Betaproteobacteria Pseudomonas sp. BcW159 14 99 FJ889609.1 Gammaproteobacteria Methylophilus methylothrophus 13 98–99 AB193724.1 Betaproteobacteria Pseudomonas sp. HC2-16 9 99 JF312964.1 Gammaproteobacteria Stenotrophomas maltophila R551-3 8 99 NR_074875.1 Gammaproteobacteria Duganella sp. S21012a 4 97–98 AB495351.1 Betaproteobacteria Pseudomonas ﬂuorescence LMG14674 4 99 GU198124.1 Gammaproteobacteria Acidovorax sp.BSB421 1 99 Y18617.1 Betaproteobacteria Curvibacter gracilis 1 99 AB109889.1 Betaproteobacteria Dechloromonas sp. SIUL 1 99 AF170356.1 Betaproteobacteria Herbaspirillum sp. PIV.34.1 1 95 AJ505863.1 Betaproteobacteria Methylotenera mobilis 1 94 AB698738.1 Betaproteobacteria Flavobacterium sp. WB2.1-78 1 93 AM167559.1 Flavobacteria Pseudomonas sp. BXFJ-8 1 99 EU013945.1 Gammaproteobacteria Pseudomonas veronni 1 99 AB334768.1 Gammaproteobacteria Clone Library Day 14 Pseudomonas ﬂuorescens LMG 14674 14 98–99 GU198124.1 Gammaproteobacteria Dechloromonas hortensis MA-1 13 98–99 NR_042819.1 Betaproteobacteria Methylophilus rhizosphaerae strain 103a 8 98–99 AB698737.1 Betaproteobacteria Methylophilus methylothrophus 7 99 AB193724.1 Betaproteobacteria Curvibacter gracilis strain 7-1 4 97–98 NR_028655.1 Betaproteobacteria Methylomonas clara strain DSM 6330 4 96–99 HF564897.1 Betaproteobacteria Janthinobacterium sp. HC3-3 2 96–99 JF312973.1 Betaproteobacteria Janthinobacterium sp. SON-1402 2 96–99 JX196629.1 Betaproteobacteria Sphingopyxis sp. BZ3 0 2 99 GQ131578.1 Alphaproteobacteria Uncultured bacterium clone 1C226551 2 96 EU799001.1 Betaproteobacteria Uncultured Betaproteobacteria clone AEP-eGFP- 2 99 FJ511736.1 Betaproteobacteria Acinetobacter sp. K7SC-11A 1 99 JF99965.1 Gammaproteobacteria Dechloromonas sp. JD15 1 100 JN873345.1 Betaproteobacteria Dechloromonas sp. SIUL 1 98 AF170356.1 Betaproteobacteria Methylovorus glucosotrophus strain DSM68 74T 1 95 FR733702.1 Betaproteobacteria Pseudomonas sp. EC3(2012) 1 99 JX912405.1 Gammaproteobacteria Stenotrophomonas sp. Bacterial community composition and succession over time Bg23-2 1 99 HF548414.1 Gammaproteobacteria Uncultured bacterium clone 2M04 1 99 EU835430.1 Betaproteobacteria Uncultured bacterium clone EDWO7B003-11 1 96 HM066437.1 Betaproteobacteria Uncultured bacterium cloneMB50-16 1 97 JN825265.1 Betaproteobacteria Uncultured Methylobacillus clone R15-96 1 95 JF808844.1 Betaproteobacteria ( d) (Continued) 294 nal of Applied Microbiology 117, 286--301 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Microbio Bacteria in drinking water bioﬁlms I. Douterelo et al. I. Douterelo et al. ( ) Closest relative in GenBank No. of clones Similarity % Accession number Bacterial group Clone Library Day 28 Comamonadaceae bacterium ED16 23 97 FJ755906.1 Betaproteobacteria Porphyrobacter sanguineus 11 99 AB062106.1 Alphaproteobacteria Stenotrophomonas maltophilia R551-3 6 99 NR_074875.1 Gammaproteobacteria Undibacterium sp. C3. 6 96 JQ417431.1 Betaproteobacteria Methylophilus methylothrophus 4 99 AB193724.1 Betaproteobacteria Oxalobacteraceae bacterium CHNTR40 4 99 DQ337591.1 Betaproteobacteria Pseudomonas sp. BFXJ-8 3 99 EU013945.1 Gammaproteobacteria Sphingomonas sp. M16 3 99 GU086440.1 Alphaproteobacteria Undibacterium sp. M4-14 3 97 HE616176.1 Betaproteobacteria Pseudomonas veronii 2 99 AB334768.1 Gammaproteobacteria Uncultured Nevskia sp. Clone T13M-B4 2 98 JN860401.1 Gammaproteobacteria Acidovorax sp. isolate G8B1 1 99 AJ012071.1 Betaproteobacteria Janthinobacterium sp. HC3-3 1 99 JF312973.1 Betaproteobacteria Janthinobacterium sp. S21124 1 99 D84576.2 Betaproteobacteria Methylobacterium marchantiae 1 99 AB698714.1 Alphaproteobacteria Methylobacterium sp. Mp3 1 99 EF015480.1 Alphaproteobacteria Nevskia ramosa 1 98 AJ001343.1 Gammaproteobacteria Nevskia sp. KNF011 1 99 AB426555.1 Gammaproteobacteria Novosphingobium sp. FND-3 1 99 DQ831000.1 Alphaproteobacteria Novosphingobium sp. HLT3-9 1 99 JX9493761.1 Alphaproteobacteria Pseudomonas ﬂuorescens 1 98 AM410631.1 Gammaproteobacteria Pseudomonas putida 1 99 EU275363.1 Gammaproteobacteria Pseudomonas sp. HC2-30 1 96 JF312926.1 Gammaproteobacteria Sphingomonas sechinoides S32312 1 99 AB649019.1 Alphaproteobacteria Uncultured Methylophilaceae bacterium 1 98 HE648207.1 Betaproteobacteria Undibacterium parvum 1 98 AM397629.1 Betaproteobacteria representatives of the Alphaproteobacteria increased in the day 28 clone library. Gammaproteobacteria, Betaproteobac- teria and Alphaproteobacteria were also the predominant phyla in a previous study, where the bacterial composi- tion of bioﬁlms obtained from the same experimental distribution system at day 28 was analysed using 454 pyrosequencing (Douterelo et al. 2013). The dominance of Proteobacteria in bioﬁlms has been previously observed in other drinking water ecosystems, for example in rubber coated valves of a nonchlorinated network (Schmeisser et al. 2003), in a nonchlorinated model system made of stainless steel and supplied with groundwater (Martiny et al. 2005), in a simulated drinking water supply system using two different materials steel and polymethylmetacry- late (Lopes et al. Bacterial community composition and succession over time 5a), suggesting that these two habitats share a limited number of bacteria and that only some free-living bacteria transported from the bulk water are able to attach to the pipe surfaces and form bioﬁlms, supporting previous observations (Doute- relo et al. 2013). Figure 9 Venn diagram of bacterial OTUs clustered with a 3% dis- tance similarity threshold, showing the number of OTUs shared by the clone libraries from days 7, 14 and 28. Number of OTUs in day 7 = 12; number of OTUs in day 14 = 14; number of OTUs in day 28 = 20; total richness of all groups = 32; number of OTUs shared between day 7 and day 14 = 7; number of OTUs shared between day 7 and day 28 = 7; number of OTUs shared between day 14 and day 28 = 5; total shared richness = 5. From the sequencing data, we can deduce that bacteria related to Pseudomonas spp., Janthinobacterium spp. and Methylophilus spp. were involved in the initial process of attachment to the pipe surface (Fig. 7). The occurrence of these bacteria in the initial phase of bioﬁlm formation can be explained due to their enhanced capability, when compared with other planktonic bacteria, of producing EPS when they establish contact with a surface and/or molecules required for cell-to-cell communication (Don- lan 2002; Huq et al. 2008). Pseudomonas spp. is one of the most abundant bacteria found in water distribution systems (Martiny et al. 2005), and several species of Pseu- domonas, like Ps. ﬂuorescens, are capable of synthesizing surfactants involved in cell-to-cell communication (Sim- oes et al. 2003); others like Ps. aeruginosa can produce alginate and exopolysaccharides when in contact with different surfaces (Huq et al. 2008). Martiny et al. 2003, when studying long-term succession of bioﬁlms in a model DWDS supplied with groundwater and using tur- bulent ﬂow conditions (ﬂow velocity of 007 m s1), also found that bacteria related to Pseudomonas spp. were predominant in the initial attachment of cells to stainless steel. As a consequence, we can conclude that Pseudomonas spp. is an ubiquitous bacteria involved in the process of bioﬁlm formation in DWDS made of dif- ferent materials and under diverse hydraulic conditions. We observed that after the initial colonization of a few bacterial species (from day 0 to 7), the modiﬁed surface of the pipe was attractive for other species (Table 2). Bacterial community composition and succession over time 2009) and in a pilot distribution system made of cement-lined cast iron (Mathieu et al. 2009). This study corroborates the predominance of this bacterial group for bioﬁlms up to 28 days old in DWDS made of HDPE, a representative material used in distributions systems worldwide, supplied with surface water and with a chlorine disinfectant residual. representatives of the Alphaproteobacteria increased in the day 28 clone library. Gammaproteobacteria, Betaproteobac- teria and Alphaproteobacteria were also the predominant phyla in a previous study, where the bacterial composi- tion of bioﬁlms obtained from the same experimental distribution system at day 28 was analysed using 454 pyrosequencing (Douterelo et al. 2013). The dominance of Proteobacteria in bioﬁlms has been previously observed in other drinking water ecosystems, for example in rubber coated valves of a nonchlorinated network (Schmeisser et al. 2003), in a nonchlorinated model system made of stainless steel and supplied with groundwater (Martiny et al. 2005), in a simulated drinking water supply system using two different materials steel and polymethylmetacry- late (Lopes et al. 2009) and in a pilot distribution system made of cement-lined cast iron (Mathieu et al. 2009). This study corroborates the predominance of this bacterial group for bioﬁlms up to 28 days old in DWDS made of HDPE, a representative material used in distributions Additional OTU-based analysis of the clone libraries conﬁrmed a gradual increase in species richness and diversity over time during the 28 days of material accu- mulation (Fig. 8). In agreement with our results, previous research on bioﬁlms showed that only a limited number of pioneer micro-organisms are able to initially attach to a surface, change the characteristics of the surface and start to form bioﬁlms. However, several of these studies were carried out on glass slides (Jackson et al. 2001; Paris et al. 2007; Dogru€oz et al. 2009), which do not reﬂect realistic conditions in DWDS, or in a model DWDS made of stainless steel supplied with groundwater and without disinfectant residual (Martiny et al. 2003). We have conﬁrmed that the process of bacterial bioﬁlm succession occurs under realistic conditions in chlori- nated drinking water networks, using a full-scale HDPE test loop facility, and we were also able to identify the 295 Microbiology 117, 286--301 © 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology. Bacteria in drinking water bioﬁlms I. Douterelo et al. Bacterial community composition and succession over time 0 OTUs Number of Sequences 0 81 19·50 1·81 15·00 1·61 1·85 2·32 11·75 17·00 2·26 2·48 33·00 42·50 72 85 N Day 7 Day 14 Day 28 H’ Chao I H’ Chao I Distance 0·03 Distance 0·05 10 20 30 40 50 60 70 80 2 4 6 8 10 12 14 16 18 20 Figure 8 Rarefaction curves at 97% of sequence similarity showing observed OTUS for the three clone libraries (samples from days 7, 14 and 28). ( ) Day 7; ( ) Day 14; ( ) Day 28. 0 OTUs Number of Sequences 0 10 20 30 40 50 60 70 80 2 4 6 8 10 12 14 16 18 20 Day 14 7 2 0 5 2 3 Number of OTUs in Day 7 = 12 Number of OTUs in Day 14 = 14 13 Day 28 Day 7 Day 14 7 2 0 5 2 3 Number of OTUs in Day 7 = 12 Number of OTUs in Day 14 = 14 Number of OTUs in Day 28 = 20 Number of OTUs shared between Day 7 and Day 14 = 7 Number of OTUs shared between Day 7 and Day 28 = 7 Number of OTUs shared between Day 14 and Day 28 = 5 Total richness of all groups = 32 Total shared richness = 5 13 Day 28 Day 7 Day 14 7 Number of Sequences Number of OTUs in Day 7 = 12 Number of OTUs in Day 14 = 14 Number of OTUs in Day 28 = 20 Total richness of all groups = 32 Figure 8 Rarefaction curves at 97% of sequence similarity showing observed OTUS for the three clone libraries (samples from days 7, 14 and 28). ( ) Day 7; ( ) Day 14; ( ) Day 28. Number of OTUs shared between Day 7 and Day 14 = 7 Number of OTUs shared between Day 7 and Day 28 = 7 Number of OTUs shared between Day 14 and Day 28 = 5 Total shared richness = 5 bacteria involved in the initial development of bioﬁlms in this type of DWDS. We also observed that bacterial community ﬁngerprints were clearly different between bulk water and bioﬁlms in pre- and postﬂushing samples (Fig. Bacterial community composition and succession over time After 2 weeks of bioﬁlm development, clones related to Dechloromonas spp. become highly abundant. Pseudomo- nas spp. losses its predominance, and new clones related to other species such as Curvibacter, spp. Methylomonas 296 f Applied Microbiology 117, 286--301 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology. I. Douterelo et al. Bacteria in drinking water bioﬁlms 15 Day 7 Day 14 Day 28 Bioflim depth (µm) Area fraction 10 5 0 –5 –10 –15 15 10 5 0 –5 –10 –15 15 10 5 0 –5 –10 –15 0·000 0·002 0·004 0·006 0·008 0·010 Area fraction 0·000 0·002 0·004 0·006 0·008 0·010 Area fraction 0·000 0·002 0·004 0·006 0·008 0·010 Figure 10 Analysis of confocal images showing changes in cell coverage over time. The graphs represent the bioﬁlm relative area fraction of ﬁve ﬁelds of view of three plastic inserts obtained from PWG coupons. Fields of view below the ﬂuorescence threshold are not represented in the graph. 15 Day 7 Day 14 Day 28 Bioflim depth (µm) Area fraction 10 5 0 –5 –10 –15 15 10 5 0 –5 –10 –15 15 10 5 0 –5 –10 –15 0·000 0·002 0·004 0·006 0·008 0·010 Area fraction 0·000 0·002 0·004 0·006 0·008 0·010 Area fraction 0·000 0·002 0·004 0·006 0·008 0·010 Area fraction Area fraction Area fracti Area fraction Area fraction surface and/or to attach to a growing bioﬁlm. From the data obtained in this study, we can conclude that species related to Pseudomonas spp., Janthinobacterium spp. and Methylophilus spp. were primary colonizers of the HDPE pipe surface and that other species such as Dechloromonas spp., Curvibacter spp. and Sphingopyxis spp. were proba- bly secondary colonizers. spp. and Sphingopyxis spp. were now present. Finally on day 28, higher OTUs richness and diversity were detected (Table 2), and members of genera such as Undibacterium spp., Porphyrobacter spp., Nevskia spp. and Sphingomonas spp. became represented in the clone library (Fig. 7). It has been hypothesized that these secondary colonizers are able to attach to a growing bioﬁlm aided by their capabil- ity to use the metabolites excreted or the remains gener- ated by the ﬁrst colonizers (Szewzyk et al. 2000). Applied Microbiology 117, 286--301 © 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology Bacterial community composition and succession over time In agreement with the DNA-based results, the analysis of confocal microscopy z-stack images obtained from steady state conditions showed that on day 7 only a limited number of cells attached to the coupon surface, making their detection difﬁcult. Cell coverage increased substan- tially after another week of development in the system, and from day 14 to 28, the area covered by the bioﬁlm increased up to three times. The role of most of the bacteria detected in drinking water bioﬁlms remains uncertain. For example, Dechloro- monas spp., which was predominant in the clone library of day 14, are widely distributed in the environment (Vigliotta et al. 2010) and are known perchlorate-reduc- ing bacteria (Oosterkamp et al. 2011), but their metabolic or physiological function in drinking water bioﬁlms is not well known. Chlorate-reducing bacteria have been detected in DWDS in the USA, associated with releases of ammonium perchlorate by military- and aerospace- related industries (Urbansky 2002). Their presence in our chlorinated system might be related to their capability of reducing nitrate instead of chlorate, but this assumption needs further investigation (Oosterkamp et al. 2011). Similarly, Methylophylus spp., which was highly abundant in the three clone libraries of this study and also in water meters from a DWDS (Hong et al. 2010), can oxidize methyl compounds and can possible carry out denitriﬁca- tion, but the particular function that makes them so abundant in the early stages of bioﬁlm formation has not been clariﬁed yet. Some of the bacterial genera detected in our clone libraries have been previously found in other drinking water-related ecosystems; for example, Porphyrobacter spp., Sphingopyxis spp. and Undibacterium spp. were detected in bioﬁlms of a membrane ﬁltration system in a treatment plant (Kwon et al. 2011). Revetta et al. 2010, using RNA to develop 16S rRNA-based clone libraries, showed that metabolically active Proteobacteria such as Porphyrobacter spp. and Sphingomonas spp. were present in bulk water samples from a chlorinated drinking water network. However, from most of these studies, it was not clear at which stage in the process of bioﬁlm formation these planktonic bacteria were able to attach to the pipe We were able to identify bacterial groups involved in initial attachment to HDPE pipes and subsequent adhesion 297 Bacteria in drinking water bioﬁlms I. Douterelo et al. logical composition of the bioﬁlms at the early stages of their formation but that they might inﬂuence the physical structure (e.g. Inﬂuence of hydraulic regimes on bioﬁlm development and mechanical stability Based on ﬁngerprinting analysis (T-RFLPs), the hydraulic regimes did not affect the bacterial community structure during the ﬁrst 28 days of bioﬁlm development (Fig. 3). This suggests that independently of the hydraulic regime, similar planktonic species were involved in the process of surface attachment and initial bioﬁlm formation. Never- theless, these results need to be interpreted with caution due to the impossibility of obtaining enough DNA for fur- ther 16S rRNA ampliﬁcations from all the samples on days 3 and 7 under varied ﬂow conditions and the fact that ﬁn- gerprinting techniques only yield information about the most abundant members of microbial communities. Another factor which might inﬂuence our results is that we have used a common reservoir to feed the three loops; as a consequence, the similar physico-chemical and microbio- logical characteristics of the water circulating within the system might contribute to the lack of inﬂuence of the hydraulic regimes in our experimental system. The enhanced capability for producing EPS in particular polysaccharides and/or adaptative physical structures like extracellular appendages in response to ﬂuctuating ﬂows might contribute to the mechanical stability of bioﬁlms and might aid certain types of bacteria in remaining attached to a surface after an increase in ﬂow velocity (Sim- oes et al. 2010). Accordingly, Liu et al., 2002 observed that micro-organisms respond to shear forces by producing stronger bioﬁlms under higher hydrodynamic shear forces and bioﬁlms become more porous and weaker under low shear forces. In addition, it has been proven that quorum- sensing mechanisms are involved in the attachment and detachment of microbial cells from bioﬁlms (Donlan 2002) and these mechanisms of communication might be enhanced under variable ﬂow conditions. This study high- lights that under representative hydraulic conditions, the strength of bioﬁlm attachment to pipe surfaces is higher under varied ﬂow conditions which can be translated into lower risk of water discolouration. By ﬂushing, we removed attached micro-organisms from the pipes together with particles such as iron and manga- nese which are most likely conﬁned within bioﬁlms, as can be seen from turbidity and metal concentration changes after ﬂushing (Fig. 2). Combining the T-RFLPs results from pre- and postﬂushing bioﬁlm samples (Fig. 5) and the water turbidity data (Fig. 2) obtained during ﬂushing, it can be suggested that the hydraulic regimes did affect the mechanical stability of bioﬁlms. Bacterial community composition and succession over time shape and distribution on pipes) of the bio- ﬁlm and potentially also their biochemical composition (i.e. amount of polysaccharides, lipids, etc.). Several bioﬁlm-related studies showed that high shear stress and turbulent ﬂow conditions can promote the development of more compact bioﬁlms due mainly to the production of extracellular polymers; this trend was observed for example in a bioﬁlm airlift suspension (Kwok et al., 1998), using bench-scale bioﬁlm-monitoring reactors (Manuel et al., 2010) and in single-species bioﬁlms consisting of Ps. ﬂuo- rescens (Pereira et al., 2002). The observed inﬂuence that different hydraulic regimes had on the cohesiveness of bio- ﬁlms developed in our full-scale DWDS agrees with obser- vations made in a rotary disk reactor by Abe et al., (2012) that a more cohesive bioﬁlm structure can be more resis- tant to external shear stress and detachment. of cells to growing bioﬁlms in chlorinated drinking water networks, but further study into their speciﬁc functions and their role within bioﬁlms is still necessary to move for- ward in this research ﬁeld and hence to deliver strategies to manage and control bioﬁlm development in DWDS. In addition, longer studies would be needed to evaluate the succession of bacterial communities in more mature bio- ﬁlms and to establish if at some point the bioﬁlm reaches a dynamic stability or equilibrium. f Applied Microbiology 117, 286--301 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology. 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https://openalex.org/W2622404422	http://eprints.lse.ac.uk/80398/1/Jimenez_Martinez_Making%20Chile%20visible%20purpose.pdf	English	null	Making Chile Visible: Purposes, Operationalisation and Audiences from the Perspective of Nation Branding Practitioners	Geopolitics	2,017	public-domain	12,361	César Jiménez-Martínez Making Chile visible: purposes, operationalisation and audiences from the perspective of nation branding practitioners Article (Accepted version) (Refereed) Original citation: Jiménez-Martínez, César (2017) Making Chile visible: purposes, operationalisation and audiences from the perspective of nation branding practitioners. Geopolitics. pp. 1-23. ISSN 1465-0045 DOI 10 1080/14650045 2017 1329724 1465-0045 DOI: 10.1080/14650045.2017.1329724 DOI: 10.1080/14650045.2017.1329724 DOI: 10.1080/14650045.2017.1329724 Original citation: Jiménez-Martínez, César (2017) Making Chile visible: purposes, operationalisation and audiences from the perspective of nation branding practitioners. Geopolitics. pp. 1-23. ISSN 1465-0045 O Abstract Chile has become the paradigm of nation branding in Latin America, employing branding initiatives to try to shake off the uncomfortable past of Augusto Pinochet’s dictatorship, as well as to create a ‘new’ image that fits into a globalised and neoliberal world. Whilst there has previously been some analysis of Chile’s branding efforts, the viewpoints of the local actors involved in these initiatives have largely been ignored. This article addresses this issue, examining the tensions faced by different individuals who have taken part in nation branding in the country. Drawing on interviews with some of these individuals, this article examines three areas of tension: (1) the conflicting purposes guiding the practice of nation branding, (2) the difficulties around the operationalisation of nation branding and (3) the controversies regarding the intended audiences for their efforts. © 2017 Taylor & Francis Group, LLC © 2017 Taylor & Francis Group, LLC This version available at: http://eprints.lse.ac.uk/80398/ Available in LSE Research Online: June 2017 LSE has developed LSE Research Online so that users may access research output of the School. Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Users may download and/or print one copy of any article(s) in LSE Research Online to facilitate their private study or for non-commercial research. You may not engage in further distribution of the material or use it for any profit-making activities or any commercial gain. You may freely distribute the URL (http://eprints.lse.ac.uk) of the LSE Research Online website. This document is the author’s final accepted version of the journal article. There may be differences between this version and the published version. You are advised to consult the publisher’s version if you wish to cite from it. Introduction In September 2008 I joined –along with five other colleagues– Ogilvy Public Relations Chile to work as an account executive on a new and ambitious project to promote Chile abroad. The stakes were high and exciting. Our client, the Export Promotion Bureau ProChile, told us that, although located in a peripheral or semi-peripheral geopolitical position,1 Chile was a country with great potential, with resources such as stunning geography, political stability and high quality food exports, as well as a good economic performance since the mid-1980s. We were informed that Chile was invisible to most people, and that such invisibility was detrimental to Chile’s future economic prospects. Consumers were unaware of the strength of Chilean exports and investors did not have enough information about the opportunities offered by the country. We had been hired to change that. As part of the newly formed Proyecto Chile –Imagen País (Chilean National Image Project), we were responsible for implementing a strategy to make Chile better 1 known in seven countries considered ‘priority markets’: the United States, the United Kingdom, Spain, Germany, Mexico, Japan and China. The strategy was guided by the nation branding hexagon proposed by British consultant Simon Anholt,2 who had been recently hired by the Chilean government. The hexagon focuses on six indicators of a national brand: exports, governance, investment and immigration, culture and heritage, people and tourism.3 Using those indicators as guides, we organized press trips for foreign journalists and bloggers; we sought potential stories that could portray Chile in a positive light for international news media, and we proudly highlighted the fact that Quantum of Solace, then the most recent James Bond movie, had been partially filmed in Chile. The project was supposed to last several years and incorporate more ‘priority markets’ over time. However, only six months later it was over. A new private but publicly funded organisation, Fundación Imagen de Chile (Image of Chile Foundation), had been created to develop and manage a narrative designed to make Chile more visible all over the world. Although short-lived, my experience working at Ogilvy Public Relations Chile marked the beginning of a personal journey. Over the next two years I worked on two smaller projects promoting specific characteristics of Chile abroad, before starting to critically engage with this topic from an academic viewpoint. Introduction It also signalled the beginning of a series of public debates among local politicians, businessmen and authorities about how to construct and project abroad a positive national image or marca país (‘country brand’ in Spanish, the term locally used to refer to nation branding).4 Such brand did not intend to merely encompass a territory, but also a set of political, economic and cultural characteristics that allegedly made Chile unique. Significantly, it was claimed that a good brand had the supposed potential of improving Chile’s position in the international arena. Hence, concerns about Chile’s international reputation were predominantly framed as discussions about nation branding. Various domestic episodes –such as the earthquake of March 2010, the rescue of 33 Chilean miners in October 2010 and the performance of the 2 2 national football team in several sporting tournaments– were evaluated in terms of their potential to positively or negatively affect the ‘Chile brand’.5 Despite the social, political and economic significance attached to nation branding in Chile, which has been noticed by foreign observers,6 the viewpoints of the actors behind local nation branding initiatives have been largely neglected.7 Early nation branding studies –both critical works as well as those aimed at practitioners– were dominated by case studies from the United States and Western Europe. The global spread of nation branding led various authors to examine examples from Asia, Africa and Eastern Europe.8 Latin America was predominantly ignored in the literature. Whilst recent studies have paid attention to the region,9 they have mostly provided an analysis of media coverage, documents and specific campaigns. Therefore, this article seeks to address this neglected topic, shedding light on the tensions that some of the individuals constructing and projecting a particular version of Chile through nation branding have faced for the last decade. The discussion has five sections. The first looks at the theoretical aspects, and suggests that nation branding is a manifestation of Dijkink’s ‘geopolitical visions’.10 Hence, I argue that the concept of visibility is particularly useful for analysing the purposes, operationalisation and intended audiences of the individuals involved in nation branding. The historical context is then considered to further understand the emergence and embrace of nation branding in Chile. I next examine the difficulties of making a single national brand visible to individuals and organisations with diverse and sometimes opposite goals. Nation Branding, Geopolitical Visions and Visibility Nation Branding, Geopolitical Visions and Visibility Along with public diplomacy, nation branding is the most recent incarnation of the task of projecting a positive image of a nation abroad. Whilst there are competing definitions of what nation branding is, there is some consensus that it is the use of marketing and advertising techniques to enhance the reputation of a nation.11 Since the late 1990s, governments from all over the world have spent hefty sums of money engaging in various initiatives that promise to re-build and project ‘new’ or ‘updated’ versions of national identity, in order to advance political, economic and or cultural agendas.12 Nation branding advocates claim that the globalisation of capitalism and the spread of communication technologies made the international image or reputation of a nation as important as economic or military power.13 Van Ham argues that the increasing relevance of nation branding signals a shift from geopolitics and power to an emphasis on symbols and influences.14 Yet nation branding does not necessarily imply a complete departure from geopolitics. Rather, it introduces a new level of complexity for their analysis.15 Nation branding can be understood as a manifestation of what Dijkink calls ‘geopolitical visions’, that is, ‘any idea concerning the relation between one’s own and other places, involving feelings of (in)security or (dis)advantage (and/or) invoking ideas about a collective mission or foreign policy strategy’. Whilst works for practitioners dominated the early nation branding literature,16 in recent times an increasing number of studies have addressed this phenomenon from a more critical viewpoint. Studies have drawn on theoretical concepts and perspectives from sociology, media and communications, anthropology, and international relations, among other fields.17 These analyses have made substantial contributions, highlighting how nation branding initiatives commodify national identity and craft a fairly homogenous version of the nation, which masks diversity and internal conflicts.18 In this article, I do draw on that critical literature. However, I depart from these works theoretically, proposing that the concept of visibility can be helpful when examining nation branding as geopolitical visions in practice. Approaching nation branding through the concept of visibility means taking into account not only the visible –campaigns, slogans, taglines, Whilst works for practitioners dominated the early nation branding literature,16 in recent times an increasing number of studies have addressed this phenomenon from a more critical viewpoint. Introduction I outline then some of the tensions cited by practitioners in regard to the operationalisation of nation branding. Finally, I discuss some of the controversies they faced in relation to the audiences to whom direct their efforts. 3 3 Nation Branding, Geopolitical Visions and Visibility Studies have drawn on theoretical concepts and perspectives from sociology, media and communications, anthropology, and international relations, among other fields.17 These analyses have made substantial contributions, highlighting how nation branding initiatives commodify national identity and craft a fairly homogenous version of the nation, which masks diversity and internal conflicts.18 In this article, I do draw on that critical literature. However, I depart from these works theoretically, proposing that the concept of visibility can be helpful when examining nation branding as geopolitical visions in practice. Approaching nation branding through the concept of visibility means taking into account not only the visible –campaigns, slogans, taglines, 4 videos–, but also the dynamism and nuance of the practices, perceptions and beliefs of the individuals behind nation branding efforts.19 The concept of visibility is often mentioned in the literature, for instance, when claiming that nation branding initiatives enable nations to ‘achieve greater visibility’.20 However, it is rarely theorised. Only in the last fifteen years have various theorists and researchers from sociology, urban studies, gender studies, and media and communications focussed on scrutinising what visibility is and what its implications are. Thompson has studied how technological advances have contributed to the development of a ‘new visibility’, with the production of a greater number of images that can potentially achieve a wider geographical reach.21 Significantly, Thompson observes that visibility, particularly through the media, ‘has become a principal means by which social and political struggles are articulated and carried out’.22 Visibility has become a political concern,23 ‘a right frequently and sometimes violently claimed; a right that all sorts of people feel entitled to obtain’.24 Such perspectives understand visibility as a means to achieve political recognition and representation. Similarly, nation branding advocates claim that the visibility of the nation is not only a right, but a necessity.25 Lacking a well-defined national brand means being left behind in the global competition for capital, tourism and political influence. The argument follows that governments must not only struggle for the visibility of the nation, but also control the terms of such visibility, managing its construction and projection to achieve their goal. Hence, nation branding proposes geopolitical ‘visions of order’ to maximise potential advantages and mitigate risks in the international arena.26 Yet visibility is not merely a source of recognition, but also a form of surveillance. Nation Branding, Geopolitical Visions and Visibility Being seen can be a way to be policed and subject to discipline and control.27 Foucault famously stated that visibility was ‘a trap’28 when writing about the Panopticon, a prison model in which inmates do not know whether or not they are being observed by guards. stated that visibility was ‘a trap’28 when writing about the Panopticon, a prison model in which inmates do not know whether or not they are being observed by guards. Furthermore, recognition and control are not in direct opposition to each other. They may in fact overlap. For instance, the development of a nation brand seeks to enhance Furthermore, recognition and control are not in direct opposition to each other. They may in fact overlap. For instance, the development of a nation brand seeks to enhance 5 visibility, and at the same time to discipline the nation and its inhabitants into forms of behaviour suitable for the global market.29 Hence, as Brighenti holds, ‘a way of seeing is a way of recognising and, at the same time, controlling’.30 Despite the political implications of visibility, Banet-Weiser observes that practitioners and some theorists have increasingly shifted their attention from the politics of visibility towards the economies of visibility.31 Whist the politics of visibility focus on how traditionally marginalised groups seek visibility as a means to produce further political – and geopolitical– change, the economies of visibility understand visibility as an end in itself. From this perspective, visibility is a way to facilitate the transaction of specific products –such as bodies, goods or, in this case, nation brands– in a market. When viewed from a predominantly economic perspective, questions about the purposes, modes of operationalisation as well as targeted audiences –the what, how and for whom– of those seeking visibility are often overlooked.32 Hence, a critical engagement with the concept of visibility may stress the (geo)political aspect of nation branding. Indeed, a thorough examination of visibility means addressing not only what is seen –logos, marketing campaigns, slogans–, but also sociocultural contexts, beliefs as well as the practices of the individuals taking part in the production of nation branding.33 As Voirol observes, ways of doing are also ways of seeing.34 The use of visibility as a theoretical framework highlights the need to scrutinise the discourses and viewpoints of the actors producing nation branding initiatives. Nation Branding, Geopolitical Visions and Visibility The critical nation branding literature has increasingly addressed the perspectives of the individuals engaged in these initiatives in a series of settings.35 Furthermore, as Kaneva suggests, nation branding studies should adopt a materialist perspective in order to ‘uncover the structural conditions and processes which underlie the ascendance of nation branding to a hegemonic status in contemporary international relations’.36 Some of those conditions and processes are outlined in the next section, which provides some historical context to further understand the emergence and embracing of nation branding in Chile. 6 6 Context: Nation Branding in Chile Whilst nation branding originated in the United Kingdom and the United States, elites from ‘emerging’ or ‘developing’ nations –such as those from Latin America– have enthusiastically followed the advice of nation branding advocates and experts.37 For the governments of Peru, Colombia, Mexico and Chile, among others, nation branding has represented a relatively cheap way to enhance the visibility of their nations abroad in order to favourably shape geopolitical visions. Local authorities have attempted to leave behind perceptions of exoticism, dictatorial governments, failed economies, or pre- modernity in order to attain political, cultural but mostly economic goals.38 The positive reception of nation branding in Latin America has to be examined through the prism of broader historical processes. Despite various episodes of internal debate and contestation, nation building in Latin America was traditionally a top-down process. States constructed, fostered –often violently– and projected a sense of homogeneous national identity amongst heterogeneous and geographically dispersed peoples. Latin American states have tried to maintain control over specific versions and visions of what supposedly constitutes the authentic nations that they claim to represent.39 In Chile, having secured independence from Spain in the early 19th century, local political and economic elites selected the representative features of the newly-formed nation, abandoning those considered undesirable.40 Nation branding is compatible with these historical aspirations. Despite claims made by nation branding advocates about the need to involve the general population to identify the ‘core idea’ –or, using Dijkink’s terminology, the ‘collective mission’– of a nation,41 the final decisions regarding what constitutes the national brand are taken by a narrow group of individuals. It is expected that the rest of the population merely adapt to the decisions taken by elites, disciplining and adjusting their behaviour to contribute to the enactment of a national portrayal appealing to foreigners.42 7 Attempts to construct a positive image of Chile for foreigners can be found in the 19th century. These attempts were primarily driven by economic goals. State-sponsored chronicles written by Chilean authors sought to attract potential Western European immigrants, in the hope that these immigrants would contribute to the development of the southern regions of the country. Publicly and privately funded campaigns promoting Chile abroad were pitched at increasing saltpetre sales. Context: Nation Branding in Chile This mineral became one of the pillars of the Chilean economy at the end of the 19th century.43 Yet the seeds of the current attempts to construct and project a version of Chile for foreigners can be found in the mid-1980s, when the local Export Promotion Bureau, ProChile, developed a campaign to boost exports to the United States, the United Kingdom, Spain, Japan and Singapore, through the depiction of Chile as an economically open and low-risk nation.44 An emphasis on economic virtues, underpinned by the neoliberal turn taken by Chile in the late 1970s, aimed to counteract the political isolation faced during the military dictatorship of Augusto Pinochet.45 With the end of Pinochet's dictatorship in 1990, Chile’s new authorities attempted to project a fresh image of their country, to secure its re-entry into the global arena, ridding itself of any connection with the Pinochet regime. For the Seville Expo 1992, the Chilean pavilion had an iceberg as its main attraction. The iceberg attempted to differentiate Chile from the rest of Latin America, depicting the country as a cold, formal and serious.46 With the end of Pinochet's dictatorship in 1990, Chile’s new authorities attempted to project a fresh image of their country, to secure its re-entry into the global arena, ridding itself of any connection with the Pinochet regime. For the Seville Expo 1992, the Chilean pavilion had an iceberg as its main attraction. The iceberg attempted to differentiate Chile from the rest of Latin America, depicting the country as a cold, formal and serious.46 Significantly, the new democratically elected Chilean government sought to distance itself from the previous regime in political terms, whilst keeping the same economic model. In the mid-2000s, the Chilean government hired foreign marketing and advertising specialists to develop a proposal for a marca país or nation brand.47 A motivation to create such a brand was the need to strengthen exports and increase investment in the light of several free trade agreements in negotiation, such as those with the United States and the European Union. As mentioned earlier, one of the advisors was Simon Anholt, the British branding consultant who coined the term ‘nation-brands’ –originally with a 8 8 hyphen– in the late 1990s.48 In 2008, Anholt began work for the government of Michelle Bachelet. His hiring also resulted in the inclusion of Chile in Anholt’s Nation Brand Index (NBI). Context: Nation Branding in Chile Anholt travelled all over the country in a series of well-publicised encounters with local politicians, businessmen, academics and artists, where he stressed the urgent need to develop a national brand to surmount the supposed invisibility of Chile in the international arena.49 In 2009 the Chilean authorities launched Fundación Imagen de Chile (Image of Chile Foundation), a private but publicly funded organisation to develop and sustain a narrative designed to make Chile more visible in the global imaginary.50 Whilst officials working at the foundation have stressed that their work is for the long term, its active campaigns have reflected the concerns of the government in power. During the administration of Sebastián Piñera, who led the country between 2010 and 2014, the foundation developed the campaign ‘Chile is good for you’ –‘Chile hace bien’ in Spanish, which means both ‘Chile is good for you’ and ‘Chile works well’–,51 which emphasised the country’s political stability, stunning geography and the resiliency of its people. This campaign was scrapped with the return of Michelle Bachelet to the Presidency in 2014, and replaced by another one that did not have a specific slogan, which stressed Chile as a trustworthy country, moving towards progress, with a mixture of tradition and modernity and an extreme geography.52 Notably, most of the features that have defined the global phenomenon of nation branding are present in the case of Chile: the need to shake off an uncomfortable past; the creation of a ‘new’ image which could fit into a perceived globalised, neoliberal world; the hiring of foreign experts for this task; and the development of new state-sponsored institutions that attempt to construct, enhance and manage the visibility of a particular version of the nation. Indeed, Chile has been considered a success story that should be imitated by other Latin American governments, such as Brazil.53 9 9 A Methodological Note As stated earlier, the viewpoints of the individuals engaged in nation branding in Chile have largely been neglected in the literature. To address this gap, I conducted seven semi- structured interviews with individuals who have taken part in the development and implementation of nation branding initiatives in Chile.54 All of them worked for the main public and private organisations that have been involved in nation branding initiatives in the country during the last decade: Image of Chile Foundation, ProChile, Ogilvy Public Relations and FutureBrand. Some of the interviewees have worked in both the public and private sector at different times. The interviews were conducted in Spanish, between March 2013 and September 2016. All interviewees requested anonymity, as most of them have remained involved directly or indirectly in the implementation of nation branding. Hence, I have changed their names and I mention their affiliation –or previous– at the time of the interview. My contact with some of them was facilitated by the fact that, as mentioned earlier, between 2008 and 2010 I worked as an account executive for Ogilvy Public Relations Chile on different nation branding projects. Indeed, I experienced directly some of the issues raised by the interviewees, which are addressed later in this article. I conducted a thematic analysis of the seven interviews to identify some of the main subjects raised by the informants and to examine how they understood these topics.55 I do not claim that this is an exhaustive study with such a limited number of respondents. It should be seen as the starting point for wider debates about nation branding in Chile as well as Latin America. I grouped the topics from the analysis of the interviews into three interrelated areas that follow the previous theoretical discussion about visibility. The areas of tension address the purposes (the what), operationalisation (the how) and the intended audiences (for whom) of nation branding initiatives in Chile. More concretely, the topics I examine are: What was the image of Chile that the interviewees were trying to make visible through nation branding initiatives? How did they operationalize the construction and projection of such image? To whom were they trying to communicate that particular national brand? 10 Purposes: What Image of Chile should be made visible? The first tension raised by the interviewees relates to the version of Chile that they tried to make visible. As discussed earlier, there have been various incarnations of nation branding in Chile, including the early attempts by ProChile in the mid 2000s, and the different campaigns carried out by the Fundación Imagen de Chile since its inception in 2009. Despite the differences in approaches and the different aims of the organisations involved in nation branding, all interviewees concluded that the main driving force behind nation branding was the attainment of economic goals. All interviewees stressed that features such as political stability, stunning geography and the warmth of the Chileans were equally important, but were relevant insofar as they were beneficial for Chile’s economy. Geography was important for tourism; political stability was required for foreign investment; and the character of Chileans mattered as evidence of a supposed entrepreneurial spirit. Interviewees agreed that their task was to portray Chile mostly as an economic unit, which produced raw materials, was suitable for foreign investment, and was an attractive tourist destination for foreigners. Fernando, from Fundación Imagen de Chile, observed that diplomats often instructed their embassy and consular communications teams to sell Chile abroad (personal communication, All interviewees stressed that features such as political stability, stunning geography and the warmth of the Chileans were equally important, but were relevant insofar as they were beneficial for Chile’s economy. Geography was important for tourism; political stability was required for foreign investment; and the character of Chileans mattered as evidence of a supposed entrepreneurial spirit. Interviewees agreed that their task was to portray Chile mostly as an economic unit, which produced raw materials, was suitable for foreign investment, and was an attractive tourist destination for foreigners. Fernando, from Fundación Imagen de Chile, observed that diplomats often instructed their embassy and consular communications teams to sell Chile abroad (personal communication, 2014). Such emphasis shows the previously discussed economies of visibility at play. Purposes: What Image of Chile should be made visible? Indeed, nation branding is a manifestation of what has been called ‘commercial nationalism’ or ‘economic nationalism’, that is, the primacy of economic practices as markers of nationhood, as well as the adoption of an economic viewpoint to evaluate the legitimacy of institutions.56 Indeed, nation branding is a manifestation of what has been called ‘commercial nationalism’ or ‘economic nationalism’, that is, the primacy of economic practices as markers of nationhood, as well as the adoption of an economic viewpoint to evaluate the legitimacy of institutions.56 Despite the consensus that Chile had to be shown primarily as an economic unit, different actors disagreed on the features that should be emphasised or masked in order to generate economic benefits. A former ProChile official and a former Ogilvy Public Relations Chile executive recalled that the first attempt to produce a nation brand for Chile was signalled by the adoption of the tagline ‘Chile, All Ways Surprising’ (‘Chile, Sorprende 11 11 Siempre’ in Spanish), developed by the branding company InterBrand in 2005. The logo and respective tagline aimed to show a ‘new’ Chile, with stars that supposedly signalled not only the beauty of the Chilean sky, but most significantly, a continuously moving and innovative business oriented nation which respected its past.57 The depiction of a nation as a mixture of tradition and modernity is far from original. The Janus-like face of nationhood, with ‘backward’ nations looking for stability in their past while at the same time embracing modernity,58 has become a staple of nation branding campaigns all over the world.59 Significantly, the excitement that the ‘All Ways Surprising’ campaign tried to communicate was soon subject to criticism within the government, which perceived it to have the potential to undermine the goals of various state agencies. As David, former official of ProChile, recalled: That brand [Chile, All Ways Surprising] was launched amidst lots of hype. There was tons of stationery ready and so on, but after a couple of months, the Foreign Investment Committee asked ProChile to remove the tagline. They said that the last thing an investor was going to find attractive was a country full of surprises. They preferred a country that was predictable, reliable and boring. The surprises worked mainly for tourists. So we ended up keeping the logo with the stars, but the tagline was scrapped (personal communication, 2013). Purposes: What Image of Chile should be made visible? David’s quote describes the supposed unsuitability of ‘All Ways Surprising’ to attract investment.60 It is also illustrative of a phenomenon that was cited by most interviewees. Despite their agreement about the relevance of carrying out nation branding initiatives for the attainment of economic purposes, interviewees stressed the difficulty of coordinating the various public and private entities, with their own individual goals, when putting these initiatives into practice. As mentioned earlier, the literature has examined how nation branding practitioners in various settings have faced several challenges –especially among themselves – when putting their initiatives into practice.61 Significantly The Fundación Imagen de Chile was created to coordinate nation branding work so mitigating controversies regarding Chile’s nation brand, but this proved difficult sometimes. Fernando, a former Fundación official, 12 recalled that during the global media frenzy after the 2010 rescue of 33 Chilean miners, the then President Sebastián Piñera wanted to change the campaign name ‘Chile is good for you’ to ‘Do it the Chilean way’ –the motto was in English language only–, which were supposedly Barack Obama’s words when discussing the successful rescue with Piñera. According to Fernando, various senior officials resisted Piñera’s efforts not only because ‘Chile is good for you’ was about to be launched, but also because they feared that ‘Do it the Chilean Way’ would become a source of parodies and jokes within Chile: Can you imagine how people would have mocked that sentence every time something did not work? It could have been very harmful in the long term (personal communication, 2014). The examples above highlight that, despite agreement about the supposed benefits of nation branding, harmony and coordination amongst the parties involved in these efforts are the exception rather than the rule. Hence, at the heart of nation branding lies a paradox: the promise to make visible a single and coordinated version of the nation, albeit with the involvement of individuals and organisations that actually have diverse and often opposite goals.62 Unsurprisingly, the promise of a coordinated version of the nation is rarely fulfilled. At best, it is possible to agree on making shallow statements. This is true for Chile’s current nation brand, which holds that the country has a diverse geography, a mixture of tradition and modernity, and people who are trustworthy and committed to progress.63 These features are hardly exclusive to Chile. Purposes: What Image of Chile should be made visible? Indeed, most interviewees openly expressed their doubts about the possibility of constructing and managing the visibility of a coordinated image of Chile. At the centre of these doubts was the realisation that it was almost impossible to reach a complete consensus regarding national identity. As Marcela, a former Fundación Imagen de Chile official stated: It is difficult to project outwards issues that have not been agreed internally. That is why I believe that the Fundación had a relevant role in bringing into the public domain the discussion about the kind of country that we are, where we want to go and what kind of future projects we have. And this narrative cannot be totalitarian, because there are spaces in which there simply is no agreement (personal communication, 2013). 13 13 This quote shows that finding the alleged ‘core idea’ or ‘collective mission’ of a nation, as encouraged by nation branding enthusiasts,64 is, in practical terms, an extremely difficult and perhaps even unattainable task. Nonetheless, the recognition that agreement may be impossible does not necessarily result in enrichment of debates about national identity, as proposed by some critical authors.65 As Marcela points out, in the Chilean case, discussions about what the nation is are still framed as a top-down process, managed by a state-sponsored organisation –‘the Fundación had a relevant role’. Furthermore, the final decision of what supposedly constitutes –and what does not– the Chilean national identity made visible by the brand remains in the hands of the foundation. Hence, paraphrasing Dayan, variability is ‘accepted but only within limits’, in this case, the limits imposed by Fundación Imagen de Chile.66 Operationalisation: How to Make Chile Visible? The second source of tension highlighted by interviewees relates to the operationalisation of nation branding. Despite the abundance of literature aimed at practitioners,67 the interviewees stated that there were no clear guidelines on how to implement as well as how to evaluate nation branding. I recall that, when working at Ogilvy Public Relations, we lacked clear instructions on how to empirically apply Anholt’s Nation Brand Hexagon.68 Given that the standard practice amongst public relation practitioners in our team was to produce press releases and measure success monitoring media coverage, we used the six sides of the hexagon as thematic categories to propose potential news stories and to classify media monitoring of Chile. Hence, whilst we adhered to Anholt’s model – at least superficially–, our practices hardly explored any new grounds, given that we settled on an established set of procedures that the team felt comfortable with. The literature often describes how nation branding practitioners, driven by commercial imperatives, increasingly make decisions about national identity that were previously made by the state.69 However, the interviews showed that the operationalisation of nation branding and the prevalence of a commercial viewpoint were not entirely dependent on 14 practitioners. Outcomes such as slogans, videos or taglines were sometimes the product of political and financial pressures unrelated to purely promotional concerns. For instance, the 2010 launch of the previously mentioned campaign ‘Chile is good for you’ was marked by a promotional video uploaded to YouTube and broadcast in several European countries.70 As Marcela, former Fundación Imagen de Chile official stated, that video –along with various other campaign outcomes – was primarily produced as a response to a particular set of political requirements and expectations seeking to justify the work and budget of the recently established foundation: ‘Chile is good for you’ was made because there was an expectation that the foundation had to deliver several products: a logo, a slogan and an important televisual advertisement. So it was begrudgingly that we produced a video in order to meet that expectation, do you understand? This is related to some political issues, regarding the distribution of the work between different state institutions, so it is not only about how you articulate Chile's communication strategy (personal communication, 2013). As Marcela revealed, the state agencies funding the work of Fundación Imagen de Chile expected outcomes traditionally associated with branding and advertising, such as promotional logos and taglines. Operationalisation: How to Make Chile Visible? The fact that local politicians demanded precisely those outcomes reveals the level of dissemination and normalisation of the language and logics of branding and advertising within the state.71 Indeed, requirements such as those mentioned by Marcela are a manifestation of the increasingly popular adoption of corporate techniques by the public sector, spurred on by a growing demand for enhanced evaluation and measurement instruments, particularly concerning the reputation of organisations.72 Most interviewees also admitted that, despite the rhetoric of nation branding enthusiasts, there are no clear parameters to systematically measure and evaluate the development and management of the image of a nation. Hence, activities such as press report preparation, media coverage monitoring, and the production of advertising and marketing materials have become the de facto ways of evaluating and justifying the costs of nation branding initiatives. Significantly, although nation branding advocates claim that their goal is making the nation stand out in a global competitive 15 market, the fact that nation branding practitioners emulate their practices across various settings, makes the outcomes of these initiatives extremely similar all over the world.73 Notably, some nation branding practitioners were explicit in their dissatisfaction with the language and format of advertising. Depictions using standard marketing language were described as ‘inauthentic’. Their criticisms were not isolated. Simon Anholt has become an outspoken critic of the practices and promises of nation branding, albeit only for the purpose of re-labelling his work as ‘competitive identity’.74 According to Fernando, former Fundación Imagen de Chile official, the perfectionism of videos such as ‘Chile is good for you’ made difficult the visibility of the authentic nation: The aesthetic and the audiovisual strategy of ‘Chile is good for you’ shows a stunning Chile, with unique landscapes, exceptional natural resources and food delicacies, while, for example, the documentaries of ‘Living Atlas Chile’ show the truth. I mean, salmon fishing is difficult and fishes are not that beautiful. So the ‘Living Atlas’ does not mean that we are ugly or anything like that, but it wants to show the truth, without a professional camera and that perfect advertising language (personal communication, 2014). Operationalisation: How to Make Chile Visible? The ‘Living Atlas Chile’ (‘Atlas Vivo de Chile’), mentioned above by Fernando, was a series of short documentary films produced by Fundación Imagen de Chile in 2013 that aimed to show the lives of real Chileans.75 According to Marcela, who was involved in the development of the documentaries, the films attempted to enhance the visibility of ‘ordinary’ Chileans. Hence, the ‘Atlas’ intended to address one of the main drivers behind the country’s nation branding efforts, namely the fact that ‘no one can picture a Chilean’.76 However, both Marcela and Fernando stated that officials within the foundation had heated discussions about the production of this series, due to, again, a lack of clarity regarding the approach to take and potential evaluation methods. The ‘Living Atlas Chile’ (‘Atlas Vivo de Chile’), mentioned above by Fernando, was a series of short documentary films produced by Fundación Imagen de Chile in 2013 that aimed to show the lives of real Chileans.75 According to Marcela, who was involved in the development of the documentaries, the films attempted to enhance the visibility of ‘ordinary’ Chileans. Hence, the ‘Atlas’ intended to address one of the main drivers behind the country’s nation branding efforts, namely the fact that ‘no one can picture a Chilean’.76 However, both Marcela and Fernando stated that officials within the foundation had heated discussions about the production of this series, due to, again, a lack of clarity regarding the approach to take and potential evaluation methods. Significantly, the different approaches taken on how to portray Chile in the ‘Living Atlas’ and ‘Chile is good for you’ led to the development of at least two parallel and disconnected accounts of the nation: one aimed at locals and one aimed at foreigners, both with radically different formats and messages. The invention of one single nation brand applicable across a variety of settings, and which could be embraced by both locals Significantly, the different approaches taken on how to portray Chile in the ‘Living Atlas’ and ‘Chile is good for you’ led to the development of at least two parallel and disconnected accounts of the nation: one aimed at locals and one aimed at foreigners, both with radically different formats and messages. The invention of one single nation brand applicable across a variety of settings, and which could be embraced by both locals 16 and foreigners, proved to be an almost impossible task. Operationalisation: How to Make Chile Visible? In other words, the poetics of nation branding were halted significantly by the politics of its operationalisation.77 Audiences: For whom should Chile be made visible? The third and final tension highlighted by the interviewees refers to the audiences to whom nation branding efforts should be directed. The prevalence of the economies of visibility over the politics of visibility was particularly clear in this regard. Most interviewees did not speak of ‘audiences’, ‘publics’ or ‘countries’ to which nation branding initiatives had to be targeted. Instead, they spoke of ‘markets’. As mentioned in the introduction, that was the term employed when I worked in nation branding projects in the late 2000s. The reference to markets confirms that nation branding is a manifestation of ‘economic’ or ‘commercial nationalism’. Indeed, one of the promises made by nation branding advocates is the levelling of the international arena, namely, a redrawing of geopolitics. According to this argument, nation branding facilitates small and medium-sized nations located far from traditional power centres, and with limited material resources, to punch ‘above their weight’.78 However, claims of empowerment and levelling the field are misleading, because nation branding rarely, if ever, challenges power relations in the international arena. Nation branding does not merely aim to make visible the supposed authenticity –as long as it can be a source of profit– of a nation. As a manifestation of geopolitical visions, it also situates nations in specific places in the world, praising and criticising nations according to alleged universal parameters of behaviour and political and economic achievements.79 That is the role of rankings such as the already mentioned Nation Brand Index (NBI) as well as the Country Brand Index (CBI) by FutureBrand,80 to which the Chilean authorities have subscribed. In both indexes nations annually move upwards or downwards in the international arena, depending on how ‘well’ or ‘poorly’ their brand has performed. Significantly, the rankings demonstrate how visibility can be a source of both recognition and surveillance. Nation branding not only promises countries to be 17 recognised. Audiences: For whom should Chile be made visible? It also seeks to discipline and control them, pushing them to embrace behaviours deemed as correct or desirable.81 Nation branding highlights the asymmetry of visibility relations among countries.82 Most nation branding consultants are based in the United Kingdom and the United States, with governments in the global south as clients.83 Indeed, the aforementioned rankings promoted by consultants reinforce the idea that the world is divided into ‘core’ and ‘peripheral’ nations between which unequal power relations exist.84 The methodology used for the NBI and the CBI places much weight on interviews with individuals predominantly based in Western nations.85 In turn, ‘peripheral’ or ‘emerging’ nations such as Chile end up targeting most of their nation branding efforts at ‘core’ ones, such as the United States, the United Kingdom, Germany or Japan. Given the attention of ‘core’ nations is portrayed as a zero-sum game, nation branding advocates hold that competition for attention among nations is natural.86 Hence, nation branding is also a manifestation of the competition state, with the aim of ‘maintaining and promoting competitiveness in a world marketplace and multi-level political system’.87 Chile is ranked traditionally around number 35 of 50 nations in the NBI88 and is often regarded as the fourth Latin American nation brand in the Country Brand Index of FutureBrand.89 Whilst the stated aspiration of most interviewees was to gradually move Chile upwards to the top rankings, they admitted that this goal is probably going to take years or even decades to attain. Interviewees also recognised that most of Chile’s nation branding efforts were directed at ‘core’ nations from Western Europe and East Asia, as well as the United States. Whilst there have been specific campaigns targeted at audiences within the region90 -and Marcela, former official at Fundación Imagen de Chile observed that ‘Juan Gabriel [Valdés, first Executive Director of the foundation] originally wanted to focus efforts on improving relations with Peru, Bolivia, and better position Chile in South America’– Chile’s initiatives are rarely focussed on Latin America. Indeed, shortly afterwards the Chilean government announced in 2014 that 18 18 local nation branding efforts were going to include the continent,91 Carlos, an official from ProChile, stated: I really don’t like what they [at Fundación Imagen de Chile] are doing. We should be focussing on our markets, on our exports, and not on solving issues with our neighbours. Audiences: For whom should Chile be made visible? That is an important issue, but I don’t think we should currently direct the limited resources we have for nation branding to do that. There are other state departments that are much more experienced and can do it better. Carlos admits that nation branding practitioners in Chile lack both the expertise and resources to deal with the fraught relationships with neighbouring countries. Similarly, Pedro, from Ogilvy Public Relations, held that Chile ‘should not be eager to please everyone’ (personal communication, 2014). Yet the country has a history of tense relationships with its neighbours, particularly Peru and Bolivia, due to conflicts that date back to the ‘War of the Pacific’ at the end of the 19th century. Indeed, in the last decade, both have taken Chile to the International Court of Justice to solve border disputes.92 Both Peru and Bolivia notably accompanied their legal efforts with communication campaigns that aimed to win public support for their legal arguments.93 Chile was a latecomer in developing communication efforts, partly because its focus on using nation branding to advance an economic agenda led the country to enhance its visibility almost exclusively before the eyes of ‘core’ nations or ‘priority markets’.94 The tension between the politics of visibility and the economies of visibility are at the core of Chile’s nation branding efforts. Nation branding advocates stress that a focus on the brand displaces the supposed aggressiveness of nationalism.95 Yet nation branding is ill equipped to deal both with conflict and the more complex intricacies of regional politics. Despite its promises of redrawing geopolitics through the empowerment of small and medium-sized states, nation branding does not provide answers to more traditional geopolitical concerns, such as the territorial disputes affecting Chile. Indeed, the geopolitical vision proposed by nation branding initiatives, which predominantly places weight on economic concerns, have arguably encouraged Chile to relate mostly to ‘core’ The tension between the politics of visibility and the economies of visibility are at the core of Chile’s nation branding efforts. Nation branding advocates stress that a focus on the brand displaces the supposed aggressiveness of nationalism.95 Yet nation branding is ill 19 nations, to the detriment of most of Latin America. Audiences: For whom should Chile be made visible? There are signs that the Chilean authorities are trying to correct this situation, engaging with other nations within the region.96 Significantly, such efforts have been labelled as ‘public diplomacy’ rather than ‘nation branding’.97 However, it is unclear whether or not these initiatives will be substantially different to nation branding ones. The boundaries between nation branding and public diplomacy are extremely blurred.98 Hence, it can perfectly be the case that some of the shortcomings of nation branding previously discussed end up being reproduced, albeit under a different name. Concluding Remarks This article has examined the viewpoints of nation branding practitioners in Chile, a country that since the mid-2000s has carried out various initiatives to try to shake off the uncomfortable past of Augusto Pinochet’s dictatorship, creating a ‘new’ image that fits into a globalised and neoliberal world. The case of Chile shows how nation branding is a manifestation of the struggles for visibility that characterise contemporary societies,99 with nations competing to achieve recognition, while at the same time becoming subjects of external control.100 Significantly, understanding nation branding within the prism of geopolitical visions and visibility, as suggested in this article, invites an examination not only of what can be seen –that is, the outcomes of specific campaigns–, but also of the beliefs and practices of the individuals behind nation branding efforts. The interviews conducted for this article shed light on the complexity of nation branding, and some of its implications for the study of geopolitics, from an empirical perspective. They corroborated how nation branding is understood as a series of practices driven and framed by mostly economic goals. The interviewees stressed three areas of tension, which highlighted the disputes surrounding the what, the how and for whom of nation branding: (1) the conflicting purposes guiding the practice of nation branding, (2) the difficulties around the operationalisation of nation branding and (3) the controversies regarding the intended audiences for their efforts. The three areas of tension show that nation branding is far from being an orchestrated effort or a neoliberal conspiracy. which highlighted the disputes surrounding the what, the how and for whom of nation branding: (1) the conflicting purposes guiding the practice of nation branding, (2) the difficulties around the operationalisation of nation branding and (3) the controversies regarding the intended audiences for their efforts. The three areas of tension show that nation branding is far from being an orchestrated effort or a neoliberal conspiracy. 20 Practitioners continuously face struggles in order to construct and project versions of national identity. Significantly, the issues raised by the interviewees point out how nation branding has severe structural flaws that make the delivery of its promises impossible. I highlight three. Firstly, nation branding advocates promise to create and give visibility to an orderly and unique portrayal of the nation, to make it stand out in the global market. Concluding Remarks Indeed, in the case of Chile, the interviewees agreed there was consensus that the country was projected predominantly as an economic unit. However, the different aims and agendas of all the organisations trying to develop portrayals of Chile, as well as the pressures they faced from other actors within the state, made the coordination of such portrayals an almost insurmountable task. Secondly, practitioners admitted that they operate on shaky grounds, without clear guidance on how to empirically conduct and evaluate their efforts. With no clear path of operationalisation and the difficulty of reaching consensus in regards to what make visible of the nation, when agreement amongst the actors was reached, shallow and unoriginal versions of national identity constituting the ‘brand’ were put forward. Thirdly, whilst nation branding promises to level the international arena, giving more opportunities to small and medium-sized nations to advance their agendas, in practice it is underpinned on geopolitical visions that reproduce asymmetrical power relations, with ‘peripheral’ nations struggling to capture the attention of ‘core’ ones. Furthermore, nation branding provides no answer to more traditional geopolitical concerns, such as territorial sovereignty disputes. It is quite possible that the significant flaws in nation branding as well as an absence of concrete results will reduce its influence amongst governments in the near future, particularly those from ‘emerging’ or ‘developing’ nations. This does not mean that the task of developing portrayals of the nation for foreigners will be abandoned. As stated earlier, the concept of public diplomacy has been increasingly used in Chile, particularly in connection with the fraught relationships with neighbouring countries. Public diplomacy is traditionally understood as a task of ministries of foreign affairs rather than public relations managers. Hence, its practitioners have gained more respectability given diplomacy is traditionally understood as a task of ministries of foreign affairs rather than public relations managers. 1 For further discussion see, for instance, P. Alexandre et al (2015). Brief Analyses of Chilean Geopolitics, in Coleção Meira Mattos - Revista Das Ciências Militares, 9(34), 129-145; R. Benedikter & K. Siepmann (Eds.) (2015). Chile in Transition: Prospects and Challenges for Latin America’s Forerunner of Development (Dordrecht: Springer); R. Ortega (2015), La geopolítica y geoestrategia chilena: ¿presente en el discurso y ausente en la práctica?, in Cuadernos de Trabajo ANEPE, 2, 1-19; I. Strodthoff (2014). Chile and Australia: Contemporary Transpacific Connections from the South (New York: Palgrave Macmillan). Concluding Remarks Hence, its practitioners have gained more respectability given 21 that they are portrayed as part of the diplomatic body of a government rather than as communication consultants or propagandists.101 In practice that reframing may not necessarily be a clear departure from nation branding efforts, given that the term public diplomacy often denominates the very same activities currently carried out by nation branding practitioners.102 Furthermore, nation branding should be seen more as a manifestation rather than the cause of the increasing understanding of the nation in mostly economic terms, as well as the embracing of branding and advertising language and techniques by the state.103 Hence, while the phenomenon of nation branding may soon disappear,104 the conditions that facilitated its emergence and dissemination remain. 7 Some significant exceptions being Aronczyk, Branding the Nation (note 3); C. Avendaño Manell (2008). Relaciones estratégicas, comunicación internacional: El caso Chile (Villa María: Eduvim); M.C. Prieto Notes 2 Simon Anholt is often called ‘the father’ of nation branding, after he coined the term ‘nation-brands’ in his article Nation-brands of the twenty-first century in 1998. The Journal of Brand Management, 5(6), 395– 406. Years later, he distanced himself from the concept and preferred to discuss ‘competitive identity’. For further details, see S. Anholt (2007), Competitive identity: The new brand management for nations, cities and regions (Basingstoke: Palgrave). 2 Simon Anholt is often called ‘the father’ of nation branding, after he coined the term ‘nation-brands’ in his article Nation-brands of the twenty-first century in 1998. The Journal of Brand Management, 5(6), 395– 406. Years later, he distanced himself from the concept and preferred to discuss ‘competitive identity’. For further details, see S. Anholt (2007), Competitive identity: The new brand management for nations, cities and regions (Basingstoke: Palgrave). 3 See also M. Aronczyk, (2013). Branding the Nation (Oxford: Oxford University Press). 3 See also M. Aronczyk, (2013). Branding the Nation (Oxford: Oxford University Press). 4 There have been various examples of op-ed pieces written during the last decades seeking to justify the need for a nation brand for Chile. For example, Y. Gallardo (2015, June 17). Opinión: Caso Vidal, un golpe a la marca Chile. In El Mostrador. URL: http://www.elmostrador.cl/mercados/2015/06/17/opinion-vidal- golpe-a-la-marca-chile/; M. Gómez (2016, October 27). Promoviento la marca Chile en Estados Unidos, in Cooperativa. URL: http://opinion.cooperativa.cl/opinion/economia/promoviendo-la-marca-chile-en- estados-unidos/2016-10-27/063806.html 5 See, for instance, E. Chadwick (2015, March 10). Chile y su imagen país: el riesgo de desaparecer del mapa, in T13. URL: http://www.t13.cl/blog/tarjeta-bip/chile-y-su-imagen-pais-el-riesgo-de-desaparecer- del-mapa’; C. Jiménez-Martínez (2013). Chile’s quest to improve its image abroad, in Place Branding and Public Diplomacy, 9(4), 279–290. 5 See, for instance, E. Chadwick (2015, March 10). Chile y su imagen país: el riesgo de desaparecer del mapa, in T13. URL: http://www.t13.cl/blog/tarjeta-bip/chile-y-su-imagen-pais-el-riesgo-de-desaparecer- del-mapa’; C. Jiménez-Martínez (2013). Chile’s quest to improve its image abroad, in Place Branding and Public Diplomacy, 9(4), 279–290. 6 To give some examples, the case of Chile is referenced in Aronczyk, Branding the Nation (note 3); C. Browning, (2015). Nation branding, national self-esteem, and the constitution of subjectivity in late modernity. Foreign Policy Analysis, 11(2), 195–214; K. Dinnie (2008). Nation branding: Concepts, issues, practice (Oxford: Butterworth-Heinemann). 22 Larraín, (2011). Branding the Chilean Nation. Socio-Cultural Change, National Identity and International Image. Leiden University, URL: https://openaccess.leidenuniv.nl/handle/1887/18141 8 See, for example, Aronczyk, Branding the Nation (note 3); J. Bátora (2005). Public diplomacy of small and medium-sized states: Norway and Canada. Discussion Papers in Diplomacy, (97), 1–26, URL: http://www.clingendael.nl/sites/default/files/20050300_cli_paper_dip_issue97.pdf; G. Bolin & P. Ståhlberg (2010). Between community and commodity: Nationalism and nation branding. In A. Roosvall & I. Salovaara-Moring (Eds.), Communicating the nation: National topographies of global media landscapes, pp. 79–101 (Goteborg: Nordicom); I. Hall (2012). India’s New Public Diplomacy. Asian Survey, 52(6), 1089–1110, URL: http://www.jstor.org/stable/10.1525/as.2012.52.6.1089; N. Kaneva (2012). Who can play this game? The rise of nation branding in Bulgaria, 2001-2005. In N. Kaneva (Ed.), Branding post- communist nations: Marketizing national identities in the “new” Europe, pp. 99–123 (New York and London: Routledge); N. Kaneva & D. Popescu (2014). “We are Romanian, not Roma”: Nation Branding and Postsocialist Discourses of Alterity. Communication, Culture & Critique, 7(4), 506–523; R. Saunders, (2015). Mediating new Europe-Asia: Branding the post-socialist world via the Internet. In J. Morris, N. Rulyova, & V. Strukov (Eds.), New media in new Europe-Asia, pp. 13 S. Anholt. Competitive identity (note 2); W. Olins (2002). Branding the nation: The historical context. Journal of Brand Management, 9(4/5), 241–249; P. van Ham, (2001). The rise of the brand state: The postmodern politics of image and reputation. Foreign Affairs, 80(5), 2–6. 14 Van Ham (note 13), 3. Larraín, (2011). Branding the Chilean Nation. Socio-Cultural Change, National Identity and International Image. Leiden University, URL: https://openaccess.leidenuniv.nl/handle/1887/18141 3 See also M. Aronczyk, (2013). Branding the Nation (Oxford: Oxford University Press). 143–166 (Abingdon, Oxon: Routledge); P. Surowiec (2017). Nation branding, public relations and soft power: Corporatising Poland (London: Routledge). 9 For instance, D. Buarque, (2015). One Country, Two Cups — The International Image of Brazil in 1950 and in 2014: A Study of the Reputation and the Identity of Brazil as Projected by the International Media During the Two FIFA World Cups in the Country. International Journal of Communication, 9, 1300–1318, URL: http://ijoc.org/index.php/ijoc/article/view/3351/1368; C. Jiménez-Martínez, (note 5); F. Lossio Chávez, (2014). La necesaria fantasía de la Marca Perú. In G. Portocarrero (Ed.), Perspectivas sobre el nacionalismo en el Perú, pp. 23–38 (Lima: Red para el desarrollo de las ciencias sociales en el Perú); Prieto Larraín (note 6); J. Sanin (2016). Colombia was passion: Commercial nationalism and the reinvention of Colombianness. In Z. Volcic & M. Andrejevic (Eds.), Commercial nationalism: Selling the nation and nationalizing the sell, pp. 46–64 (Basingstoke: Palgrave Macmillan); C. Villanueva Rivas (2011). The rise and fall of Mexico’s international image: Stereotypical identities, media strategies and diplomacy dilemmas. Place Branding and Public Diplomacy, 7(1), 23–31. 10 G. Dijkink (1996). National identity and geopolitical visions: Maps of pride and pain (London: Routledge), 11. 11 Aronczyk, Branding the Nation (note 3); E. Castelló & S. Mihelj (2017). Selling and consuming the nation: Understanding consumer nationalism. Journal of Consumer Culture, 1–19; N. Kaneva, (2011). Nation Branding: Toward an Agenda for Critical Research. International Journal of Communication, 5, 117–141, URL: http://ijoc.org/index.php/ijoc/article/view/704; N. Kaneva (2016). Nation branding and commercial nationalism: Notes for a materialist critique. In Z. Volcic & M. Andrejevic (Eds.), Commercial nationalism: Selling the nation and nationalizing the sell, pp. 175–193 (London: Palgrave Macmillan); Surowiec, Nation branding, public relations and soft power: Corporatising Poland (note 8). 12 See, for instance, the cases examined in Aronczyk, Branding the Nation (note 3); Bátora (note 8); Bolin & Ståhlberg (note 8); Hall (note 8); Kaneva (note 8); Saunders (note 8); S. Mains (2015). From Bolt to brand: Olympic celebrations, tourist destinations and media landscapes. In S. Mains, J. Cupples & C. Lukinbeal (Eds.). Mediated geographies and geographies of the media, pp. 329-348 (Dordrecht: Springer); K. Valaskivi (2013). A brand new future? Cool Japan and the social imaginary of the branded nation. In Japan Forum, 25(4), 485-504; S. Wood (2017). Rebranding the nation: Germany’s image politics. In International Politics, 54(2), 161-181. The list is not by any means comprehensive. 13 S. Anholt. 15 C. Marklund (2016). The Nordic Model on the Global Market of Ideas: The Welfare State as Scandinavia’s Best Brand. Geopolitics, 1–17. 31 S. Banet-Weiser (2015). Keynote address: Media, markets, gender: Economies of visibility in a neoliberal moment. The Communication Review, 18(1), 53–70; see also N. Kaneva (note 19). 3 See also M. Aronczyk, (2013). Branding the Nation (Oxford: Oxford University Press). Competitive identity (note 2); W. Olins (2002). Branding the nation: The historical context. Journal of Brand Management, 9(4/5), 241–249; P. van Ham, (2001). The rise of the brand state: The postmodern politics of image and reputation. Foreign Affairs, 80(5), 2–6. 23 18 Aronczyk, Branding the Nation (note 3); Kaneva (notes 8 and 11). 18 Aronczyk, Branding the Nation (note 3); Kaneva (notes 8 and 11). 19 For instance, Kaneva has used the concept of visibility to examine the mediated construction of femininity in post-socialist nations, in N. Kaneva (2015) Mediating Post-Socialist Femininities, Feminist Media Studies, 15(1), 1-17. 19 For instance, Kaneva has used the concept of visibility to examine the mediated construction of femininity in post-socialist nations, in N. Kaneva (2015) Mediating Post-Socialist Femininities, Feminist Media Studies, 15(1), 1-17. 20 Volcic & Andrejevic (note 17), p. 604. 16 Kaneva (note 11) 17 See, for instance, Aronczyk, Branding the Nation (note 8); Bolin & Ståhlberg (note 8); G. Bolin & P. Ståhlberg (2015). Mediating the Nation-State: Agency and the Media in Nation-Branding Campaigns. International Journal of Communication, 9, 3065–3083; Browning (note 4); Castelló & Mihelj (note 11); Kaneva (note 6 and 9); Kaneva & Popescu (note 8); Saunders (note 6); Surowiec, Nation branding, public relations and soft power: Corporatising Poland (note 8); Z. Volcic, & M. Andrejevic (2011). Nation branding in the era of commercial nationalism. International Journal of Communication, 5(1), 598–618, URL: http://ijoc.org/index.php/ijoc/article/view/849/544. 33 Based on Dayan (note 24); Voirol (note 23). 34 Voirol (note 23), p. 102. 35 Aronczyk, Branding the Nation (note 3); Bolin & Ståhlberg (note 8); Kaneva (note 11); L. Kulcsár & Y.- O.Yum (2012). One nation, one brand? Nation branding and identity reconstruction in post-communist Hungary. In N. Kaneva (Ed.), Branding post-communist nations: Marketizing national identities in the “new” Europe, pp. 193–212, (London: Routledge); Surowiec, Nation branding, public relations and soft power: Corporatising Poland (note 8). 35 Aronczyk, Branding the Nation (note 3); Bolin & Ståhlberg (note 8); Kaneva (note 11); L. Kulcsár & Y.- O.Yum (2012). One nation, one brand? Nation branding and identity reconstruction in post-communist Hungary. In N. Kaneva (Ed.), Branding post-communist nations: Marketizing national identities in the “new” Europe, pp. 193–212, (London: Routledge); Surowiec, Nation branding, public relations and soft power: Corporatising Poland (note 8). 33 Based on Dayan (note 24); Voirol (note 23). 20 Volcic & Andrejevic (note 17), p. 604. 21 J.B. Thompson, ‘The new visibility’, Theory, Culture & Society, 22/6 (2005), pp. 31-51. 22 Ibid, p. 49. 23 O. Voirol (2005). Les Luttes pour la Visibilité - Esquisse d’une problématique. La Découverte, Réseaux, 1(129–130), 89–121. 24 Dayan, D. (2013). Conquering visibility, conferring visibility: Visibility seekers and media performance. International Journal of Communication, 7, 137–153, URL: http://ijoc.org/index.php/ijoc/article/view/1966/845, p. 139. 25 S. Anholt. Competitive identity (note 2); K. Dinnie. Nation branding: Concepts, issues, practice (note 6); W. Olins (note 13); Szondi, G. (2008). Public diplomacy and nation branding: Conceptual similarities and differences. Discussion Papers in Diplomacy, URL: http://www.clingendael.nl/sites/default/files/20081022_pap_in_dip_nation_branding.pdf 26 G. Dijkink (1996). National identity and geopolitical visions: Maps of pride and pain (note 10). 27 A. Brighenti (2010). Visibility in social theory and social research (Basingstoke: Palgrave Macmillan). 28 M. Foucault (1979). Discipline and punish: The birth of the prison (Harmondsworth: Penguin), p. 200. 29 Aronczyk, Branding the Nation (note 3); Browning (note 6); S.C. Jansen (2008). Designer nations: Neo- liberal nation branding – Brand Estonia. Social Identities, 14(1), 121–142; N. Kaneva (2012). Nation branding in post-communist Europe: Identities, markets, and democracy. In Branding post-communist nations: Marketizing national identities in the “new” Europe, pp. 3–22 (New York: Routledge). 30 Brighenti. Visibility in social theory and social research (note 22), p. 58. 31 S. Banet-Weiser (2015). Keynote address: Media, markets, gender: Economies of visibility in a neoliberal moment. The Communication Review, 18(1), 53–70; see also N. Kaneva (note 19). 24 32 Banet-Weiser (note 31). 33 Based on Dayan (note 24); Voirol (note 23). 50 Aronczyk, Branding the Nation (note 3); Jiménez-Martínez, (note 5). 36 Kaneva (note 11), p. 177. 54 The interviews were with “David”, from ProChile in 2013; “Marcela”, from Fundación Imagen de Chile, in 2013; “Pedro”, from Ogilvy Public Relations, in 2014; “Rodrigo”, from Ogilvy Public Relations, in 2014; “Fernando”, from Fundación Imagen de Chile, in 2014; “Carlos”, from ProChile, in 2016; “Diego”, from FutureBrand, in 2016. 54 The interviews were with “David”, from ProChile in 2013; “Marcela”, from Fundación Imagen de Chile, in 2013; “Pedro”, from Ogilvy Public Relations, in 2014; “Rodrigo”, from Ogilvy Public Relations, in 2014; “Fernando”, from Fundación Imagen de Chile, in 2014; “Carlos”, from ProChile, in 2016; “Diego”, from FutureBrand, in 2016. 55 J. Attride-Stirling (2001). Thematic networks: an analytic tool for qualitative research. Qualitative Research, 1(3), 385–405; J. Fereday, & E. Muir-Cochrane, E. (2006). Demonstrating rigor using thematic analysis: A hybrid approach of inductive and deductive coding and theme development. International Journal of Qualitative Methods, 5(1), 80–92; Hsieh, H.-F., & Shannon, S. E. (2005). Three Approaches to Qualitative Content Analysis. Qualitative Health Research, 15(9), 1277–1288. 56 Castelló & Mihelj (note 11); Kaneva (note 11); Volcic & Andrejevic (note 17); Volcic, Z., & Andrejevic, M. (2015). Introduction. In Z. Volcic & M. Andrejevic (Eds.), Commercial nationalism: Selling the nation and nationalizing the sell, pp. 1–13, (Basingstoke: Palgrave Macmillan). 57 Abarca Lucero (note 49); Álvarez Caselli (note 39); Dinnie. Nation branding: Concepts, issues, practice (note 6); Prieto Larraín (note 7). 58 T. Nairn (1997). Faces of nationalism: Janus revisited (London: Verso). For an examination of the tension between tradition and modernity in Latin America, see N. García Canclini (2001). Consumers and citizens: Globalization and multicultural conflicts (Minneapolis: University of Minnesota Press). 59 See the examples in Aronczyk, Branding the Nation (note 3). 59 See the examples in Aronczyk, Branding the Nation (note 3). 60 Various authors, such as Álvarez Caselli (note 39); Jiménez-Martínez (note 5) have also mentioned the story that David recalled. 61 Aronczyk, Branding the Nation (note 3). 61 Aronczyk, Branding the Nation (note 3). 62 This paradox is further examined in C. Ooi (2004). Poetics and Politics of Destination Branding: Denmark. Scandinavian Journal of Hospitality and Tourism, 4(2), 107–128. 63 See https://marcachile.cl/english/the-brand-chile/ 63 See https://marcachile.cl/english/the-brand-chile/ 64 Aronczyk, Branding the Nation (note 3). 64 Aronczyk, Branding the Nation (note 3). 65 See, for instance, Aronczyk, Branding the Nation (note 3) and Lossio Chávez (note 9). 66 D. Dayan, (2008). Beyond media events: Disenchantment, derailment, disruption. In M. E. Price & D. 36 Kaneva (note 11), p. 177. 36 Kaneva (note 11), p. 177. 37 G. Turner (2016). Setting the scene for commercial nationalism: The nation, the market, and the media. In Z. Volcic & M. Andrejevic (Eds.), Commercial nationalism: Selling the nation and nationalizing the sell, pp. 14–26 (Basingstoke: Palgrave Macmillan). 38 See Buarque, (note 9); Jiménez-Martínez (note 5); Lossio Chávez, (note 9); Prieto Larraín (note 7); Sanin (note 9); Villanueva Rivas (note 9). 38 See Buarque, (note 9); Jiménez-Martínez (note 5); Lossio Chávez, (note 9); Prieto Larraín (note 7); Sanin (note 9); Villanueva Rivas (note 9). 39 For further details, see P. Álvarez Caselli (2009). Chile MR: la difícil ecuación histórica entre identidad, imagen y marca-país. Revista DISEÑA, 1, 50–61. Retrieved from http://www.revistadisena.com/chile-mr- la-dificil-ecuacion-historica-entre-identidad-imagen-y-marca-pais/; J. Itzigsohn & M. vom Hau (2006). Unfinished imagined communities: States, social movements, and nationalism in Latin America. Theory and Society, 35(2), 193–212; J. Larraín, (2000). Identity and modernity in Latin America (Malden, Mass.: Polity Press). 40 Larraín. Identity and modernity in Latin America (note 39). 41 Aronczyk, Branding the Nation (note 3); Dijkink (1996). National identity and geopolitical visions: Maps of pride and pain (note 10). 42 Aronczyk, Branding the Nation (note 3); Kaneva, (note 11); Kaneva & D. Popescu (note 8) 44 C. Macario (1998). Chile: De las políticas de subsidio a las exportaciones a las políticas de desarrollo de la competitividad. Santiago. Retrieved from http://archivo.cepal.org/pdfs/1998/S9800548.pdf 45 Prieto Larraín (note 7). 46 J. Abarca Lucero (2009). Public diplomacy and qualitative jump: The case of chile. Tripodos, 24, 53–61. Retrieved from http://www.raco.cat/index.php/Tripodos/article/view/129434/178817; Jiménez-Martínez (note 5); Prieto Larraín (note 7). 47 Abarca Lucero (note 46); Prieto Larraín (note 7). 47 Abarca Lucero (note 46); Prieto Larraín (note 7). 48 Anholt (note 2). 49 Aronczyk, Branding the Nation (note 3). 50 Aronczyk, Branding the Nation (note 3); Jiménez-Martínez, (note 5). 50 Aronczyk, Branding the Nation (note 3); Jiménez-Martínez, (note 5). 51 Jiménez-Martínez (note 5). 25 53 L. Chagas de Moura, (2013). A Marca Brasil: O poder da imagem e a construção da identidade competitiva. Ministério das Relações Exteriores, Brasília, Brasil. 53 L. Chagas de Moura, (2013). A Marca Brasil: O poder da imagem e a construção da identidade competitiva. Ministério das Relações Exteriores, Brasília, Brasil. 52 Aronczyk, Branding the Nation (note 3); Bakit, M. (2014, November 8). Canciller Heraldo Muñoz explica la renovada estrategia de Imagen de Chile: Nueva “Marca País” pondrá énfasis en relación vecinal y reformas. Ministerio de Relaciones Exteriores de Chile, URL: http://www minrel gov cl/minrel/site/artic/20141111/pags/20141111111516 html; Jiménez-Martínez (note 52 Aronczyk, Branding the Nation (note 3); Bakit, M. (2014, November 8). Canciller Heraldo Muñoz explica la renovada estrategia de Imagen de Chile: Nueva “Marca País” pondrá énfasis en relación vecinal y reformas. Ministerio de Relaciones Exteriores de Chile, URL: http://www.minrel.gov.cl/minrel/site/artic/20141111/pags/20141111111516.html; Jiménez-Martínez, (note 4); R. Merino (2010, November 10). Chile hace bien. In Marca Chile, URL: https://marcachile.cl/noticias/chile-hace-bien/; ProChile (n.d.). Chile hace bien, URL: http://www.prochile.gob.cl/int/peru/wp-content/blogs.dir/42/files_mf/1384869827FichaChileEsp.pdf 67 Among the many works in this field, I can mention Anholt. Competitive identity (note 2); K. Dinnie. Nation branding: Concepts, issues, practice (note 6); L. Echeverri, C. Estay-Niculcar, C. Herrera & J. Santamaría (2013). Desarrollo de marca país y turismo: El caso de estudio de México, Estudios y perspectivas en turismo, 22(6), 1121-1139; Olins (note 13). For a more comprehensive review, please refer to Kaneva (note 11). 96 Bakit (note 52). 96 Bakit (note 52). 96 Bakit (note 52). 88 See http://www.gfk.com/es-cl/insights/press-release/ranking-anholt-nation-brand-index-latam/ 36 Kaneva (note 11), p. 177. Dayan (Eds.), Owning the Olympics: Narratives of the new China, pp. 391–403 (Ann Arbor, MI: University of Michigan Press), p. 392. 26 68 Anholt. Competitive identity (note 2). 69 Aronczyk, Branding the Nation (note 3). 69 Aronczyk, Branding the Nation (note 3). 70 The video is available at https://www.youtube.com/watch?v=yZq4N5ekKD8 70 The video is available at https://www.youtube.com/watch?v=yZq4N5ekKD8 71 L. Moor (2007). The rise of brands (London: Bloomsbury). 72 M. Power (2000), ‘The audit society – second thoughts’, International Journal of Auditing, 4/1, pp. 111- 119. See also M. Power et al (2009), ‘Reputational risk as a logic of organizing in late modernity’, Organization Studies, 30/2-3, pp. 301-324. 73 Aronczyk, Branding the Nation (note 3). 74 S. Anholt. Competitive identity (note 2). 75 Available at http://www.livingatlaschile.com/ 76 Aronczyk, Branding the Nation (note 3), 134. 77 Based on Ooi (note 62). 77 Based on Ooi (note 62). 78 A critical examination of this argument can be found in Marklund, p.3 (note 15); Turner (note 37). 78 A critical examination of this argument can be found in Marklund, p.3 (note 15); Turner (note 37 79 Aronczyk, Branding the Nation (note 3); see also, A. Kantola (2010). The disciplined imaginary: The nation rejuvenated for the global condition. In A. Roosvall & I. Salovaara-Moring (Eds.), Communicating the nation: National topographies of global media landscapes, pp. 237–254 (Goteborg: Nordicom); M. Kunczik (2002). Globalisation: News media, images of nations and the flow of international capital with special reference to the role of rating agencies. Journal of International Communication, 8(1), 39–79. 80 The rankings are available at http://nation-brands.gfk.com/ and http://www.futurebrand.com/country- brand-index 80 The rankings are available at http://nation-brands.gfk.com/ and http://www.futurebrand.com/country- brand-index 81 Based on Brighenti. Visibility in social theory and social research (note 27). 83 Aronczyk, Branding the Nation (note 3). 83 Aronczyk, Branding the Nation (note 3). 84 See I. Wallerstein (2004). World-systems analysis: An introduction (Durham: Duke Universit 85 See http://www.gfk.com/es-cl/insights/press-release/ranking-anholt-nation-brand-index-latam/; http://cbramericalatina.com/en/ 85 See http://www.gfk.com/es-cl/insights/press-release/ranking-anholt-nation-brand-index-latam/; http://cbramericalatina.com/en/ 86 See Anholt. Competitive identity (note 2); Dinnie. Nation branding: Concepts, issues, practice (note 6); Olins (note 13). 86 See Anholt. Competitive identity (note 2); Dinnie. Nation branding: Concepts, issues, practice (note 6); Olins (note 13). 87 P.G. Cerny (2010). The competition state today: From raison d’État to raison du Monde. Policy Studies, 31(1), 5–21, pp. 6. 87 P.G. Cerny (2010). The competition state today: From raison d’État to raison du Monde. Policy Studies, 31(1), 5–21, pp. 6. 94 Brito (note 93); Jiménez-Martínez (note 5). 97 Aguirre Azócar & Villalobos (note 93). 97 Aguirre Azócar & Villalobos (note 93). 98 Theorists and practitioners have proposed competing definition of what nation branding and public diplomacy are. For example, see Kaneva (note 11); E. Gilboa (2008) Searching for a theory of public diplomacy, in The ANNALS of the American Academy of Political and Social Science, 616(1), 55-77; G. Szondi (2008) Public diplomacy and nation branding: conceptual similarities and differences, in Discussion Papers in Diplomacy, Netherlands Institute of International Relations ‘Clingendael’, October 2008; Volcic, & Andrejevic (note 14). 99 Thompson (note 21). 99 Thompson (note 21). 89 See http://cbramericalatina.com/en/ 91 Bakit (note 52). 91 Bakit (note 52). 91 Bakit (note 52). 92 Jiménez-Martínez (note 5); Prieto Larraín (note 7). 92 Jiménez-Martínez (note 5); Prieto Larraín (note 7). 93 D. Aguirre Azócar & J. Villalobos (2016). La ofensiva de Bolivia y Chile y la diplomacia pública mediada. Relaciones Internacionales, 89(2), 17–46; J.I. Brito (2015, July 30). Diplomacia pública. In La Tercera, URL: http://www.latercera.com/noticia/diplomacia-publica/; R. Jara (2015, October 7). Juicio en La Haya: La "batalla" comunicacional entre Chile y Bolivia tras el fallo de la Corte. In Emol, URL: http://www.emol.com/noticias/Nacional/2015/10/06/753232/Juicio-en-La-Haya-La-batalla- comunicacional-entre-Chile-y-Bolivia-tras-el-fallo-de-la-Corte.html 94 Brito (note 93); Jiménez-Martínez (note 5). 95 van Ham (note 13). 95 van Ham (note 13). 89 See http://cbramericalatina.com/en/ 90 For example, in 2012 the Chilean tourism board put a series of posters in Lima’s main airports. Those posters were taken down after the Peruvian media reported that residents were supposedly unhappy to see the Chilean ads. See C. Cáceres (2012, May 12). Sacan publicidad de Chile en aeropuerto de Lima para "evitar conflictos", in Emol, URL: http://www.emol.com/noticias/economia/2012/05/10/539914/gobierno- retira-publicidad-turistica-de-chile-en-aeropuerto-de-lima-para-evitar-conflicto.html 36 Kaneva (note 11), p. 177. 27 88 See http://www.gfk.com/es-cl/insights/press-release/ranking-anholt-nation-brand-index-latam/ 89 See http://cbramericalatina.com/en/ Snow, P. Taylor (Eds.) Routledge Handbook of Public Diplomacy, pp. 19-23 (New York: Routledge); J. Melissen (2005). The new public diplomacy: between theory and practice, in Melissen, J. (Ed.) The new public diplomacy: soft power in international relations, pp. 3-27 (New York: Palgrave Macmillan); J. Pamment (2013) New public diplomacy in the 21st century: a comparative study of policy and practice (London: Routledge). 103 Castelló & Mihelj (note 11); Kaneva (note 11); Moor. The rise of brands (note 71). 102 See note 98. 104 Based on Aronczyk, Branding the Nation (note 3). 104 Based on Aronczyk, Branding the Nation (note 3). 100 Based on Brighenti. Visibility in social theory and social research (note 27). 100 Based on Brighenti. Visibility in social theory and social research (note 27). 100 Based on Brighenti. Visibility in social theory and social research (note 27). 101 American diplomat Edmund Gullion proposed the current meaning of public diplomacy in 1965. He was looking for a term to describe the activities carried out by the American government aimed at foreign publics during the Cold War. The most obvious choice was ‘propaganda’, but the negative associations carried by this word caused him to discard it. The choice of a new concept was not only a matter of semantics. It also legitimised and raised the status of this ‘new’ practice. Public diplomacy practitioners gained more respectability because they were associated with diplomats rather than with public relations professionals; the concept justified the creation or empowerment of institutions exclusively dedicated to this task, such as the United States Information Agency; and it facilitated the task of differentiating these activities from similar ones carried out by the enemy, claiming that, while the United States was doing public diplomacy, the Soviet Union was misleading people and hiding the truth through propagandistic machinery. For further details see G. Cowan, N. Cull (2008) Public Diplomacy in a changing world, in The ANNALS of the American Academy of Political and Social Science, 616(1), 6-8; N. Cull (2008) Public Diplomacy: taxonomies and histories, in The ANNALS of the American Academy of Political and Social Science, 616(1), 31-54; N. Cull (2008) Public Diplomacy before Guillion: the evolution of a phrase, in N. 28 29
https://openalex.org/W3186865852	https://pureadmin.qub.ac.uk/ws/files/245495603/Silva_Vergara_Perucich2021_Article_DeterminantsOfUrbanSprawlInLat.pdf	English	null	Determinants of urban sprawl in Latin America: evidence from Santiago de Chile	SN social sciences	2,021	cc-by	17,611	Queen's University Belfast - Research Portal: Queen's University Belfast - Research Portal: Li k t bli ti d i Q ' U i it B lf t R h P t Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal Queen s University Belfast Research Portal: Link to publication record in Queen's University Belfast Research Portal y Link to publication record in Queen's University Publisher rights Copyright 2021 the authors. This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. g Copyright 2021 the authors. Copy g 0 e au o s This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/ which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited Take down policy Th R h P Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. Open Access This research has been made openly available by Queen's academics and its Open Research team. We would love to hear how access to this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Determinants of urban sprawl in Latin America: evidence from Santiago de Chile Silva, C., & Vergara-Perucich, F. (2021). Determinants of urban sprawl in Latin America: evidence from Santiago de Chile. SN Social Sciences. https://doi.org/10.1007/s43545-021-00197-4 Determinants of urban sprawl in Latin America: evidence from Santiago de Chile Silva, C., & Vergara-Perucich, F. (2021). Determinants of urban sprawl in Latin America: evidence from Santiago de Chile. SN Social Sciences. https://doi.org/10.1007/s43545-021-00197-4 General rights C f g Copyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. Determinants of urban sprawl in Latin America: evidence from Santiago de Chile Silva, C., & Vergara-Perucich, F. (2021). Determinants of urban sprawl in Latin America: evidence from San de Chile. SN Social Sciences. https://doi.org/10.1007/s43545-021-00197-4 Published in: SN Social Sciences Document Version: Publisher's PDF, also known as Version of record Open Access Thi h Open Access This research has been made openly available by Queen's academics and its Open Research team. We would love t this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Download date:24. Oct. 2024 SN Soc Sci (2021) 1:202 https://doi.org/10.1007/s43545-021-00197-4 SN Soc Sci (2021) 1:202 https://doi.org/10.1007/s43545-021-00197-4 ORIGINAL PAPER * Cristian Silva c.silva@qub.ac.uk Abstract Urban sprawl has been widely discussed in regard of its economic, political, social and environmental impacts. Consequently, several planning policies have been placed to stop—or at least restrain—sprawling development. However, most of these policies have not been successful at all as anti-sprawl policies partially address only a few determinants of a multifaceted phenomenon. This includes processes of extended suburbanisation, peri-urbanisation and transformation of fringe/belt areas of city-regions. Using as a case study the capital city of Chile—Santiago—thirteen determinants of urban sprawl are identified as interlinked at the point of defining Santiago’s sprawling geography as a distinctive space that deserves planning and policy approaches in its own right. Unpacking these determinants and the policy context within which they operate is important to better inform the design and implementation of more comprehensive policy frameworks to manage urban sprawl and its impacts. Keywords Urban sprawl · Urban development · Suburbanisation · Planning policy · Santiago de Chile Determinants of urban sprawl in Latin America: evidence from Santiago de Chile Cristian Silva1 · Francisco Vergara‑Perucich2 Received: 13 January 2021 / Accepted: 8 June 2021 © The Author(s) 2021 1 School of Natural and Built Environment, Queen’s University Belfast, David Keir Building, Stranmillis Road, Belfast BT9 5AG, UK 2 Chair of the Centre for the Production of the Space, Universidad de Las Américas, Campus Providencia, Manuel Montt 948, Providencia, Chile Francisco Vergara‑Perucich jvergara@udla.cl Introduction SDG target 11.6 indicates that a substantial reduction of ‘environmental impact of cities, including by paying spe- cial attention to air quality and municipal and other waste management’, must be achieved by 2030. Target 11.a makes an explicit claim on the importance of having ‘positive economic, social, and environmental links between urban, peri-urban and rural areas’ by strengthening planning at national and regional levels. Target 11.b advocates for having efficient mitigation and adaptation plans to climate change and resilience to disasters by strengthening local governments (UN 2017). However, in this scenario, one of the main threats that cities face to achieve these goals is ‘urban sprawl.’ Traditionally related to low-density suburbia, urban sprawl can also be ‘compact’ (Ståhle 2008), spatially diverse and functionally multifaceted (Dunham- Jones and Williamson 2009,). In all these instances, the presence of different inter- stitial spaces that contribute to land fragmentation also become constitutive of urban sprawl (Phelps and Silva 2017).f Urban sprawl differentiates from ‘suburbia’ (characterised by the production of low-density residential neighbourhoods) and ‘peri-urbanisation’ (mainly focussed on the transformation of fringe-belt areas and the urban–rural transect), as it defines a specific pattern of urban growth that embraces both processes of suburbanisation and peri-urbanisation, but also dispersion of infrastructure interspersed with farm- ing and vacant lands. The understanding of urban sprawl used in this paper adheres to Sieverts’ notion of the Zwischenstadt (Sieverts 2003), wherein all the aforemen- tioned elements configure a distinctive geographical space in which rural, industrial, infrastructural, residential, and interstitial lands coexist; a geography of interrela- tion between the proper city and the open countryside (ibid: 2003). While Sievert’s Zwischenstadt clarifies the geographical scope of urban sprawl, it is characterised by the scattered expansion of residential developments, encroachment of rural lands, environmental fragmentation, high rates of car-dependency, inorganic expansion of urban boundaries, the presence of different interstitial spaces, concentration of peripheral poverty, and saturation of local services (Altieri et al. 2014; Jaret et al. 2009; Phelps and Silva 2017; Romero and Órdenes 2004). There is a broad-based consensus that urban sprawl is an unsustainable pattern of urban development with impacts elucidated in what Bruegmann (2005) identified in the first, second, and third ‘anti-sprawl campaigns’. These impacts include the costs of sprawl, social problems, environmental objections, limits to urban growth, car-dependency, ineffi- cient land-uses, encroachment of rural lands, sociospatial segregation, and effects at global scales linked to climate change (Bruegmann 2005). Introduction Cities have emerged as keystones within global policies towards sustainable devel- opment. United Nations (UN) projections indicate that urban population will be increased by 2050 (UN 2017). Cities are identified as one of the main drivers of Francisco Vergara‑Perucich jvergara@udla.cl Francisco Vergara‑Perucich jvergara@udla.cl 1 School of Natural and Built Environment, Queen’s University Belfast, David Keir Building, Stranmillis Road, Belfast BT9 5AG, UK 2 Chair of the Centre for the Production of the Space, Universidad de Las Américas, Campus Providencia, Manuel Montt 948, Providencia, Chile Vol.:(0123456789) Page 2 of 35 SN Soc Sci (2021) 1:202 202 environmental change and energy consumption (Gargiulo and Russo 2017; Watts 2017). Against this backdrop, the UN’s New Urban Agenda (UN Habitat III 2017) has framed ambitious global goals for sustainability in which cities are highlighted as pathways to sustainable development. This aspiration is captured within the Sus- tainable Development Goals (SDGs)—specifically the SDG11—clearly aligned with social and environmental sustainability theories. SDG target 11.6 indicates that a substantial reduction of ‘environmental impact of cities, including by paying spe- cial attention to air quality and municipal and other waste management’, must be achieved by 2030. Target 11.a makes an explicit claim on the importance of having ‘positive economic, social, and environmental links between urban, peri-urban and rural areas’ by strengthening planning at national and regional levels. Target 11.b advocates for having efficient mitigation and adaptation plans to climate change and resilience to disasters by strengthening local governments (UN 2017). However, in this scenario, one of the main threats that cities face to achieve these goals is ‘urban sprawl.’ Traditionally related to low-density suburbia, urban sprawl can also be ‘compact’ (Ståhle 2008), spatially diverse and functionally multifaceted (Dunham- Jones and Williamson 2009,). In all these instances, the presence of different inter- stitial spaces that contribute to land fragmentation also become constitutive of urban sprawl (Phelps and Silva 2017).f environmental change and energy consumption (Gargiulo and Russo 2017; Watts 2017). Against this backdrop, the UN’s New Urban Agenda (UN Habitat III 2017) has framed ambitious global goals for sustainability in which cities are highlighted as pathways to sustainable development. This aspiration is captured within the Sus- tainable Development Goals (SDGs)—specifically the SDG11—clearly aligned with social and environmental sustainability theories. Introduction Further impacts include correlations between car-dependency and higher rates of obesity (Wu et al. 2020), traffic congestion, inner-city decay, high rates of energy consumption, transportation costs, air pollution, reduction of environmental services, inefficient land-uses, and adverse impacts on social capital inter alia (Hamidi and Zandiatashbar 2019). SN Soc Sci (2021) 1:202 Page 3 of 35 202 Since the 1920s, several planning policies have been implemented to address these impacts, usually translating into restrictions to urban growth such as the ‘urban limits’ (Grimes and Liang 2009), ‘green belts’ (Dockerill and Sturzaker 2019), and ‘urban growth boundaries’ (Grimes and Liang 2009). However, in addition to being ineffective, these planning approaches, in many cases, have leapfrogged develop- ment and land fragmentation (Amati 2016). Alternatively, narratives of retrofitting and compactness have emerged as more preventive; ‘these ideas form something of a Zeigeist in architectural, design and planning circles’ (Phelps 2015, p. 43) for attracting city-like functions and qualities. These policies have fed anti-sprawl nar- ratives along with ‘the rise of new planning mantras such as “sustainable”, “com- pact” or “zerocarbon” development’ (Phelps 2012, p. 172). However, the underlying problem is that urban sprawl is much a product of modernists-inspired nations where their capital cities reflect projects of national development. This modernisation is seen as being driven by the values of the neoliberal agenda that has reached trans- national implications since these values ‘have become embedded in the politics and praxis of governments, institutions and organisations—at all levels of spatial governance—around the globe’ (Boland et al. 2017). Thus, despite the apparently ‘natural’ (or spontaneous) emergence of sprawling growth, ‘it has been thoroughly planned as a result of interventions by all ties of government’ (Phelps 2015, p. 40). Against all predictions, ‘urban sprawl has become a global phenomenon as an outcome of growing population and rapid urbanization’ (Pawe and Saikia 2020, p. 226). As such, urban sprawl remains one of the most longstanding patterns of urban development and a permanent structure of cities in the aftermath of the WWII. Despite this, urban sprawl manifests differently between regions. Studies conducted in Europe describe urban sprawl as more continuous residential expansion and com- pact peri-urban developments (Ståhle 2008), highly influenced by public policies; ‘in Paris, for example, large parts of suburban settlements consisted of high-density houses that were directly built by governmental bodies (…). Introduction Although empirical studies in this regard have been conducted in China (You and Yang 2017; Li and Li 2019), North America (Barrington and Millard 2015), Europe (Oueslati et al. 2015; Pirotte and Madre 2011) and Middle East (Masoumi et al. 2018; Bagheri and Tousi 2018), not much has been researched about the Latin American context and the manner in which it contributes to wider debates on urban sprawl beyond studies on its impacts (Romero and Órdenes 2004). Taking the case of the capital city of Chile—Santiago—the questions raised in this paper point at what the determinants of urban sprawl are, and how they influence Santiago’s urban development. It is hypothesised that planning responses are unable to articulate the whole spectrum of interlinked determinants that drive the sprawling growth of cities, and have difficulties to operate within the specific policy context in which urban sprawl unfolds. What is found in the case of Santiago, is that at least thirteen interlinked determinants drive Santiago’s urban sprawl, thus posing significant challenges for urban governance. On this basis, this paper contributes to a more comprehensive understanding of urban sprawl from a Latin American perspective. Although the term ‘urban sprawl’ has its origins in the Anglo-Saxon literature, case studies from the Global South can contribute to further insights on how urban sprawl is mani- fested. This is particularly sensitive in Latin America in which sprawling growth reveals clear signs of sociospatial segregation (Cox and Hurtubia 2020), and are often a subject of standardised planning solutions unable to articulate the cultural, historical, socioeconomic, political, and geographical singularities. In the next section the meanings of urban sprawl are discussed to situate its mul- tifaceted character and the need to unpack its determinants. This provide insights about urban sprawl in both conceptual and empirical terms that are pulled together in a subsequent section wherein the determinants of Santiago’s urban sprawl are examined. The conclusions underline the determinants of Santiago’s sprawl and the varied ways they define its sprawling suburbia. The values of institutional cross- sectoral coordination are also highlighted but most importantly, the need of fram- ing more strategic planning responses where ‘the problematic for urban politics is (…) not a question of growth versus no growth—as posited in the classic growth machine model—but rather what kind of growth’ (Phelps 2015, p. 45). The contribution of this paper is twofold. Introduction This procedure was not common in the United States, were private-market, single-family home was the norm’ (Patacchini et al. 2009, p. 126). In China, urban sprawl is characterised as leapfrog development in which rural villages, informal settlements, farming spaces and infrastructure of transport coexist with the formal expansion of cities (Deng and Huang 2004). In Africa, urban sprawl also takes the form of leapfrogged develop- ment and ‘occur in nucleated or clustered pattern where physical development pro- ceeds at the peripheries of cities, often characterised by concentration of people of particular social class or income group’ (Cobbinah and Aboagye 2017, p. 233). Here, ‘physical development is patchy, dotted and spread out, creating discontinuity in effective urban development’ (ibid: 233). In the Australasian context urban sprawl takes a more America form ‘characterized by detached houses, domestic carports (usually for two cars or more), roads, street lights, car sale yards, fast-food outlets, shopping malls with large car-parking areas and inner highways’ (Silva 2019a, b, p. 2). As tackling the causes of urban sprawl relates to more structural elements of our societies—including market-driven politics, population growth, the culturally rooted ‘suburban dream’ and improvements on regional infrastructure and housing afford- ability (Willing and Pojani 2017)—addressing the determinants of sprawl seems to Page 4 of 35 SN Soc Sci (2021) 1:202 202 be feasible. In this paper, the notion of ‘determinant’ is used to analyse the specific factors that explain sprawling growth from a planning perspective—including quan- titative, qualitative and context-dependent factors—as well as policy-based causes. Although empirical studies in this regard have been conducted in China (You and Yang 2017; Li and Li 2019), North America (Barrington and Millard 2015), Europe (Oueslati et al. 2015; Pirotte and Madre 2011) and Middle East (Masoumi et al. 2018; Bagheri and Tousi 2018), not much has been researched about the Latin American context and the manner in which it contributes to wider debates on urban sprawl beyond studies on its impacts (Romero and Órdenes 2004). Taking the case of the capital city of Chile—Santiago—the questions raised in this paper point at what the determinants of urban sprawl are, and how they influence Santiago’s urban development. be feasible. In this paper, the notion of ‘determinant’ is used to analyse the specific factors that explain sprawling growth from a planning perspective—including quan- titative, qualitative and context-dependent factors—as well as policy-based causes. Situating urban sprawl The meanings of urban sprawl are contested and differ between morphological, instrumental and policy-based research. In an attempt to define the geographi- cal context of urban sprawl, empirical research suggests that urban sprawl is an all-embracing geography that involves processes of suburbanisation (Charmes and Keil 2015), peri-urbanisation (Ravetz and Loibl 2011) and transformation of fringe- belt areas (Whitehand and Gu 2017). Urban sprawl describes a morphology largely characterised by residential neighbourhoods, high levels of car-dependency, single land-uses, and lack of physical continuity (Jaret et al. 2009). This includes land fragmentation and dispersion of infrastructures such as airports, energy plants, and industries (Altieri et al. 2014; Trubka et al. 2010). Urban sprawl is also accepted to embrace developments beyond suburbia, including settlement of different sorts such as ‘exurbia’ (Bruegmann 2005), ‘technoburbs’ (Fishman 1987), ‘edge cities’ (Garreau 1991), `edgeless cities’ (Lang 2003), ‘satellite towns’ (Abubakar and Doan 2017) and ‘post- suburbs’ (Phelps and Wood 2011), all part of Sieverts’ Zwischen- stadt (2003): a distinctive geography of transition between the proper city and the open countryside (Sieverts 2003). Similarly, Rauws and de Roo’s notion of the ‘third type of landscape’ (2011) is something ‘which cannot solely be understood in terms of progressive intensification of urban functions in the rural environment’ (Rauws and de Roo 2011, p. 269), but ‘be distinguished in terms of its own specific dynam- ics and characteristics (…), a spatial system in its own right’ (ibid: 270). Instrumental studies propose a ‘sprawl index’ to identify patterns of urban sprawl by combining different indicators (Wolman et al. 2005). Here, urban sprawl is defined as ‘a pattern of land use in an urbanised area exhibiting low levels of some combination of eight distinct dimensions: density, continuity, concentration, cluster- ing, centrality, nuclearity, mixed uses, and proximity’ (Galster et al. 2001, p. 685). From this viewpoint, there is not just ‘one’ urban sprawl but ‘many’, as it can differ from region to region regarding differences in these criteria (Li et al. 2020). Dif- ferent areas of the same city can also show different levels of sprawling growth, in which some areas can be more (or less) sprawling than others. Hess (2001) argues that what can be seen as a compact city can be a very sprawling one in the future: ‘it is the trend in population density, rather than current population density, that determines whether a city is sprawling or not. Introduction First, it provides further elements to ensure a more comprehensive understanding of urban sprawl and its policy context. These findings link to debates around the production of the space under neoliber- alised nation-states and the contradictions of the ‘growth machine’ (Phelps 2012). Second, the paper unpacks the set of interlinked determinants of urban sprawl that allows the formulation of specific policies to address them more in a more strategic manner. The paper seeks to shed light on understanding urban sprawl beyond simpli- fications and anti-growth narratives, and how the case of Santiago can better inform the design and implementation of planning solutions aligned with the UN’s New Urban Agenda and the SDG11 targets. SN Soc Sci (2021) 1:202 Page 5 of 35 202 202 Situating urban sprawl A city becoming less densely popu- lated through time is said to be sprawling, even if it is currently quite densely popu- lated in comparison to other cities’ (Hess et al. 2001, p. 6). On this basis, urban sprawl cannot be reduced to a static view of a city. Instead, it must be seen as ‘a continuous process of urban transformation which functions more like a verb than a noun’ (Silva 2019a, b, p. 58), and can illustrate different dynamics, even within the same city-region. As such, urban sprawl can also be observed in small towns and villages—making it a non-exclusive condition of megacities (Horn and van Eeden 2018)—which raises further reflections around its intrinsic negative connotation. Page 6 of 35 SN Soc Sci (2021) 1:202 202 According to theoretical research, the underlying assumption that urban sprawl is an ‘urban’ issue limits its implications at regional levels (Osman et al. 2016). Urban sprawl is usually filtered through urban theories focussed on intensive processes of ‘the production of the space’ (Brenner and Elden 2009) as synonym of ‘built-up’ space. This leaves aside the varied spectrum of rural, undeveloped, vacant and inter- stitial spaces that lie between developments (Silva 2019a, b). However, urban sprawl is corseted by existing (and somehow obsolete) analytical containers of the ‘urban’ that minimise its comprehension from rural and regional theories. This undermines the inter-institutional relevance of sprawling processes ‘that are administratively urban but retain their rural structure and characteristics’ (Qian and Wong 2012, p. 409). The policy context of urban sprawl Urban sprawl is associated with processes of extended suburbanisation started in North America within the economic recovery after the WWII. Indeed, the term ‘urban sprawl’ appeared in the 1940s (Gillham 2002) to explicate the American sub- urbanisation driven by the increasing use of private cars and the expansion of the interstate motorway system (Soule 2006). It has been demonstrated that oil compa- nies strongly campaigned for the use of private cars (and thus, oil), the extensions of motorways, the reduction of public transport, and the consolidation of the ‘subur- ban dream’ as synonym of personal freedom (Phelps 2015; Hamel and Keil 2015). Notably, since the 1970s urban sprawl—rather than being natural or spontaneous—it has been thoroughly planned (Gottdiener 1977) as the unintended consequence of underlying politics of modern capitalism (Beck et al. 2003). Here, the environmental and social effects of sprawl ‘have been as much a product of the state as of the pri- vate sector’ (Phelps 2015, p. 4), given that public and private sectors have become barely distinguishable. In this context, the policy factors that promulgated urban sprawl relate to land-use conversion, population change, traffic and vehicle miles travelled, energy consumption and fiscal measures (Nelson 1999). i Since its origins urban sprawl has been evolving in terms of its spatial, economic, social, and functional composition, different studies have examined the emergence of suburban employment nodes, emerging patterns of polycentricity, multi-func- tionality, as well as economic and functional self-sufficiency (Phelps and Wood 2011; Burger and Meijers 2012). The latter determines the ‘post-suburban’ nature of suburbia characterised by a more balanced employment and residential linkage (Phelps 2015). These changes were detected by Calthorpe and Fulton (2001), who observed that ‘since their creation, the suburbs have been evolving and changing. From bedroom communities to edge cities, the trend has been toward more complex and complete places’ (Calthorpe and Fulton 2001, p. 198). The policy context of urban sprawl Similarly—and on the basis of how Oslo has defined its sprawling space—Røe and Saglie (2011) put for- ward a model for urban sustainability through the consolidation of suburban ‘minici- ties; a constellation of interconnected districts characterised by a mix of residential SN Soc Sci (2021) 1:202 Page 7 of 35 202 land-uses, workplaces, housing typologies, public amenities, transportation and public spaces with comparatively higher indicators of urban quality (Røe and Saglie 2011).i land-uses, workplaces, housing typologies, public amenities, transportation and public spaces with comparatively higher indicators of urban quality (Røe and Saglie 2011).i Accordingly, it is possible to indicate that there is no single definition of urban sprawl as they differ from morphological, instrumental, and policy-based research. It is also apparent that urban sprawl operates through belated implementation of land- use plans that leave policies of control behind de facto urbanisations (Sharifi et al. 2014; Phelps 2012). Although urban sprawl somehow mimics the morphologies of the spatially homogeneous ‘suburbia’, it also describes a random assemblage of shapes, infrastructures and functions that suggest a diverse space of experimenta- tions in planning beyond bi-dimensional zoning (Gallent and Shaw 2007). In light of these conceptual gateways, the operational understanding of urban sprawl used in this paper relates to the geographical scope proposed by Sieverts’ Zwischenstadt (2003), which situates urban sprawl as the multifaceted and distinc- tive space of transition between the consolidated city and the open countryside. This (in-between) space of transition varies from and one region to another regarding how different policy frameworks define ‘the city’ and ‘the rural’ and in essence, the magnitude and scope of the ‘in-between.’ Nonetheless, bounding these geographies becomes significant considering its practical implications in planning (Fig. 1). Fig. 1 Geographical scope of urban sprawl (author’s diagram) Fig. 1 Geographical scope of urban sprawl (author’s diagram) Page 8 of 35 SN Soc Sci (2021) 1:202 SN Soc Sci (2021) 1:202 202 Physical and geographical determinants In the case of China, Li et al. (2020) demonstrated that driving forces of urban sprawl differ across spatial scales. The authors argue that transport and water sup- ply influence at prefectural and county levels, while the influence of GDP, non- agricultural population growth, institutional arrangements and foreign investments are at a regional level. The study concluded that the hierarchical governance and the socioeconomic and political determinants assume greatest significance (Li et al. 2020). In a geographical analysis of American cities, physical constraints for con- tinuous development and the value of open space are the main determinants of urban sprawl. It relates to the concentration of employment at the ‘central business district’ (CBD). The latter is surrounded by suburbia that results from the trade-off between convenient commuting, affordable housing and the low-cost of private water-wells facilitating the scattered concentration of houses where there is an aquifer from water is pumped (Burchfield et al. 2006). Determinants of urban sprawl The literature about the determinants of urban sprawl is specific to the context where it is examined. In European cities, these determinants relate to population growth, housing demands, land-market constraints as well as improvements in infrastructure (Oueslati et al. 2015; Pirotte and Madre 2011). It is noteworthy that these studies are limited to monocentric city models with clear correlations between land fragmenta- tion and income growth (that derives into higher rates of car use). Considering the extant debate about determinants of urban sprawl, these can be classified into physi- cal and geographical, social, and policy-based determinants. Socioeconomic determinants Urban sprawl is also associated with the political implications derived from sub- urban ideals about freedom and private property (Vicenzotti and Qviström 2018). This is also inextricably linked with mobility and economic security, as well as the image of a ‘good living’ defined by detached or semi-detached homes with private gardens in a rural or suburban living environment (Bontje 2004). Correspondingly, Carrion and Irwin (2004) associate urban sprawl with individuals’ decisions even as commercial and industrial developments at fringe-belt areas become clustered, whereas residential developments become fragmented and dispersed. This disper- sion is partly encouraged by perceived urban ills—such as crime or low school quality—and discretionary decision-making (Carrion and Irwin 2004). Similarly, there is a correlation between perceived rates of crime and land affordability. This, in turn, encourage preferences on suburban areas (Qian and Wong 2012). Accord- ing to empirical research, sociodemographic, economic, political and cultural forces define the primary causes of urban sprawl. These include population growth, trans- portation, industrialisation and economic development, rapid urbanisation, land-use SN Soc Sci (2021) 1:202 Page 9 of 35 202 changes of agricultural lands, migration, income growth, and flexible planning restrictions (Rosni and Noor 2016). An extensive study carried out in Switzerland at municipal level found that deter- minants of urban sprawl are linked to prices of agricultural lands, value of land for construction, population growth, land accessibility, federal tax, changes in employ- ment growth, number of homeowners, income, and commuting rates by private and public transport. The study concluded that ‘the highest contribution to urban sprawl is to be expected in highly accessible areas that are not yet fully urbanised’ (Weilen- mann et al. 2017, p. 481). Accessibility clearly leads ‘to an increased density along a rural–urban gradient which basically follows the level of accessibility’ (ibid: 481) and uncoordinated planning decisions (Phelps 2012). Policy‑based determinants SN Soc Sci (2021) 1:202 Page 10 of 35 202 Table 1 General determinants of urban sprawl Physical and geographical determinants Scale of urban expansion Transport and water supply Population growth Physical constraints (hills, bodies of water, etc.) Presence of water aquifers Socioeconomic determinants Suburban ideals (affordability, detached home, tranquillity) Desire of private property Proximity to nature/countryside Perceived urban ills (crime, low-quality schooling, others) Economic growth and industrialisation Income and employment growth Investments in infrastructure and motorisation (car-depend- ency) Rapid urbanisation Foreign investments Prices of agricultural lands / value of land for construction Taxation on developable lands / windfall gain Policy-based determinants Policies of growth control Locational policies (preference for outer developments) Preference for unregulated areas (e.g. conurbations) Discretionary decision-making (muddling through) Technocratic decision-making on political goals Flexible land-restriction Policies of land-use conversion Institutional fragmentation Asymmetrical urban–rural governance Policies over rural lands Accessibility to rural lands and natural assets Policies on windfall compensation Distribution of private lands dispersion of employment awl Scale of urban expansion Transport and water supply Population growth Physical constraints (hills, bodies of water, etc.) Presence of water aquifers Suburban ideals (affordability, detached home, tranquillity) Desire of private property Proximity to nature/countryside Perceived urban ills (crime, low-quality schooling, others) Economic growth and industrialisation Income and employment growth Investments in infrastructure and motorisation (car-depend- ency) Rapid urbanisation Foreign investments Prices of agricultural lands / value of land for construction Taxation on developable lands / windfall gain Policies of growth control Locational policies (preference for outer developments) Preference for unregulated areas (e.g. conurbations) Discretionary decision-making (muddling through) Technocratic decision-making on political goals Flexible land-restriction Policies of land-use conversion Institutional fragmentation Asymmetrical urban–rural governance Policies over rural lands Accessibility to rural lands and natural assets Policies on windfall compensation Distribution of private lands dispersion of employment Policy-based determinants Policy‑based determinants Empirical studies conducted in Lagos, Nigeria, identified spatial policies as the main determinants of urban sprawl, influencing the characteristics of neighbour- hoods, proximity to water bodies, and economic growth. These results indicate that institutional factors such as the skewed distribution of private land, the high costs of undeveloped land, and flexible regulations also influence sprawling growth along- side accessibility as well as land-use change of forests and farmlands (Braimoh and Onishi 2007). ) In a similar vein, Li and Li (2019) found that arrangements for land-use con- version, enhancement of industrialisation and population controls stimulate urban sprawl in large Chinese cities. Institutional fragmentation allows the formation of coalitions between local government and enterprises, thus driving GDP growth and urban sprawl (Li and Li 2019). Local villagers also end up getting attracted to land-use conversion as the windfall compensation is higher than farming income. Finally, planners operate as ‘technical actors’ who justify decisions made by local or provincial officials to prioritise outer developments (Fang and Pal 2016). Stud- ies conducted in Central and Easter Europe indicate that urban sprawl relates to the preference of new urbanisations in unregulated areas—such as conurbation zones— as they expand the municipal boundaries and new developments indirectly leverage from closer consolidated services (Spórna 2018). As discussed, the determinants of urban sprawl correlate to geographical restrictions but primarily social, planning and policy factors (Table 1). To an extent, these determinants denote natural or regulatory constraints that affect the nature of urban sprawl itself. In addition to the fact that some such constraints are social or regulatory constructions which can be unmade—or are natural features that could be ‘improved’ or ‘reclaimed’ to allow sustainable develop- ment—further inspection is needed given the context-dependent nature of these determinants and because the Latin American context has not been examined in a similar way. Context of analysis: justifying Santiago de Chile The analysis presented in this paper is based on urban sprawl in Santiago de Chile, focussed on the south area which is described as the most important axis for suburbani- sation. The city exemplifies the patterns of urban sprawl observed in other Latin Ameri- can cities characterised by sprawling growth around transport infrastructure (Inostroza et al. 2013), peripheral concentration of large social housing developments (Coq-Huelva and Asián-Chaves 2019), consolidation of peri-urban (upper class) gated communities SN Soc Sci (2021) 1:202 Page 11 of 35 202 (Roitman and Phelps 2011) and fragmented expansion linked to both ‘auto-construc- tion’ and privatised implementation of housing and infrastructure (Monkkonen et al. 2018; Heinrichs and Nuissl 2015). As part of the Latin American context, Santiago is a good example of the significance of normative planning and sociopolitical acceptance of extended suburbanisation (De Mattos et al. 2014). This case is used to identify the determinants of urban sprawl, and as a case study for abstraction and generalisation of conclusion around the main topic of this paper (Flyvbjerg 2006; Burawoy 1991). The capital city of Santiago has been subject of aggressive housing policies over the last 40 years with important consequences for social and spatial segregation (Borsdorf et al. 2007). This city reflects planning rationales adjusted to facilitate urban growth, land privatisation, and centralisation of social housing supply, all elements of a neoliberal planning approach (Vergara and Boano 2020). Santiago mainly describes a monocentric city-model with at least four stages of urban development: (a) the historical city, (b) the consolidated city, (c) the suburban and (d) the exurban. The suburban and the exurban comprise the sprawling geogra- phy of Santiago, characterised by a wide spectrum of interstitial spaces, large-scale housing developments and a few peripheral centralities—including high-streets, warehouses, shopping centres and civic centres—that entails an emerging polycen- tric structure (Truffello and Hidalgo 2015). This sprawling geography is clearly delimitated by the Americo Vespucio ring road which encircles the city to connect most peripheral communes and separates suburban sprawl from the consolidated and historical city (Fig. 2). Fig. 2 Map of Santiago, its four stages of urban growth and boundaries (Author’s map) Fig. Research design, methodological approach and methods The findings of this paper are the outcome of a study-case research conducted between 2012 and 2017, in which a mixed methodological approach was used to articulate qualitative and quantitative data (Tashakkori and Creswell 2007). The methods included documents review, site visits, morphological analysis and map- ping, as well as analysis of semi-structured interviews with relevant actors. The ana- lytical approach is based on the examination of policy conflicts and stakeholders’ understandings of Santiago’s urban development, which made it possible to ascer- tain how planning policies give effect to suburban sprawl in Santiago. p g p gf p g Document review (Bowen 2009) implied secondary research and institutional policies, legal norms, central development plans and regulations, urban design projects, municipal development plans, and historical records covering Santiago’s urban growth. Thematic analysis and critical review of these documents allowed for the identification of key themes around the different determinants of urban sprawl (Gavin 2008). This information was collated with empirical data from official sta- tistical databases (INE 2017) and information provided by 56 semi-structured inter- views—conducted between 2014 and 2016—with a range of actors selected by their first-hand knowledge of Santiago’s urban development (Stender 2017; Galletta 2013). Interviewees were carried out with planners and designers of large urban pro- jects in Santiago, policy-makers and politicians (central/local), metropolitan authori- ties, social and environmental organisations, residents, academics, as well as private developers. These interviews were designed in accordance with the sponsors’ ethical codes of conduct; key informants were anonymised, so that the respondents could be candid, without any fear of social, professional or political repercussions. Thematic analysis was implied to codify data into four categories: (a) Policy-based determi- nants, (b) Economic determinants, (c) Infrastructural (and physical) determinants and (d) conceptual (hermeneutic) determinants, all linked to contents of policy doc- uments (Evans and Lewis 2018). Site visits and direct observations were conducted between 2014 and 2016 to assess the spatial quality and ongoing suburbanisations. These observations were based on Rayback’s approach (2016), who suggests visual records (photographs) and measurements to establish commonalities and differences, and corroborate whether an area of analysis meets the study goals (Rayback 2016). Photographs pro- vided technical accuracy, flexibility of perspectives, and mobility to obtain specific views of the area of analysis. Context of analysis: justifying Santiago de Chile 2 Map of Santiago, its four stages of urban growth and boundaries (Author’s map) Page 12 of 35 SN Soc Sci (2021) 1:202 202 Santiago consists of 36 communes—each one with its own local authority—with a population of 7.112.808 inhabitants representing 40.5% of the country’s popula- tion (INE 2017). The analysed area comprises thirteen communes characterised by the location of large social housing developments over the last 40 years (Tapia 2011). In total, the analysed communes sum a population of 2.665.039 inhabitants, representing 37.47% of the total population of Santiago. In terms of social housing developments, Santiago records a total of 222.024 units between 1979 and 2002, in which the analysed communes concentrate a total of 159.680 units that accounts for 71.92% of the overall metropolitan stock (Fig. 3). Despite the expansion of the urban area, only 1.87% of the city’s population lives in a functionally self-sufficient neigh- bourhoods mainly concentrated in the city centre (Correa-Parra et al. 2020). Fig. 3 Map of Santiago, the southern communes and their population and social housing units (Author’s map based on INE 2017; Tapia 2011; Hidalgo 2007 and Fernandez 2008) Fig. 3 Map of Santiago, the southern communes and their population and social housing units (Author’s map based on INE 2017; Tapia 2011; Hidalgo 2007 and Fernandez 2008) SN Soc Sci (2021) 1:202 Page 13 of 35 202 202 202 Determinants of Santiago’s urban sprawl Santiago de Chile shares common patterns of urban development with most Latin American cities (Flores et al. 2017; Herzog 2015; Rojas, et al. 2013). In Chile, this urban development has been invoked as ‘urban dispersion’ (Heinrichs et al. 2009), ‘urban fragmentation’ (Link 2008), ‘metropolitan expansion’ (De Mattos 1999), ‘extended suburbanisation’ (De Mattos 2001) and ‘dispersed urban expansion’ (Ducci and Gonzalez 2006). Morphological studies on the peri-urban areas, how- ever, have confirmed this development as urban sprawl (Rojas et al. 2013). Specifi- cally, the development has been elucidated as a fragmented space characterised by the presence of varied neighbourhoods, interstices, and a sustained expansion to outer zones (Gainza and Livert 2013). Santiago’s sprawl has been debated since the 1980s after the enactment of The National Policy of Urban Development (NPUD/1979), which declared that urban land was a non-scarce resource and abolished the ‘urban limit’, ‘allowing urban sprawl and doubling the metropolitan area of cities, such as Santiago’ (Vergara and Boano 2020, p. 6). The underlying politics behind the NPUD related to a radical shift from previous centre-left/socialist regimes—in which access to land was con- sidered a right—towards a neoliberal approach whereby land was commodified and managed under market-driven dynamics (Barton et al. 2012). A first amendment to the NPUD/1979 was issued in 1985. However, it did not change the essential features of the 1979 policy apart from giving more relevance to the state to foster urban development through a subsidiary approach (Gross 1991). Following the 2010 earthquake, Chile’s president Sebastian Piñera ordered the conformation of a techni- cal board to elaborate a new Urban Development Policy. A new version of this pol- icy was published in 2014 by the Ministry of Housing and Urbanisation (MINVU 2014c), with clear statements about promoting a sustainable urban development. However, the policy does not strongly place ‘urban sprawl’ as an issue. Instead, it focuses on determining how the impacts of the urban expansion can be absorbed via compensation and promotion of inner densification. In Sect. 2 (economic develop- ment), objective 2.3, point 2.3.5., the policy states: To establish a framework of special obligations to new areas of urban expan- sion, in order to ensure their responsibility on the externalities. Also, to estab- lish a framework of incentives to densification projects to address the impacts over the public space and urban functions in accordance to the law (MINVU 2014c, p. 35). Research design, methodological approach and methods These are deemed evidence—specifically on physi- cal infrastructure, environmental and spatial attributes, land-uses, day-to-day activi- ties, accessibility, and quality of surroundings—to contrast maps and written records (Collier and Collier 1986), and provide data on the morphological and material composition of Santiago’s suburban sprawl (Roberts 2016). Morphological analysis included mapping the suburban expansion of Santiago over the last 40 years follow- ing a Conzenian approach where building patterns, open space, land subdivisions, streets, vegetation, and land land-uses were documented (Kropf 2011). Page 14 of 35 SN Soc Sci (2021) 1:202 202 Policy‑based (and regulatory) determinants A first range of determinants relates to the Chilean constitutional arrangement— defined at the end of the 1970s (Inzulza and Gatica 2019)—in which a set of legal norms frames the planning system. These norms enforce the state to be prescrip- tive about planning and delimitating the role of planners as ‘administrative techni- cians’—professionals who approve or reject planning applications on the basis of these norms. The evaluation of projects in their own merits (or in a case-by-case basis) and in light of ‘the public interest’—which is the essential divide with the British planning practice (Cirianni et al. 2013)—have an intricate legal framework that is highly debated regarding the standards for implementing projects that effec- tively contribute to the public interest. Legal norms aim to consolidate common standards, legal certainty, and fair assessment while being efficient in the use of scarce resources. Additionally, these norms shape the planning system as a ‘regula- tor’ of private initiatives, leaving the role of the state as subsidiary of low-income families. Determinants of Santiago’s urban sprawl In the NPUD/2014, a clear assumption is made that inner densification contributes to sustainable development. This is acknowledged by highlighting some census data around increments in the housing stock over the past 20 years, a higher construc- tion rate of apartments in comparison to detached houses, and a reduction in the rate of expansion in major cities. Apart from minor changes, the sprawling pattern has been a longstanding trend with permanent impacts at environmental, economic, and social levels (Romero and Órdenes 2004). There is also a correlation between sprawling growth and environmental, residential, and sociospatial segregation SN Soc Sci (2021) 1:202 Page 15 of 35 202 (Romero et al. 2012; Sabatini et al. 2009). The latter is recognised by NPUD/2014 as a major challenge ‘caused by decades of advances in reducing the housing short- age from a quantitative approach, without paying attention to the location of housing and access to basic public goods’ (MINVU 2014c, p. 9). However, this policy has come under strong criticism due to its lack of conceptual accuracy around the notion of ‘segregation’ and its practical implications in planning. Besides overlooking criti- cal problems of land-management, land-market, and land policy (Sabatini 2015), it lacks concrete mechanisms to control capital gain (and windfall gain) resulting from uncontrolled urban growth (Trivelli 2015). Finally, the NPUD/2014 lacks the legal status as the ‘General Ordinance of Urbanism and Constructions’ (MINVU 2014b) regulates the public and private operations on the urban space, and must first be changed to ensure the NPUD/2014 possesses appropriate power to leverage urban development. In this policy context, unpacking the determinants of Santiago’s suburban sprawl becomes relevant, as sprawling growth appears as a less controlled process in planning (Cortés and Iturra 2019). In this paper, at least thirteen determinants are identified and sorted by policy-based, economic, infrastructural, and conceptual determinants. Planning policies, regulations, and social housing developments Low-income groups and maximisation of public benefits defined the supply of low- cost housing in cheaper lands beyond urban boundaries, thus allowing families to become ‘landowners’ and beneficiaries of public benefits (Valdivia et al. 2012). The financial scheme was a ‘subsidy to the house’ to complement families’ sav- ings, which has had a recognised quantitative significance in reducing the housing shortage. However, the massive delivery of subsidies defined a huge concentration of poverty highly dependent on public transport (Hidalgo 2019). This policy was Page 16 of 35 SN Soc Sci (2021) 1:202 202 reinforced by implementing repetitive housing typologies, including the ‘caseta sanitaria’ (sanitary hut) composed of a basic toilet and installations for mounting a kitchen; over time, families would gradually build the rest. Urban services were not implemented, resulting in a vast concentration of clusters of basic shelters. Housing extensions were informally done and after a few years, the resulting suburbia was a patchwork of roads, farming areas, and semi-informal neighbourhoods concentrated in the southern peripheries of La Pintana, San Bernardo, Puente Alto, Padre Hur- tado, Maipu, and El Bosque. The situation of La Pintana is described as follows: The area “El Castillo” originally had about 25,000 poor residents. The over- crowding was horrible, and crime exponentially grew. Current social houses are about 30m2 with a 110m2 site, but these “sanitary huts” were only 2.5m2 of basic services and the rest was informally finished by the families. So, peo- ple built everything wrong and with wrong materials. In many cases those small houses had up to 12 people! and without common spaces. It triggered an endless chain of problems including domestic violence, crime and others that now we all need to deal with (interview with a member of the Agricultural Cooperative José Maza, La Pintana, June 2014). The centralised nature of planning also contributes to Santiago’s sprawl as central authorities decide the place for new social housing developments. These are often in peripheral areas immediately after communal boundaries. Thus, local plans are continuously updated to incorporate new urbanisations driven by the MINVU. The creation of future housing areas distinguishes between zones of ‘extension’ (con- tinuous growth) and zones of ‘expansion’ (discontinuous growth) (MINVU 1998), where the latter are peri-urban (or rural) communes that act as ‘receivers’ of new housing developments. The cases of Lampa, Buin, Maria Pinto, or Melipilla have been included into metropolitan plans since 1997. Planning policies, regulations, and social housing developments Local authorities ‘consider these operations unfair; short-term centralised decisions that transfer saturation of infra- structure to local authorities’ (interview with local planner at Municipality of Lo Espejo, May 2014). Peripheral municipalities tend to reject this concentration of poverty, which is attributed to political tensions around central-local political alignment: ‘this is problematic when central authorities have an average of 8000 families per year with approved subsidies but without available land’ (interview with local planner at Municipality of La Pintana, May 2014). This tension is perceived as critical when compared with technical instruments, thereby ‘confirming that Santiago’s sprawl- ing management is more a political exercise rather than a technical practice’ (inter- view with senator for 8th zone of East Santiago, May 2014). These decisions imply a reduction of rural assets, the inorganic expansion of communal boundaries, con- centration of peripheral poverty, and saturation of local services, all of which are elements of sprawling growth. Regulatory weaknesses Santiago’s urban sprawl also relates to various regulatory weaknesses which allows for uncontrolled growth to occur. Real estate firms command their projects through SN Soc Sci (2021) 1:202 Page 17 of 35 202 these weaknesses to implement larger housing developments and maximise profit- ability. As an example, for social housing, the regulation requires an additional study of transport impact for projects of 250 car-parking lots or more (MINVU 2003), which means extra investments in roads capacity. Therefore, developers submit (adjacent) projects of ‘249’ parking lots to avoid this obligation: One of the consequences of extending the city trough this exercise is that neighbourhoods end up with narrow streets and lack of parking space. So, on- street parking becomes the norm…cars block the access to ambulances, rub- bish collection, police, fire engines, etc. Large residential areas have been cre- ated in the peripheries through these 249-car-parking projects and we cannot reject them because “they are legal”. This is why in Chile we say that “made the law, so, made the trick” (interview with local planner at Municipality of Cerrillos, May 2014). Despite being ‘legal’, these operations extend the city through the implementation of low-density neighbourhoods with no proper transport infrastructure and end up in segregated areas that amplify the sprawling character of suburban communes. The law 3516/1980 At the end of the 1980s and supported by the law Nº3.516 (1980), upper-class fami- lies started to move to peri-urban zones stimulated by the creation of the ‘Parce- las de Agrado’ [Plots of Pleasure]. This regulation allows residential urbanisations beyond the urban limit in 5000 m2 sites. Although a half-hectare plot will not consti- tute a land for agricultural exploitation, it would preserve the rural landscape despite its residential functions (Ministry of Agriculture 1980). Generally, the resulting landscape is a cluster of half-hectare neighbourhoods characterised by detached sin- gle-family houses placed in rural settings, equipped with urban infrastructure (pipes, sewage, electricity, water supply, and collection of domestic rubbish), and connected to the city by roads. Generally, there is no public transport, which means an intensi- fied used of private cars. i To develop a ‘Parcela de Agrado’, developers must obtain a favourable report from the Ministry of Agriculture (one of the few powers of agricultural authorities) confirming the absence of negative impacts, residential land-uses, and land size. These are the few restrictions in comparison to traditional inner suburbia, which label ‘Parcelas de Agrado’ as ‘free of restriction zones’ (Borsdorf and Hidalgo 2005). These peripheral zones are an ambiguous scenario of governance as the (sub) urbanisation process occurs on rural lands in possession of the Ministry of Agri- culture. However, extra regulation cannot be exerted as it becomes ‘residential’ and thus, part of the MINVU realm. Exceptional circumstances for further plot subdivi- sions of less than 5000 m2 can only be suggested by the Ministerial Regional Secre- tary of Agriculture (SEREMI) intended for social housing: This process relies almost entirely on the SEREMIs, and it is discretionary to each regional authority during a presidential period. This is a very attractive Page 18 of 35 SN Soc Sci (2021) 1:202 202 position as it serves as a resort of political progression. Thus, new urbanisa- tions—both ‘Parcelas de Agrado’ and social housing—tend to be approved and extend the city with almost no regulation (interview with Professional Advisor at SEREMI of Agriculture 2010-2014, May 2014). position as it serves as a resort of political progression. Thus, new urbanisa- tions—both ‘Parcelas de Agrado’ and social housing—tend to be approved and extend the city with almost no regulation (interview with Professional Advisor at SEREMI of Agriculture 2010-2014, May 2014). The relocation of slums One longstanding determinant of Santiago’s sprawl relates to the eradication of slums from central areas. This process has been operational since the 1980s, when the city witnessed the highest rate of informal occupations (Swatson 1998). These occupations were then eradicated to southern communes; an operation with a double purpose: providing houses to low-income families and liberating central areas for profitable real estate projects. During the 1990s, this policy was intensified to trans- form landownership while justifying the implementation of social programmes: Poor people were moved here to have a house, then they wanted to go to their workplaces at the city centre. The only connection was the Santa Rosa Ave- nue, and was not enough. So, many gardeners, carpenters, builders, nannies and others from La Pintana lost their jobs because it takes so long to arrive on time. These plans of eradication with poor urban standards extended the city and left these people behind…all for the sake of becoming a ‘landowner’ (interview with local planner, Municipality of La Pintana, May 2014). The overall outcome is a dispersed expansion of the city through the concentra- tion of poverty in the southern peripheries. This increased the spatial segregation that transformed Santiago into one of the most segregated cities in Latin America (Sabatini et al. 2009). The law 3516/1980 The ‘Parcelas de Agrado’ is spreading around the metropolitan region, configuring a ‘pseudo-(sub)urbanisation’—identified as ‘the infiltrated city’ (Naranjo 2009)—that has significantly reduced proper farming lands and blurred the urban–rural transect that has even encompassed mall outer towns (Boccardo 2011). Institutional asymmetries Rural policies have a very limited influence in controlling the encroachment of rural land by the urban expansion. In addition, agricultural authorities are often politically corseted by the views of experts and the public: Rural authorities are in a political position in which rejecting urbanisations is unpopular among real estate firms. Additionally, mass media always shows that land and housing prices are increasing because there is a scarcity of land within the city (interview with Professional Advisor at the SEREMI of Agri- culture 2010-2014, May 2014). For agricultural authorities, cross-sectorial coordination becomes crucial in negoti- ating irreversible impacts of urbanisation: ‘This is a challenging process that mainly relies on the leadership capacities of public authorities and agreements among polit- ical coalitions, which confirms the political dimension of the Chilean planning sys- tem’ (Interview with the Director of the INIA, Ministry of Agriculture, May 2104). It is noteworthy that the asymmetric relationship between urban and rural authorities correlates to the unidirectional implementation of housing projects on rural lands, causing the reduction of rural assets and contributing to the sprawling extension of the city. The ‘urban limit’ This containment policy was implemented in 1960 through the ‘Intercommunal Regulator Plan’ (PRI). The purpose was to set the city’s boundary and restrict urban- isations beyond the specified limit. However, the urban limit has been constantly modified to incorporate new developments since the 60s. For some, the ‘urban limit’ is a disturbing tool as the division of a land in ‘urban’ and ‘rural’ by an arbitrary line increases its value without any condition or extra-cost for the owner (windfall gain). Additionally, land subdivision encourages landlords to alter functions of those portions outside the limit since their profitability increases with real estate projects. Therefore, the ‘urban limit’ has a paradoxical effect: while it attempts to prevent uncontrolled growth and protect agricultural lands, it also creates a perverse incen- tive for incorporating the countryside in trying to modify norms and functions SN Soc Sci (2021) 1:202 Page 19 of 35 2 202 (López 1981). As an undesirable side-effect, low-income families must be relocated further away on cheaper lands. Detractors of the ‘urban limit’ consider its abolition as panacea for rectifying land-market distortions, increasing the land stock, and simultaneously decreasing land prices. Accordingly, the ‘urban limit’ was abolished in 1979 to allow devel- opment in any location within the regional space (Fig. 4). However, landlords did not sell their lands and kept them undeveloped to catch value over time. Santiago’s peri-urban space entered into an unprecedented land-banking exercise that cre- ated rings of unaffordable lands around the city. Thus, low-income families were located in remote rural areas without proportionated responses in transportation, employment, and services (Rodríguez y Sugranyes 2004). In this process, Santia- go’s fringes became fragmented and shaped by low-quality neighbourhoods inter- spersed with expensive rural lands. In 1994, this ‘urban limit’ was finally reinstated (PRMS/1994), including zoning for future housing developments (MINVU 1998). Fig. 4 Evolution of Santiago’s expansion and the urban limit (authors’ map based on data from Observa- torio de Ciudades UC) Fig. 4 Evolution of Santiago’s expansion and the urban limit (authors’ map based on data from Observa- torio de Ciudades UC) Page 20 of 35 SN Soc Sci (2021) 1:202 202 The implementation of urban limits in Santiago—as not being supported by con- trols on the land-market—contributed to encroachments of adjacent rural lands. Conversely, its absence created market distortions that encouraged leapfrogged development, thereby enhancing sprawling growth. Planned outer developments Santiago’ sprawl is also increased by the creation of planned zones for urbanisation beyond the urban limits. The ‘Conditioned Urban Development Zones’ (ZODUCs), ‘Conditioned Urban Development Projects’ (PDUCs) and ‘Prioritised Areas of Urban Development’ (AUDPs) are examples of large-scale urban developments. Introduced by the Metropolitan Plan of 1997, these zones comprehend 300 hec- tares for residential purposes mainly (MINVU 2014a). In the northern metropolitan space, the implementations of ZODUCs have transformed the historic rural land- scape into a constellation of upper-class residential clusters with unambiguous signs of gentrification (Inzulza and Galleguillos 2014). These developments are identified as planned ‘gated communities’, around high-quality private roads that connect the zones with the business districts of Lo Barnechea and Vitacura (Salazar 2010). Vari- ous real estate firms initially bought the lands and obtained approval for urbanisation in the communes of Colina and Chicureo in the north, and Pudahuel in the west inter alia (Fig. 5). SN Soc Sci (2021) 1:202 Page 21 of 35 202 202 Fig. 5 A view of a private–public space, an artificial lagoon, a shopping centre and houses at the Piedra Roja private residential complex, Colina (author’s photo 2014) Fig. 5 A view of a private–public space, an artificial lagoon, a shopping centre and houses at the Piedra Roja private residential complex, Colina (author’s photo 2014) These zones were pioneered in incorporating externalities associated to transport infrastructure (Heinrichs et al. 2009), as private developers were required to provide motorways, commercial services, light industry, public space, water plants, security and others, and configure a privatised suburban space with high levels of functional self-sufficiency. Arguably, ‘these new settlements have begun to acquire govern- ment functions and civic spaces along with greater density and mix use of devel- opments and buildings’ (Phelps 2015, p. 32). In one way or another, the ZODUCs have become a Latin American version of suburban ‘minicities’ (Røe and Saglie 2011). Their impacts are linked to leapfrogged development and a type of suburbia with clear signs of spatial segregation at metropolitan levels (Cooper and Henríquez 2010). Economic determinants For central authorities, Santiago’s sprawl relates to the costs of enabling inner lands for housing due to the land price and lack of mechanisms to internalise capital gain. Land-value capture from public investments do not have the proper legal basis to levy, which provides an advantage to private sectors. Similarly, local authorities change their development plans to enable lands for development (of further densifi- cation), which is a plus value also captured by the private sector. This translates into Page 22 of 35 SN Soc Sci (2021) 1:202 202 a restriction to the public budget with consequences on regressive investment on inner lands or the implementation of a robust urban regeneration policy (Sepúlveda and Larenas 2011). a restriction to the public budget with consequences on regressive investment on inner lands or the implementation of a robust urban regeneration policy (Sepúlveda and Larenas 2011). The absence of urban regeneration policies The absence of an urban regeneration policy stimulates the search of peripheral lands for urbanisation. Despite efforts of retrofitting—such as the programmes ‘Zonas de Renovación Urbana’ (Zones of Urban Renewal) and ‘Quiero mi Barrio’ (I Love my Neighbourhood)—a standard policy to recover obsolete industrial lands, underused areas, infrastructural zones or any type interstitial space has not been fully implemented (Páramo and López 2020). In 2012, a study of the Chilean Cham- ber of Construction (C.Ch.C.) detected numerous vacant plots of more than two hectares without restrictions for densities of around 150 people/hectare (higher than Santiago’s average of 85 people/hectare) that are irrecoverable. In 1992, a state-led regeneration programme was implemented only for central areas (Zones of Urban Renewal) to stimulate re-densification via subsidising first-time buyers (Verdugo 2003). However, this programme was not extended to other districts, intensifying the search of peripheral lands to respond to housing shortages. For developers, an additional problem relates to the historical ‘atomisation of the land’—the continuous subdivision of land in smaller properties—that impedes the reconversion of large areas: It is easier to transform an urban area when the land is not too “atomised”. This is the outcome of transforming everyone in a landowner. So, when we want to develop a project, we find the area transformed into a cluster of small- scale landlords and we must negotiate with all of them! …simply impossible. So? Better to find peripheral larger plots (interview with Honorary Advisor and real estate developer, Chilean Chamber of Construction, May 2014). An over-protection of historical low-density neighbourhoods linked to conceptual understandings of ‘identity’ and ‘place attachment’ is also a restriction for urban regeneration: We have several very low-quality areas in Santiago of two/three-story houses, functionally obsolete, abandoned, without maintenance. Do these really have any historical or architectonic value? So, the absence of a regeneration policy does not allow recovering them because we do not have legal certainty and thus, peri-urban zones remain more attractive (interview with the Coordinator of Housing and Real Estate Development at Chilean Chamber of Construction, May 2014). The absence of a regeneration policy contributes to the inorganic growth of San- tiago as it indirectly stimulates the search of peripheral lands for suburbanisation. It also does not support land-mixing for having large plots as an alternative to land-atomisation. SN Soc Sci (2021) 1:202 Page 23 of 35 202 202 No taxation on undeveloped lands For many years, the absence of any form of impact fee on private vacant lands— or internalisation of capital gain—has contributed to discontinuous urbanisations, as landowners leave suburban lands undeveloped. This has indirectly influenced the location of new housing beyond consolidated urban areas. Importantly, this absence of financial restriction was a strong stimulus to land-banking: In Chile we have a serious problem with land-banking that should be part of the tax reform. The law indicates that lands pay taxes regarding their “functions” (not loca- tion). So? landowners label their sites as “rural” to avoid taxation. This is a direct incentive from our policy, as landowners get the capital gain without paying for that. Some landowners are indeed foreign investors that see the business…they literally buy lands and keep them empty for years. Then, they sell them with no taxation at all (interview with the National Director of Urban Development, MINVU, May 2014). In many cases, these undeveloped sites are abandoned—and as not entitled for public maintenance and security—subject to physical erosion that affects the entire suburban landscape. Over a period of time, they become socially accepted as part of the landscape (Fig. 6). An effective land taxation over ‘non-agricultural’ undeveloped lands has been applied by the Internal Revenue Service (SII) only since 2017. However, the absence of mechanisms of value capture for years did not contribute to inner densification. It also forced the search of peripheral lands for social housing that contribute to sprawl. Fig. 6 An abandoned private site in La Cisterna (author’s photo 2014) Fig. 6 An abandoned private site in La Cisterna (author’s photo 2014) Page 24 of 35 SN Soc Sci (2021) 1:202 SN Soc Sci (2021) 1:202 202 Improvement of roads capacity Local authorities must request road updating to central authorities—specifically, the Ministry of Public Works (MOP)—which requires a technical justification based on urgent needs often linked to natural disasters or regional priorities. Therefore, local authorities justify investments via development projects, pointing at the improve- ment of roads capacity to avoid urban sprawl. If technical capacities are limited— which is the case for low-income communes—road plans tend to be technically poor and therefore, rejected. In this scenario, political leadership and negotiation skills become critical and highly politically sensitive: The state never relies solely on technical reports for the improvements of our roads. These improvements strongly depend on our political and techni- cal teams, and our possibilities to convince different people at central levels around common goals. However, if our mayor is in the wrong side (politically speaking), we would need to wait for the next political cycle to get support and improve our road capacity. By then, the city has been further expanded (inter- view with local planner at Municipality of Maipu, May 2014). If local authorities do not belong to the political coalition of the central government, the assessment of a municipal project can be delayed or rejected. This has significant implications at economic and social levels as road capacities in Chile are designed to support six-story housing blocks, restricting the implementation of high-rise buildings. A six-story building does not require lifts (MINVU, OGUC/2014), which makes the block affordable for low-income families and restricts the arrival of medium/high-income families that prefer lifts. Consequently, the area becomes unattractive for private developers and creates political tensions as local authorities are unable to achieve higher rates of social mobility. As seen, outdated roads do not allow further inner densification that enhance the search of outer rural lands for new housing developments. Infrastructural determinants Some suburban municipalities are known to have land capacity to increase their den- sity, but lack the road capacity to assimilate increments of car use and public trans- portation associated with densification. Regional infrastructure The expansion of railway services and of motorways to satellite tows and other regional services—such as port-ship areas—often triggers (unplanned) ‘transit- oriented development’ (TOD) that consolidates conurbation processes. Led by the Ministry of Public Works (MOP), the extension of transport infrastructure creates SN Soc Sci (2021) 1:202 Page 25 of 35 202 opportunities for developments in the in-between lands. In other cases, the MINVU approves new urbanisations in the conurbation space without proportional responses in transport infrastructure, thus illustrating a lack of cross-sector coordination between central authorities: opportunities for developments in the in-between lands. In other cases, the MINVU approves new urbanisations in the conurbation space without proportional responses in transport infrastructure, thus illustrating a lack of cross-sector coordination between central authorities: It is important to define the connectivity first. If not, new neighbourhoods will have problems of congestion and accessibility. If the MOP or the real estate firm do not update the road capacity, how traffic congestion will be absorbed? and by ‘who’? In our commune there is only one large road to connect the city with these new developments, but the MOP is instead updating other roads. For sure our one will collapse. The central authorities are in debt with the southern communes as they solve their regional issues but creating local prob- lems. We are now forced to approve housing projects over this road which is not prepared for this new demand; it is still a rural road (interview with local planner as Municipality of Padre Hurtado, June 2014). Regional connectivity influences the encroachment of rural lands as they become accessible to real estate projects, industries, and the scattered location of metropoli- tan facilities that contribute to urban sprawl. Conurbation zones As mentioned before, conurbation zones are linked to transport corridors. These areas are increasingly complex due to the unregulated coexistences of rural and urban functions inter alia. The typical landscape of Santiago’s conurbations reveals a heterogeneous amalgamation of farming lands, high and low-income residential neighbourhoods, heavy and light industries, interstitial spaces, military facilities, and geographical restrictions such as rivers, lakes, and hills. In these zones, many lands keep agricultural functions and industries, including animal breeding and milk factories that coexist with residential zones (such as those framed by the Law 3516/1980). After a while, conurbations become contested spaces where industrial noise, smell, pollution, farming, and traffic of heavy vehicles collide with residential functions: When water canals – which are open tracts – pass through a neighbourhood it is used to throw rubbish! and we cannot close them because there is a restric- tion to build over it. So, developers should keep them untouched. Thus, people have an open tract within their properties of at least two metres wide. In other cases, underground canals are pierced by tenants to get an ‘extra pipe’! water from laundry and kitchen is melted with the water used for agricultural pur- poses (interview with the President of Water Community Villa Las Rosas, La Pintana, June 2014). Amidst these tensions, the MINVU and housing developers prevail as the unintended use of chemicals and hazardous substances in agriculture impact on people’s health and thus, land-use changes from rural to residential are implemented (Fig. 7). Page 26 of 35 SN Soc Sci (2021) 1:202 202 Fig. 7 Railway lines in the conurbation between Santiago and Padre Hurtado. At the back, farming spaces and new social housing developments (author’s photo 2014) Fig. 7 Railway lines in the conurbation between Santiago and Padre Hurtado. At the back, farmin spaces and new social housing developments (author’s photo 2014) Conurbation zones are unregulated spaces in which the encroachment of rural lands is linked to regional connectivity. In these zones, there is also a mixed governance whereby both rural and urban activities take place, thus contributing to the scattered growth of Santiago. Conceptual ambiguities: what is finally the problem? Although there is abundant literature on Santiago’s metropolitan expansion (Truf- fello and Hidalgo 2015; De Mattos et al. 2014; Link 2008; Ducci and Gonzalez 2006; De Mattos 2001; Armijo 2000), there is no consensus among central authori- ties about what better describes Santiago’s pattern of urban growth (Fuentes and Pezoa 2018) and what are the policy implications: ‘there is an ambiguous interpreta- tion—at the core of the central planning practice—of what urban sprawl is. We have divergent positions—even within the same political coalition—about restraining SN Soc Sci (2021) 1:202 Page 27 of 35 202 or intensifying Santiago’s sprawl’ (interview with Director of Consultants, MOP. May 2014). While urban sprawl continues to be debated, planning solutions tend to be delayed. For some, this delay has evinced a deliberate position of ‘planning for growth’ since the mid-1970s; the outcome of a neoliberal agenda that has instru- mentalised planning to promote further sprawl and ‘has transformed urban design to make it an efficient mechanism for capital accumulation’ (Vergara and Boano 2020: (1). Here, suburban sprawl has become utilitarian for the serial production of mas- sive residential space with homogeneous aesthetics (ibid 2020) at the expense of having the environmental and social sustainability under siege (Romero and Órdenes 2004). This conceptual ambiguity contributes to urban sprawl as it restrains not only the implementation of controls over the scattered expansion, but also collaborative debates around the efficacious ways of peripheral land managing. or intensifying Santiago’s sprawl’ (interview with Director of Consultants, MOP. May 2014). While urban sprawl continues to be debated, planning solutions tend to be delayed. For some, this delay has evinced a deliberate position of ‘planning for growth’ since the mid-1970s; the outcome of a neoliberal agenda that has instru- mentalised planning to promote further sprawl and ‘has transformed urban design to make it an efficient mechanism for capital accumulation’ (Vergara and Boano 2020: (1). Here, suburban sprawl has become utilitarian for the serial production of mas- sive residential space with homogeneous aesthetics (ibid 2020) at the expense of having the environmental and social sustainability under siege (Romero and Órdenes 2004). This conceptual ambiguity contributes to urban sprawl as it restrains not only the implementation of controls over the scattered expansion, but also collaborative debates around the efficacious ways of peripheral land managing. fi As discussed, a total of thirteen determinants—clustered into policy-based, economic, infrastructural and conceptual—influences Santiago’s urban sprawl (Table 2). Conclusions Despite the implementation of urban growth boundaries, urban sprawl remains an intractable predicament for spatial planning. This is partly attributed to the diffi- culties of planning instruments to control the determinants of urban sprawl within the policy context they are manifested. The extant literature indicates that physical restrictions, socioeconomic, and policy-based determinants influence processes of urban sprawl. The empirical analysis developed in this paper confirms these deter- minants and proves that policy-based decisions critically contribute to urban sprawl. p p y y p As illustrated through the case of Santiago de Chile, urban sprawl is a complex phenomenon that is not possible to be managed through growth restrictions. Thus, unpacking its determinants appears as a plausible alternative for framing a more sustainable development. The determinants of Santiago’ urban sprawl are context- dependent, multiple and interlinked, specifically defined by legal norms, historical relocation of slums, institutional asymmetries, restrictive controls on urban growth, the creation of planned outer developments (ZODUCs), weaknesses in planning regulations, specific laws (3.516/1980), restrictions to inner densification, improve- ments in regional connectivity, as well as ambiguities around the implications of urban sprawl. All these determinants are placed at different institutional levels—and with repercussions at different spatial scales—configuring a challenging policy con- text that calls for the development of theories of urban politics beyond normative rationales. Considering the ethos of development plans, the failure in controlling sprawl sug- gests that planning in Chile has lost sight of any meaningful notion of the collec- tive interest—placed in the SGDs—and how agendas of urban sustainability can be implemented though traditional (post)democratic channels. The massive production of social housing attempted under modernist state interventions and the implementa- tion of regional/ inter-urban motorways may account for the scale of the subsequent dysfunctionalities of suburban sprawl; all contradictions in the accumulation pro- cess now having to be addressed by further rounds of state interventions—such as the new version of the NPUD/2014. This confirms planning as a political practice that has far overpassed its prescriptive ethos built upon the supremacy of technical macro-scale plans. p Further research is necessitated around the strategic articulation of the determi- nants of urban sprawl in contexts where ‘muddling through’ prevails as a mode of decision-making in land-use planning. Conceptual ambiguities: what is finally the problem? Table 2 Determinants of Santiago’s urban sprawl Policy-based determinants 1 Planning and housing policies 2 Normative gaps 3 The law 3516 (1980) 4 Relocation of slums 5 The ’urban limit’ 6 Institutional asymmetries 7 Planned outer developments Economic determinants 8 The absence of a regeneration policy 9 No taxation on undeveloped lands Infrastructural determinants 10 Improvements on roads capacity 11 Regional transport infrastructure 12 Conurbation zones Conceptual determinants 13 Ambiguities around the meanings of urban sprawl— how urban sprawl is manifested—Disparities around benefits and impacts of urban sprawl—dis- parities between growth v/s no growth Table 2 Determinants of Santiago’s urban sprawl Page 28 of 35 SN Soc Sci (2021) 1:202 SN Soc Sci (2021) 1:202 202 Conclusions As Lindblom (1999) has shown, planning as root or rational comprehensive planning is in itself difficult to achieve, as it rests mainly on piecemeal processes of decision-making, incremental implementation of long-term views and the assessment of urban projects in a case-by-case basis. The introduction of environmental studies is also relevant for examining the extent to which urban sprawl in Santiago contributes to CO2 emissions and other environ- mental issues highlighted by the SDGs—an agenda that is subscribed to by the Chilean government. It places the importance of Santiago’s sprawl and its value in forcing inter-disciplinarity in urban studies. Therefore, it is necessary for a strate- gic approach to consider cross-sectorial coordination, alignment between local and SN Soc Sci (2021) 1:202 Page 29 of 35 202 central authorities, and innovations in suburban governance. Unpacking the deter- minants of urban sprawl offers an alternative point of entry into the studies of sub- urbanisation and associated dilemmas around ’grow or not to grow’, also shedding light on how urban sprawl can be articulated as a distinctive geographical space that deserves planning and policy approaches in its own right. Acknowledgements The authors would like to thank the editors of the journal and anonymous reviewers for their insightful comments on the previous versions of this article. The authors are grateful to inter- viewees for providing data and valuable insights for this article. Author contributions The corresponding author contributed to the study conception and design. Mate- rial preparation, data collection and analysis were mostly performed by Cristian Silva with collaboration of Francisco Vergara-Perucich. The first draft of the manuscript was written by Cristian Alejandro Silva Lovera, and all authors commented on second and third versions of the manuscript. All authors read and approved the final manuscript. Funding This work was supported by the National Commission of Science and Technology of Chile, CONICYT, Grant 72110038; the FONDECYT Project Number 11180569; the School of Natural and Built Environment (SNBE), Queen’s University Belfast (QUB), Grant D820PAC. Data availability The datasets of semi-structured interviews generated during and/or analysed during the current study are not publicly available due to restrictions imposed by the ethical assessment of the research (Ethic Application ID: 5588/001, 2014, University College London) that required interviews to be anonymised so that the respondents could be frank, without fear of professional repercussions. Declarations Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest. Ethical statement The content and results presented in this paper are part of a research carried out at Uni- versity College London (UCL), approval of ethics assessment granted under the application ID: 5588/001, 2014. 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W2892146738.txt	https://zenodo.org/record/6454873/files/source.pdf	en		Long-Term Bioclimatic Modelling the Distribution of the Fire-Bellied Toad, Bombina Bombina (Anura, Bombinatoridae), Under the Influence of Global Climate Change	Vestnik zoologii	2,018	cc-by	4,329	Vestnik Zoologii, 52(4): 341–348, 2018 DOI 10.2478/vzoo-2018-0036 UDC 597.822(477.64) LONG-TERM BIOCLIMATIC MODELLING THE DISTRIBUTION OF THE FIRE-BELLIED TOAD, BOMBINA BOMBINA (ANURA, BOMBINATORIDAE), UNDER THE INFLUENCE OF GLOBAL CLIMATE CHANGE V. Tytar1, O. Nekrasova2, A. Pupina3, M. Pupins 4, O. Oskyrko5 1, 2 Schmalhausen Institute of Zoologу, NAS of Ukraine, vul. B. Khmelnytskogo, 15, Kyiv, 01030 Ukraine E-mail: oneks22@gmail.com 3,4 Departament of Ecology, Institute of Life Sciences and Technologies, Daugavpils University, Parades street, 1A, Daugavpils. LV5400 Latvia E-mail: mihails.pupins@gmail.com 3,4 Latgales Zoo, Vienibas street, 27, Daugavpils. LV5400 Latvia 5 Educational and Scientific Center “Institute of biology and medicine”, Taras Shevchenko National University of Kyiv, pr. Akademika Hlushkova, 2, Kyiv, 03022 Ukraine E-mail: sashaoskirko@gmail.com Long-Term Bioclimatic Modelling the Distribution of the Fire-Bellied Toad, Bombina bombina (Anura, Bombinatoridae), under the Influence of Global Climate Change. Tytar, V., Nekrasova, O., Pupina, A., Pupins, M., Oskyrko, O. — The article describes the potential distribution area of B. bombina and figure out the significant climatic factors of the species at a home range scale. This species is listed on Appendix II of the Bern Convention and on Annexes II and IV of the EU Natural Habitats Directive. It is protected by national legislation in many countries, occurs in many protected areas, and is listed in many national and sub-national Red Data books and lists. We collected the occurrence records and a set of climatic variables including 19 factors from 10’ resolution historical (summarizing annual trends, seasonality and extreme conditions during 1961–1990) and projected data (2050) available at the CliMond database. As a result, under climate predictions for 2050 there may be a substantial north and north-west shift of optimal habitat. Under such a scenario B. bombina populations may suffer mostly in the east and south of Ukraine. Under the modelled scenario the species representation in protected areas throughout the home range should be considered, but especially in Ukraine. K e y w o r d s : global climate change, Bombina bombina, distribution modelling, amphibians. Introduction Recent reports suggest that as much as a third of all known amphibians are in decline, many inhabiting areas far from obvious human disturbances (Stuart, 2008). Global warming and climate change have been implicated as forces likely to drive amphibian declines by significantly changing a habitat through time (e. g., 342 V. Tytar, O. Nekrasova, A. Pupina, M. Pupins, O. Oskyrko Pounds, 2001; Carey, Alexander, 2003; Raffel et al., 2013). Meta-analysis comparing responses of a wide range of different taxonomic groups to climate change across several biogeographical regions already indicate shifts in the distribution patterns of many plants and animals (e. g., Parmesan, Yohe, 2003). The range shift parallels a 0.8 °C warming over Europe during the last century, which has shifted the climatic isotherms northwards by an average of 120 km (Beniston, 1998). The Intergovernmental Panel on Climate Change (Solomon, 2007 ;Climate Change, 2014) projections forecast changes in the global climate during the 21st century even larger than those observed during the 20th century. Further temperature warming and decreasing of water availability will produce dramatic consequences for amphibians in terms of large extinctions and/or range shifts (Araújo et al., 2006). In the context of future climate change, range shifts are a key response, and can affect species representation in protected areas. Ectotherms are more likely to track their climate space compared to endotherms (Aragon et al., 2010) and major shifts in herpetofaunal assemblages caused by climate change are predicted worldwide (Lawler et al., 2010). Northward range shift would thus be reflected in either a net extinction at the southern boundary or a net colonization at the northern boundary. Because habitat suitability is shifting with climate change, such shifts could lead to population declines and high extinction risk for species that are unable to move to new appropriate conditions because of the patchy landscape and insufficient dispersal corridors (Araújo et al., 2006). The complex interaction between species distribution and climate change is yet insufficiently understood, but the increasing availability of information on the variation of environmental parameters in geographic space, species distribution data, and computation capacities during the last decade now allow large scale assessments of such relationships (Kozak et al., 2008). Relationships can be assessed by calculating ‘environmental’ or ‘ecological’ niches and their subsequent projection into geographic space (Guisan, Zimmerman, 2000). But testing the hypothesis that global warming has already caused amphibian declines is challenging because many factors could act synergistically with climate in complex ways (Pounds, 2001). In this study, we aimed at projecting the availability of suitable habitat for an endangered amphibian species, the Fire-bellied toad Bombina bombina (Linnaeus, 1761). The species is distributed in lowlands over a wide continental area: it is widespread in Poland, Lithuania, Belarus, Western Russian Federation, Ukraine, Bulgaria, Rumania and Hungary. The western border reaches the lowlands of Austria, Czech Republic and East-Germany. The northern border of distribution follows approximately the 56° degree of latitude. The most Northern European populations are in Southern Sweden, East-Denmark, Northern Germany (Schleswig-Holstein), Belarus and Southern Latvia (Drobenkov et al., 2005; Kuzmin et al., 2008). In Sweden during the 1960s, it became extinct. In western and northern Europe the species is threatened by the loss of habitat. Recent declines in northwestern Europe might also be related to climate change (Agasyan et al., 2009). Even small climatic changes could have profound effects on the abundance of the species in this area. In Latvia, for instance, the cold climate, snowless winters, cold short springs and dry hot summers are specified as negative factors based on the risks for B. bombina populations (Pupina, Pupins, 2016). Material and methods A substantial amount of species records of B. bombina are available through the Global Biodiversity Information Facility (GBIF, www.gbif.org) and HerpNet databases (www.herpnet.org). In addition, species records can be obtained from own field trips (Pupina, Pupins, 2015), museum collections or literature (Sillero et al., 2014). In total, 2303 georeferenced occurrence records of B. bombina finds were analyzed. Of these, 163 are from Latvia (Pupina, Pupins, 2015) and 866 from Ukraine (80 % personal data of O.Nekrasova: Tytar, Nekrasova, 2016; Tytar et al., 2018 a, b and from the literature: Shaitan, 1999, etc.) that cover the southern and northern boundaries of the home range of B. bombina. In recent years, species distribution modeling (SDM) has been widely used to estimate ecological requirement of particular species and to characterize and map the spatial distribution of habitat occupied by species at a landscape scale (Elith, Leathwick, 2009; Franklin, 2009; Peterson et al., 2011; Phillips, Elith, 2013). The principle of SDM is related to Hutchinson super-volume theory, and emphasizes the ecological requirement of species, especially the abiotic factors controlling species distribution (Guisan, Thuiller, 2005; Li et al., 2013). In general, many climatic factors were used as predicted variables when simulating species potential distribution at large scale with coarse resolution. According to the predicted map, we can depict the climatic niche and response curves of species. Currently, there are many algorithms in SDM technique, but Elith et al. (2006) have shown that maximum entropy model (MaxEnt) is one of the best method among an array of algorithms. The aim of this work was to simulate the potential distribution area of B. bombina and figure out the significant climatic factors of the species at a home range scale. Firstly, we collected the occurrence records. Secondly, we collected a set of climatic variables including 19 factors from 10’ resolution historical (summarizing annual trends, seasonality and extreme conditions during 1961–1990) and projected data (2050) available at the CliMond database (Kriticos et al., 2012) (table 1). These variables represent annual trends (e. g., mean annual temperature, annual precipitation) seasonality (e. g., annual range in temperature and precipitation) and extreme or limiting environmental factors (e.g., temperature of the coldest and warmest month, and precipitation of the wet and dry quarters). Finally, we used the occurrence records and cli- Long-Term Bioclimatic Modelling the Distribution of the Fire-Bellied Toad… 343 T a b l e 1 . Description of bioclimatic variables Variable acronym Bio01 Bio02 Bio03 Bio04 Bio05 Bio06 Bio07 Bio08 Bio09 Bio10 Bio11 Bio12 Bio13 Bio14 Bio15 Bio16 Bio17 Bio18 Bio19 Variable Annual mean temperature ,°C Mean diurnal temperature range (mean; period max–min), °C Isothermality, Bio02÷Bio07 Temperature seasonality, C of V Max temperature of warmest week, °C Min temperature of coldest week, °C Temperature annual range (Bio0–Bio06), °C Mean temperature of wettest quarter, °C Mean temperature of driest quarter, °C Mean temperature of warmest quarter, °C Mean temperature of coldest quarter, °C Annual precipitation, mm Precipitation of wettest week, mm Precipitation of driest week, mm Precipitation seasonality, C of V Precipitation of wettest quarter, mm Precipitation of driest quarter, mm Precipitation of warmest quarter, mm Precipitation of coldest quarter, mm matic factors as input data of MaxEnt model to simulate the potential distribution area of the species under contemporary and future climate conditions. This study is mainly concerned with the following objects: (1) identifying climatically suitable habitats for B. bombina at a home range scale; (2) estimating leading climatic requirements (niche) of B. bombina; (3) determining suitable areas for the (re)introduction and/ or conservation of the species; (4) forecast the distribution in a future climate, based on a climate change projection model for 2050. Geographical biases in the occurrence records were dampened by thinning the distribution points with OccurrenceThinner 1.03 (Verbruggen et al., 2013). Usually, researchers calculate correlation coefficients (e. g. Pearson coefficient) to avoid correlated variables and to reduce the effects of multi-colinearity in their models. However, from this type of analysis ecologically relevant variables could be excluded. Burnham and Anderson (1998) have made clear that applying correlation analysis in order to find a significant set of predictor variables will most probably expose false correlations. Fortunately, MaxEnt is able to incorporate complex dependencies between predictor variables, even in the presence of correlated variables, non-linearity, bimodality etc. Thus, all covariates were retained for the final model. The jackknife variable importance feature in MaxEnt was used to assess the relative importance of the environmental predictors in the model. We determined the relative importance of variables remaining in the model with percent contribution. MaxEnt allows the construction of response curves to illustrate the effect of selected variables on probability of occurrence. Identification of the most important predictors, and the analysis of the relations between the predictors and predicted habitat suitability, allows the description of the autecology of a species. We ran the MaxEnt models using the default setting, except for when selecting regularization values. This parameter was determined by the application of the small sample corrected variant of Akaike’s Information Criterion (AICc) implemented in ENMTools 1.3 (Warren, Seifert, 2011). Model performance was assessed using t Warren he average AUC (area under the receiver operating curve) score. AUC values >0.9 are considered to have “very good”, > 0.8 “good” and > 0.7 “useful” discrimination abilities (Swets, 1988). The logistic output format was used, because it is easily interpretable with logistic suitability values ranging from 0 (lowest suitability) to 1 (highest suitability). Better interpretation is made in most cases by defining thresholds of habitat suitability. With a reference to the classification proposed by Yang et al. (2013), five classes of potential habitats can be distinguished: unsuitable habitat (0–0.2); barely suitable habitat (0.2–0.4); suitable habitat (0.4–0.6); highly suitable habitat (0.6–0.7); very highly suitable habitat (0.7–1.0). For each model, we calculated the area of the optimal distribution, classified as highly or very highly suitable habitat (0.6–1). 344 V. Tytar, O. Nekrasova, A. Pupina, M. Pupins, O. Oskyrko Results In total, our database on B. bombina consisted 2,277 georeferenced point data. After reducing the geographical sampling bias in occurrence records their number was thinned to 598. Several regularization values were tested: 1, 1.5, 2, 2.5 and 3. The better regularization value (lower value of AICc) was 2. We ran models with 10 bootstrap replicates. The resulting potential distribution of B. bombina within its native range under contemporary climate (fig. 1) had high AUC values (0.836 ± 0.002). In terms of the bioclimatic niche, the most suitable for the species areas are located in Poland and Ukraine (fig. 2). In Ukraine areas of high or very high suitable habitat potential (> 0.6) occupy around 46 % of the country, in Poland this figure is even higher — 65 %. Together these countries accommodate 87 % of the potential for highly or very highly suitable habitat for B. bombina. At the northern border of the distribution of the toad areas of moderate suitability are found in southern Latvia. Areas of moderate suitability are found as well in Germany, Denmark and Sweden, but in these countries there are sporadic patches of even highly suitable habitat, which should favour the (re)introduction of the species. B. bombina has been successfully reintroduced to Sweden (Andren, Nilson, 1988), but the modelling suggests that the species has the chances to spread to a wider portion of the country. The MaxEnt model’s internal jackknife test of variable importance showed that the environmental variable with highest gain when used in isolation is Bio2 (Mean diurnal temperature range), which therefore appears to have the most useful information by itself. The environmental variable that decreases the gain the most when it is omitted is Bio3 (Isothermality), which therefore appears to have the most information that isn’t present in the other variables. The percent contribution of Bio2 (29.4 %) too supports the significance of this variable for contributing to the MaxEnt model. Another significant (> 10 %) contribution to the model is made by Bio8 (Mean temperature of wettest quarter, i. e., predominantly summer, 14.9 %). Further on, response curves gave an indication of the dependence of the predicted probability of presence (i. e., predicted suitability) on each of these variables as well as the range under which they reach an optimum of suitability. These probabilities are calculated for the range values of one variable, with all other 19 variables set to their average value over the set of presence localities. Upward trends for variables indicate a positive relationship; downward movements represent a negative relationship; and the magnitude of these movements indicates the Fig. 1. The potential distribution map for B. bombina under contemporary climatic conditions. The colour gradient represents high (red) to low (green) habitat suitability for the species. Long-Term Bioclimatic Modelling the Distribution of the Fire-Bellied Toad… 345 A B Fig. 2. The predicted areas of high or very high suitable habitat potential (> 0.6) for B. bombina (A — for contemporary climate conditions; B — for projected climate in 2050). Fig 3. Response curve showing how the logistic prediction changes as the environmental variable Bio2 (Mean diurnal temperature range, oC, X-axis) is varied, keeping all other environmental variables at their average sample value. The curve shows the mean response of the 10 replicate Maxent runs (red) and and the mean +/– one standard deviation (blue). strength of the relationship (Baldwin, 2009), essentially they demonstrate biological tolerances. The corresponding response curve for Bio2 shows a clear downward trend (fig. 3), representing a negative relationship between increasing values of the Mean diurnal temperature range and predicted probability of presence (i. e., habitat suitability) for B. bombina in the locality. Under future climate change warmer temperatures are expected to lead to the convergence of daytime and nighttime temperatures, meaning a reduction of the diurnal temperature range (Rohr, Raffe, 2010). These effects of temporal variation in temperature will be more pronounced in temperate regions and theoretically may turn out to enhance habitat suitability for B. bombina. Compared with the area of the most optimal habitat under current climate prediction, the predictions for 2050 show a clear north and north-west shift of such habitat. It is questionable, of course, can the toads track their climate space. This may greatly depend on their migratory abilities and how fragmented is the landscape. Once again, in terms of 346 V. Tytar, O. Nekrasova, A. Pupina, M. Pupins, O. Oskyrko Fig. 4. The potential distribution map for B. bombina under projected 2050 climatic conditions. The colour gradient represents high (red) to low (green) habitat suitability for the species. the bioclimatic niche, large suitable (> 0.6) for the species areas will continue to persist in Poland. Conditions are predicted to considerably improve for the species in Lithuania, neighbouring areas of Belarus, as well as in Latvia, where patches of highly suitable habitat conditions will appear. Moderate habitat suitability may promote the species to expand as far as Estonia and up to the latitude of Saint Petersburg in Russia (fig. 4). Optimal habitat is predicted to expand in Sweden, but is likely to shift to the north-east from its current location. If so, this may have an impact on measures for reintroducing the species. In Ukraine, however, areas of potential high or very high habitat suitability are predicted to drastically drop from 46 % of the country to 18 %, meaning there could be a net extinction of B. bombina at the southern boundary of the home range of the species. In the context of these shifts the species’ representation in protected areas can seriously be affected, particularly in the south and east of the country. Preemptive measures should be undertaken to preserve the toad and its associated habitats in the west and north-west of Ukraine, where favourable conditions are predicted to persist. Conclusions Species distribution modelling has confirmed that vast areas in Poland and Ukraine, in terms of the bioclimatic niche, are highly suitable for the Fire-bellied toad, B. bombina. Further to the north areas of moderate suitability are found in a number of countries ranging from Denmark to Latvia. The modelling suggests that prospects for the species in Sweden could be better than thought. Modelling has shown that the Mean diurnal temperature range is an important dimension of the bioclimatic niche of the toad and the expected convergence of daytime and nighttime temperatures under global climate change may favour the species. Under climate predictions for 2050 there may be a substantial north and north-west shift of optimal habitat. Under such a scenario B. bombina populations may suffer mostly in the east and south of Ukraine. Optimal habitat is predicted to expand in Sweden, but is likely to shift from its current location. This may affect ongoing efforts to reintroduce the species to the country. Under the modelled scenario the species representation in protected areas throughout the home range should be considered, but especially in Ukraine. Long-Term Bioclimatic Modelling the Distribution of the Fire-Bellied Toad… 347 This research was supported in part by the National Research Program EVIDEnT “The value and dynamic of Latvia’s ecosystems under changing climate”, subproject number 4.6. We mourn the loss of our colleague and co-author Aija Pupina and are grateful for her high-valued cooperation in the research. References Andren, C., Nilson, G. 1988. Reintroduction of the fire-bellied toad Bombina bombina in Scania, south Sweden. Memoranda Societatis pro Fauna et Flora Fennica, 64 (3), 100. 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https://openalex.org/W2326733993	https://europepmc.org/articles/pmc5042014?pdf=render	English	null	Tumor suppressor PRSS8 targets Sphk1/S1P/Stat3/Akt signaling in colorectal cancer	Oncotarget	2,016	cc-by	6,918	ABSTRACT PRSS8 is a membrane-anchored serine protease prostasin and has been shown an association with carcinogenesis. Herein we found that PRSS8 expression was significantly reduced in colorectal adenomas and adenocarcinomas. The decreased PRSS8 was well correlated with clinical stages, poor differentiation and shorter survival time of colorectal cancer. Furthermore, increase of PRSS8 led to the inhibition of colorectal cancer cell proliferation, knockdown of PRSS8 accelerated cell proliferation in vitro, and overexpressing PRSS8 retarded cancer cell growth in nude mice. Mechanistic studies revealed that PRSS8 inhibited Sphk1/S1P/Stat3/ Akt signaling pathway, in terms of inverse association between PRSS8 and Sphk1 in human colorectal cancers and in Sphk1-/- mice. In conclusion, PRSS8 acts as a tumor suppressor by inhibiting Sphk1/S1P/Stat3/Akt signaling pathway, and could be used as a biomarker to monitor colorectal carcinogenesis and predict outcomes. Tumor suppressor PRSS8 targets Sphk1/S1P/Stat3/Akt signaling in colorectal cancer Yonghua Bao1,, Kai Li2,, Yongchen Guo1,, Qian Wang3, Zexin Li4, Yiqiong Yang1, Zhiguo Chen5, Jianguo Wang4, Weixing Zhao2,5, Huijuan Zhang5, Jiwang Chen6, Huali Dong7, Kui Shen7, Alan M. Diamond7, Wancai Yang1,7 1Department of Pathology and Institute of Precision Medicine, Jining Medical University, Jining 272067, China 2Department of Pathology, the First Affiliated Hospital, Xinxiang Medical University, Xinxiang 453003, China 3Department of Immunology, Xinxiang Medical University, Xinxiang 453003, China 4Department of Surgical Oncology, the First Affiliated Hospital, Xinxiang Medical University, Weihui 453003, China 5Department of Pathology, Xinxiang Medical University, Xinxiang 453003, China 6Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA Yonghua Bao1,, Kai Li2,, Yongchen Guo1,, Qian Wang3, Zexin Li4, Yiqiong Yang1, Zhiguo Chen5, Jianguo Wang4, Weixing Zhao2,5, Huijuan Zhang5, Jiwang Chen6, Huali Dong7, Kui Shen7, Alan M. Diamond7, Wancai Yang1,7 1Department of Pathology and Institute of Precision Medicine, Jining Medical University, Jining 272067, China 2Department of Pathology, the First Affiliated Hospital, Xinxiang Medical University, Xinxiang 453003, China 3Department of Immunology, Xinxiang Medical University, Xinxiang 453003, China 4Department of Surgical Oncology, the First Affiliated Hospital, Xinxiang Medical University, Weihui 453003, China 5Department of Pathology, Xinxiang Medical University, Xinxiang 453003, China 6Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA 7Department of Pathology, University of Illinois at Chicago, Chicago, IL 60612, USA These authors have contributed equally to this work Correspondence to: Wancai Yang, email: wyang06@uic.edu Keywords: PRSS8, colorectal cancer, tumor suppressor, Sphk1, Stat3 Keywords: PRSS8, colorectal cancer, tumor suppressor, Sphk1, Stat3 Abbreviations: PRSS8, protease serine 8; Stat3, signal transducer and activator of transcription 3; Sphk1, sphingosine kinase 1; S1PR, Sphingosine-1- Phosphate receptor Abbreviations: PRSS8, protease serine 8; Stat3, signal transducer and activator of transcription 3; Sphk1, sphingosine kinase 1; S1PR, Sphingosine-1- Phosphate receptor Abbreviations: PRSS8, protease serine 8; Stat3, signal transducer and activator of transcription 3; Sphk1, sphingosine kinase 1; S1PR, Sphingosine-1- Phosphate receptor Received: December 23, 2015 Accepted: March 06, 2016 Published: March 31, 2016 Accepted: March 06, 2016 Published: March 31, 2016 Oncotarget, Vol. 7, No. 18 Oncotarget, Vol. 7, No. 18 www.impactjournals.com/oncotarget/ INTRODUCTION was significantly reduced in colorectal cancer, and the reduction of PRSS8 expression was associated with poor differentiation and poor outcome, and lead to alterations in Sphk1/S1P/Stat3 signaling, the later has been reported to be associated with colitis-associated colorectal cancer [7, 8]. was significantly reduced in colorectal cancer, and the reduction of PRSS8 expression was associated with poor differentiation and poor outcome, and lead to alterations in Sphk1/S1P/Stat3 signaling, the later has been reported to be associated with colitis-associated colorectal cancer [7, 8]. Colorectal cancer (CRC) is the third most common malignant disease and the second leading cause of cancer- related death in the United States [1], which is majorly due to cancer metastasis. Several decades effort have revealed that the development and progression of colorectal cancer is linked to the inactivation or downregulation of tumor suppressors, and activation or upregulation of oncogenes, leading to oncogenic signaling activation such as Wnt/β- catenin [2–4], inflammatory signaling [5, 6], etc. Using gene expression analysis we have found that PRSS8 PRSS8 (protease serine 8), also known as Prostasin, is a trypsin-like serine peptidase [9–11]. PRSS8 was preliminarily found highly expressed in normal prostate gland and seminal fluid, and other studies have reported that PRSS8 is overexpressed in epithelial cells of various tissues and is involved in terminal epithelial PRSS8 mRNA levels were reduced in colorectal adenoma and adenocarcinoma To determine how PRSS8 expression differed between normal and tumor colorectal tissues, we used qRT-PCR to measure PRSS8 mRNA levels in 38 pairs of colorectal cancer tissues and their adjacent non-cancer tissues. As shown in Figure 1A, PRSS8 mRNA levels were reduced about 65% in adenocarcinoma tissues compared with non-cancer tissues (p<0.01). To determine whether loss of PRSS8 occurred in the early stage of tumor progression, we used an online gene expression data set to compare 32 paired patient adenoma tissues and their adjacent normal colonic mucosa (GEO# GDS2947/202525/PRSS8). Similar to adenocarcinoma, PRSS8 mRNA was reduced about 50% in adenoma compared to normal mucosa (p<0.0001, Figure 1B). The reduction of PRSS8 in colorectal cancer was also supported by mining The Cancer Genome Atlas (TCGA) online data (http://cancergenome.nih.gov/) [21], showing that PRSS8 was significantly reduced in 215 cases of colorectal cancers in comparison to the normal colorectal mucosa (Figure 1C, p<0.0001). These finding indicated that PRSS8 mRNA levels were significantly reduced during colorectal cancer progression. The present study employed tissue microarrays of colorectal, esophageal and liver cancers as well as prostate and breast cancers, to investigate the expression of PRSS8 and its clinical significance in these cancers. Gain- and loss-of-expression studies were used to determine the biological functions of PRSS8 and underlying molecular mechanism in culture cells and in nude mice. The results generated from these experiments strongly suggested that (Continued ) Figure 1: PRSS8 expression levels were reduced in tumors and the reduction of PRSS8 was associated with poor differentiation and survival time. A. mRNA of PRSS8 (mean+/-SD) was reduced about 65% in colorectal cancer tissues with comparison to their paired non-cancer tissues. Total RNA was extracted from 38 paired frozen colorectal cancer/non-cancer tissues, and PRSS8 mRNA levels were analyzed and normalized to the corresponding levels of GAPDH, assayed by qRT-PCR. B. PRSS8 mRNA levels (mean+/-SD) were reduced about 50% in colorectal adenoma tissues with comparison to their paired non-tumor tissues. PRSS8 mRNA expression levels of colorectal adenoma were extracted from the online database (GEO Profile # GDS2947) (p<0.01, p<0.001). C. The reduction of PRSS8 in colorectal cancer was also seen in The Cancer Genome Atlas (TCGA) online data (http://cancergenome.nih. gov/), in 215 cases of colorectal cancers, compared to 22 normal colorectal mucosa (p<0.0001). Figure 1: PRSS8 expression levels were reduced in tumors and the reduction of PRSS8 was associated with poor differentiation and survival time. A. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 26780 PRSS8 is a tumor suppressor in colorectal cancer, which is through inhibiting Sphk1/S1P/Stat3 signaling pathways. differentiation. In addition, PRSS8 has the physiological and pathophysiological functions of regulating of sodium and fluid levels via proteolysis of the epithelial sodium channel, and shows important roles in the epidermal barrier function, skin phenotypes, and embryonic viability [10, 11]. More importantly, both oncologic and tumor suppressing roles of PRSS8 have been demonstrated in ovarian cancers [12, 13], for instance, one study showed that PRSS8 levels were increased in the serum of ovarian cancer patients [14], and another study showed that PRSS8 expression was decreased in chemoresistant ovarian cancer patients and chemoresistant cell line, and forced overexpression of prostasin in ovarian cancer cells greatly induced cells death [15]. Moreover, recent studies have also demonstrated tumor suppressive roles of PRSS8 on prostate, breast, bladder and gastric cancers, in which PRSS8 expression was reduced in prostate [16, 17], breast [18], bladder [19] and gastric cancers [20], due to the hypermethylation of PRSS8. While it has been studied extensively in other cancers, the effect of PRSS8 on colorectal cancer is not clear. PRSS8 mRNA levels were reduced in colorectal adenoma and adenocarcinoma mRNA of PRSS8 (mean+/-SD) was reduced about 65% in colorectal cancer tissues with comparison to their paired non-cancer tissues. Total RNA was extracted from 38 paired frozen colorectal cancer/non-cancer tissues, and PRSS8 mRNA levels were analyzed and normalized to the corresponding levels of GAPDH, assayed by qRT-PCR. B. PRSS8 mRNA levels (mean+/-SD) were reduced about 50% in colorectal adenoma tissues with comparison to their paired non-tumor tissues. PRSS8 mRNA expression levels of colorectal adenoma were extracted from the online database (GEO Profile # GDS2947) (p<0.01, **p<0.001). C. The reduction of PRSS8 in colorectal cancer was also seen in The Cancer Genome Atlas (TCGA) online data (http://cancergenome.nih. gov/), in 215 cases of colorectal cancers, compared to 22 normal colorectal mucosa (p<0.0001). (Continued ) www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 26781 Oncotarge 26782 impactjournals.com/oncotarget ure 1 (Continued ): D. PRSS8 was reduced in colorectal cancers and the cancers of esophagus, liver, breast and prostate, compare heir normal (esophagus, colon and liver) or benign lesions (breast and prostate) assayed by immunohistochemical staining using ant SS8 antibody. The reduced expression of PRSS8 was associated with poor differentiation. E. PRSS8 protein levels were associated wi survival time and disease-free period of the patients of colorectal cancers. The survival curve was generated using GraphPad Prism 5.0 Figure 1 (Continued ): D. PRSS8 was reduced in colorectal cancers and the cancers of esophagus, liver, breast and prostate, compared to their normal (esophagus, colon and liver) or benign lesions (breast and prostate) assayed by immunohistochemical staining using anti- PRSS8 antibody. The reduced expression of PRSS8 was associated with poor differentiation. E. PRSS8 protein levels were associated with the survival time and disease-free period of the patients of colorectal cancers. The survival curve was generated using GraphPad Prism 5.0,. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 26782 Table 1: The association between PRSS8 expression and the clinicopathological characteristics Clinicopathologic Variables N PRSS8 expression score P value 3 2 1 Age Patient number (N=282) 119 93 70 Median age (years) 59.0 56.5 62.0 0.822 Range 27-82 36-86 33-85 Gender 0.088 Male 143 69 (48%) 40 (28%) 34 (24%) Female 139 50 (36%) 53 (38%) 36 (26%) Lymphatic metastasis 0.157 Yes 111 41 (37%) 36 (32%) 34 (31%) No 171 78 (46%) 57 (33%) 36 (21%) Clinical stages 0.0046 1-2 149 70 (47%) 50 (34%) 29 (19%) 3-4 133 42 (32%) 44 (33%) 47 (35%) Differentiation <0.00001 High 135 74 (55%) 37 (27%) 24 (18%) Moderate 112 40 (36%) 45 (40%) 27 (24%) Low 35 5 (14%) 12 (34%) 18 (51%) N, number of patients with colorectal cancers. Table 1: The association between PRSS8 expression and the clinicopathological characteristics N, number of patients with colorectal cancers. N, number of patients with colorectal cancers. PRSS8 protein levels were reduced in colorectal cancers and the cancers of esophagus, liver, breast and prostate adenoma or non-cancer tissues, respectively. Similar to colorectal cancer, PRSS8 expression levels were reduced in esophageal cancers and liver cancers compared to normal adjacent tissues (Figure 1D). Consistent with previous studies, PRSS8 expression was reduced in breast and prostate cancers, compared to breast and prostate adenomas (Figure 1D). Moreover, PRSS8 expression was positively associated with differentiation of esophageal, liver, breast and prostate cancers (Figure 1D) To determine whether PRSS8 protein levels in colorectal cancers were also reduced, we conducted immunohistochemical staining in a colorectal cancer tissue microarray (TMA) that contained 282 cases of colorectal cancer with follow-up information. Compared to the adjacent non-cancer tissues, colorectal cancers showed significant reduction of PRSS8 expression (Figure 1D). Interestingly, the reduction of PRSS8 was positively associated with cancer differentiation. For instance, PRSS8 protein level was highly expressed in well differentiated colorectal adenocarcinomas and was lower or absent in poorly differentiated colorectal adenocarcinomas (Figure 1D, Table 1, p<0.00001). PRSS8 expression was associated with survival of colorectal cancer patients Since reduced expression of PRSS8 was seen in colorectal adenoma and adenocarcinoma (Figure 1D), we then determined whether PRSS8 expression was linked to clinicopatholgical characteristics and outcomes. As shown in Figure 1E, reduced PRSS8 was significantly associated with shorter survival time and disease-free period of patients with colorectal cancer (Figure 1E, p<0.0001). In addition, there was an inverse correlation between PRSS8 expression levels and colorectal cancer stages (Table 1, p=0.0046). Taken together, above findings strongly demonstrated the important clinical significance of PRSS8 in cancer formation, progression and clinical outcomes. Previous studies have demonstrated that PRSS8 was differentially expressed in ovarian cancer [15], prostate cancer [16, 17] and breast cancer [18]. To determine the levels of PRSS8 in other cancers, we used 4 sets of TMAs that contained 72 pairs of esophageal cancers and their adjacent non-cancer tissues, 117 pairs of liver cancers and their adjacent non-cancer tissues, 46 pairs of prostate cancers and their adjacent adenoma or non-cancer tissues, and 75 pairs of breast cancers and their adjacent www.impactjournals.com/oncotarget Oncotarget 26783 PRSS8 affected colorectal cancer cell proliferation, cell cycle biological functions of PRSS8 in colorectal cancer in vitro and in nude mice. Four human colorectal cancer cell lines were screened for PRSS8 mRNA and protein levels by qRT-PCR and immunoblotting, respectively. PRSS8 mRNA levels (Figure 2A) and protein levels (Figure 2B) were lower in SW480 and HCT116 cells and higher in HCT8 biological functions of PRSS8 in colorectal cancer in vitro and in nude mice. Four human colorectal cancer cell lines were screened for PRSS8 mRNA and protein levels by qRT-PCR and immunoblotting, respectively. PRSS8 mRNA levels (Figure 2A) and protein levels (Figure 2B) were lower in SW480 and HCT116 cells and higher in HCT8 Due to the strong correlation between PRSS8 expression levels and clinical significance, we then used gain- and loss-of-expression approaches to determine the tj l / t t nd loss-of-expression approaches to determine the lower in SW480 and HCT116 cells and higher in (Con 2: Differential expression and biological functions of PRSS8 in human colorectal cancer cell lines. A evels were analyzed by qRT-PCR and normalized to GAPDH. B. PRSS8 proteins were evaluated by immunoblotting, an d as internal control. C and D. overexpressing PRSS8 inhibited cancer cell proliferation in SW480 (C) and HCT116 cel nockdown PRSS8 by small interfering RNA accelerated cell proliferation in Caco2 (E) and HCT8 cells (F). (Continued ) Figure 2: Differential expression and biological functions of PRSS8 in human colorectal cancer cell lines. A. PRSS8 mRNA levels were analyzed by qRT-PCR and normalized to GAPDH. B. PRSS8 proteins were evaluated by immunoblotting, and β-actin was used as internal control. C and D. overexpressing PRSS8 inhibited cancer cell proliferation in SW480 (C) and HCT116 cells (D). E and F. knockdown PRSS8 by small interfering RNA accelerated cell proliferation in Caco2 (E) and HCT8 cells (F). www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 26784 Figure 2 (Continued ): G. increased expression of PRSS8 decreased the expression of Sphk1, S1P receptor (S1PR), Sgpp1, phosphorylated Stat3 (p-Stat3), and phosphorylated Akt (p-Akt) in both HCT116 and SW480 cell lines. H. knockdown of PRSS8 expression led to increase of Sgpp1, p-Stat3 and p-Akt in HCT8 and Caco2 cell lines, assayed by immunoblotting. Figure 2 (Continued ): G. increased expression of PRSS8 decreased the expression of Sphk1, S1P receptor (S1PR), Sgpp1, phosphorylated Stat3 (p-Stat3), and phosphorylated Akt (p-Akt) in both HCT116 and SW480 cell lines. H. knockdown of PRSS8 expression led to increase of Sgpp1, p-Stat3 and p-Akt in HCT8 and Caco2 cell lines, assayed by immunoblotting. cancer formation and progression besides the activation of Wnt-β-catenin signaling. A recent study has reported that persistent activation of Stat3 by the Sphk1/S1P signaling pathway is observed in chronic intestinal inflammation and colitis-associated colorectal cancer [8]. Thus, we determined whether PRSS8 was involved in the Sphk1/ S1p/Stat3 signaling. Indeed, increased expression of PRSS8 remarkably decreased the expression of Sphk1, S1P receptor (S1PR), Sgpp1, phosphorylated Stat3 (p-Stat3), and phosphorylated Akt (p-Akt) in both HCT116 and SW480 cell lines (Figure 2G), although total of Stat3 and total Akt were not changed. In contrast, knockdown of PRSS8 upregulated the expression of Sgpp1, p-Stat3 and p-Akt in HCT8 and Caco2 cell lines (Figure 2H). and Caco2 cells. SW480 and HCT116 cells, that have lower expression of PRSS8, were chosen for transfection with PRSS8 expression plasmid (pFlag-PRSS8). MTT assay showed that increasing PRSS8 expression inhibited cancer cell proliferation at 24 and 48 hours in both SW480 and HCT116 cell lines (Figure 2C, 2D, p<0.05), compared to the cells transfected with empty vector. We then determined whether PRSS8 expression was correlated with the cell cycle. Flow cytometry analysis showed that increased expression of PRSS8 led to cell cycle arrest at G1/G2 phase at both SW480 and HCT116 cells (Table 2, p<0.05, compared to the cells transfected with empty vector). To determine the effects of knockdown of PRSS8 expression in colorectal cancer cells, we decreased expression of PRSS8 of the high-expression colorectal cancer cell lines Caco2 and HCT8 using small interfering RNA (siRNA) targeting human PRSS8 (siR-PRSS8). MTT results showed that knockdown of PRSS8 significantly promoted cell proliferation (Figure 2E and 2F). The inverse correlation between PRSS8 and Sphk1 Interestingly, knockdown of PRSS8 could cause Sphk1 upregulation in HCT8 cells (Figure 3A). Moreover, increasing expression of Sphk1 in HCT8 cells led to the downregulation of PRSS8 (Figure 3B), and vice versa, knockdown of Sphk1 by small interfering RNA targeting Sphk1 in HCT116 and SW480 cells led to the upregulation of PRSS8 (Figure 3C). PRSS8 suppressed Sphk1/S1P/Stat3/Akt signaling PRSS8 expression was increased in Sphk1-/- mouse colon (lower panel), compared to the Sphk1+/+ mice (upper panel). E. Online gene profile data set (www.oncomine.com) was deeply mined and neutralized, the results showed that PRSS8 was higher and Sphk1 was lower in normal colorectal mucosa, but RPSS8 was reduced (p=1.8x10-5) and Sphk1 was increased in colorectal cancers (p=9.1x10-28, Figure 3E), exhibiting a strong negative correlation. Figure 3: Negative interaction between PRSS8 and Sphk1/S1P/Stat3/Akt signaling in colorectal cancer cells, in Sphk1 mouse models and in human colorectal cancers. A. knockdown of PRSS8 led to the upregulation of Sphk1 in HCT8 cells. B. overexpression of Sphk1 in HCT8 cells suppressed PRSS8 expression. C. knockdown of Sphk1 expression caused upregulation of PRSS8 in HCT116 and SW480 cells. D. PRSS8 expression was increased in Sphk1-/- mouse colon (lower panel), compared to the Sphk1+/+ mice (upper panel). E. Online gene profile data set (www.oncomine.com) was deeply mined and neutralized, the results showed that PRSS8 was higher and Sphk1 was lower in normal colorectal mucosa, but RPSS8 was reduced (p=1.8x10-5) and Sphk1 was increased in colorectal cancers (p=9.1x10-28, Figure 3E), exhibiting a strong negative correlation. (Continued ) www.impactjournals.com/oncotarget PRSS8 suppressed Sphk1/S1P/Stat3/Akt signaling It has been known that malignant transformation of chronic colitis is another major cause of colorectal www.impactjournals.com/oncotarget Oncotarget 26785 Table 2: Overexpression of PRSS8 caused cell cycle arrest at G1/G2 phases in human colorectal cancer cells SW480 HCT116 Vector PRSS8 Vector PRSS8 G1 (%) 36.2 53.5 34.9 53.7* G2 (%) 25.7 31.4* 7.4 30.9* S (%) 38.1 15.1 57.7 15.4 Total (%) 100.0 100.0 100.0 100.0 Note: Human colorectal cancer cells SW480 and HCT116 were transfected with PRSS8 expression plasmids. 48 hours after transfection, the cells were collected for cell cycle analysis. The results showed that increased expression of PRSS8 caused cell cycle arrest in G1/G2 phases, compared to the cells with transfection of empty vector, assayed by flow cytometry. (p<0.05). PRSS8, the cells with transfection of PRSS8 expression plasmid pFlag-PRSS8; Vector, the cells with transfection of empty vector. Note: Human colorectal cancer cells SW480 and HCT116 were transfected with PRSS8 expression plasmids. 48 hours after transfection, the cells were collected for cell cycle analysis. The results showed that increased expression of PRSS8 caused cell cycle arrest in G1/G2 phases, compared to the cells with transfection of empty vector, assayed by flow cytometry. (p<0.05). PRSS8, the cells with transfection of PRSS8 expression plasmid pFlag-PRSS8; Vector, the cells with transfection of empty vector. (Continued ) gure 3: Negative interaction between PRSS8 and Sphk1/S1P/Stat3/Akt signaling in colorectal cancer cells, in Sphk1 ouse models and in human colorectal cancers. A. knockdown of PRSS8 led to the upregulation of Sphk1 in HCT8 cells. B. erexpression of Sphk1 in HCT8 cells suppressed PRSS8 expression. C. knockdown of Sphk1 expression caused upregulation of PRSS8 HCT116 and SW480 cells. D. PRSS8 expression was increased in Sphk1-/- mouse colon (lower panel), compared to the Sphk1+/+ mice per panel). E. Online gene profile data set (www.oncomine.com) was deeply mined and neutralized, the results showed that PRSS8 was her and Sphk1 was lower in normal colorectal mucosa, but RPSS8 was reduced (p=1.8x10-5) and Sphk1 was increased in colorectal ncers (p=9.1x10-28, Figure 3E), exhibiting a strong negative correlation. Figure 3: Negative interaction between PRSS8 and Sphk1/S1P/Stat3/Akt signaling in colorectal cancer cells, in Sphk1 mouse models and in human colorectal cancers. A. knockdown of PRSS8 led to the upregulation of Sphk1 in HCT8 cells. B. overexpression of Sphk1 in HCT8 cells suppressed PRSS8 expression. C. knockdown of Sphk1 expression caused upregulation of PRSS8 in HCT116 and SW480 cells. D. www.impactjournals.com/oncotarget Oncotarget 26786 Figure 3 (Continued ): F. the interaction between PRSS8 and Sphk1 was examined by co-immunoprecipitation assay. PRSS8/Sphk1 complex was precipitated using anti-Flag or anti-Sphk1 antibodies, probed with anti-Sphk1 or anti-Flag antibodies, respectively. G. The interaction between PRSS8 and Sphk1 was also confirmed by immuno-fluorescence staining assay, showing that PRSS8 protein (p-EGFP-PRSS8, Green-fluorescence staining) was co-localized with Sphk1 protein (pEx-6-Sphk1, Red-fluorescence staining) in cytoplasm and nuclei. Figure 3 (Continued ): F. the interaction between PRSS8 and Sphk1 was examined by co-immunoprecipitation assay. PRSS8/Sphk1 complex was precipitated using anti-Flag or anti-Sphk1 antibodies, probed with anti-Sphk1 or anti-Flag antibodies, respectively. G. The interaction between PRSS8 and Sphk1 was also confirmed by immuno-fluorescence staining assay, showing that PRSS8 protein (p-EGFP-PRSS8, Green-fluorescence staining) was co-localized with Sphk1 protein (pEx-6-Sphk1, Red-fluorescence staining) in cytoplasm and nuclei. (Figure 3F, upper 2 bands). Notably, the Figure 3F lower 2 bands showed again an inverse correlation between Sphk1 and PRSS8, in which Sphk1 suppressed PRSS8 expression (in the whole lysate) and PRSS8 suppressed Sphk1 expression (in the whole lysate). The interaction between PRSS8 and Sphk1 was further confirmed by immuno- fluorescence staining assay (Figure 3G), which showed that PRSS8 protein (p-EGFP-PRSS8, Green-fluorescence staining) was co-localized with Sphk1 protein (pEx-6- Sphk1, Red-fluorescence staining) in the cytoplasm and nucleus. The negative regulation between PRSS8 and Sphk1 was also observed in Sphk1 mouse model. As shown in Figure 3D, PRSS8 expression was increased in the colon of Sphk1-/- mice, compared to Sphk1+/+ mice, assayed by immunohistochemical staining. The negative association between PRSS8 and Sphk1 was evidenced by a published gene profile data set (www. oncomine.com)[22]. The data was deeply mined and results showed that in the 65 pairs of colorectal normal and cancers, PRSS8 was higher and Sphk1 was lower in normal colorectal mucosa, but RPSS8 was significantly reduced (p=1.8x10-5) and Sphk1 was dramatically increased in colorectal cancers (p=9.1x10-28, Figure 3E), exhibiting a strong negative correlation. PRSS8 inhibited tumor growth in nude mice To determine tumor suppressive roles of PRSS8 in vivo, we established a stable PRSS8 overexpression HCT116 cell line, and injected these cells subcutaneously in nude mice. As shown in Figure 4A, overexpressing PRSS8 significantly inhibited tumor growth in mice, resulting in significant retardation of tumor size and tumor weight. To further confirm whether tumor growth inhibition was directly resulted from PRSS8, PRSS8 protein levels were assayed by immunohistochemical staining. Results showed that PRSS8 was overexpressed in HCT116 tumor cells which were transfected with PRSS8 plasmid, and that PRSS8 was almost undetectable The aforementioned gain- and loss-of-expression studies of PRSS8 and Sphk1 showed a negative regulation of PRSS8 and Sphk1/ S1P/Stat3 signaling in vitro and in vivo, leading us to determine whether there is a direct interaction between PRSS8 and Sphk1. Using co- immunoprecipitation (co-IP) assay, we co-transfected the HEK 293 with PRSS8 expression plasmid (pFlag-PRSS8) and Sphk1 expression plasmid (pEx-6-Sphk1), and then precipitated the PRSS8/Sphk1 complex with anti-Flag or anti-Sphk1 antibodies, and immunoblotted with anti- Sphk1 or anti-Flag antibodies, respectively. We found that there was a strong band between PRSS8 and Sphk1 www.impactjournals.com/oncotarget Oncotarget 26787 in the tumor cells that were transfected with empty vector (Figure 4B). or oncogenic roles in colorectal cancer progression and metastasis through suppressing of inflammatory signaling pathways. In the present study, we identified that PRSS8 expression was reduced in colorectal cancer and this reduced expression was associated with poor differentiation and late stages of tumors, and with poor outcome of colorectal cancer patients. Overexpressing PRSS8 inhibited cancer cell proliferation, led to cell cycle arrest at G1/G2 phase, and retarded cancer cell growth in nude mice. Mechanistic studies showed that PRSS8 inhibited Sphk1/S1P/Stat3 signaling. Consistent with the effects of PRSS8 on oncogenic signaling pathways in vitro, in the HCT116 cell xenografts PRSS8 also suppressed Sphk1/S1P/Stat3/Akt signaling (Figure 4C). DISCUSSION Recent studies have made significant advances in understanding colorectal carcinogenesis, demonstrating the critical roles of non-canonical Wnt signaling in colorectal cancer formation and progression, and identifying the link between chronic colitis and colorectal cancer, particularly in deciphering the roles of cytokines and their downstream transcription factors NF-kB and STAT3 [3, 5, 7, 23, 24]. These studies have also demonstrated that a chronic inflammatory microenvironment accelerates Apc mutation-initiated intestinal tumor formation in the Apc/ Muc2 double mutant mouse model of intestinal cancer [25], showing a synergistic role of Apc/Wnt/β-catenin and cytokine/Cox2 inflammatory signaling in colorectal carcinogenesis. However, it is unknown whether there are any upstream regulators that exhibit tumor suppressing Reduced expression of tumor suppressor genes by inactivation or silencing is frequently observed in late stage of colorectal cancer [26, 27], and it may be a result of tumor progression, not a driver. In this study, using five sets of variant TMAs, including colorectal, esophageal, liver, prostate, and breast cancers, we have found that PRSS8 expression was reduced in all of the five types of cancer tissues, and poorly differentiated cancers exhibited less or absent expression of PRSS8. Notably, the expression of PRSS8 was reduced in colorectal adenoma, a pre-cancer lesion. Most importantly, the reduced expression of PRSS8 was associated with shorter survival time in patients with colorectal cancers. In vivo studies Figure 4: PRSS8 inhibited colon cancer cell growth in nude mice. A. 1.5x106 stable cell lines with transfection of empty vector (Vector) or PRSS8 expression plasmid (PRSS8) were injected subcutaneously in nude mice, and the mice were sacrificed 3 weeks post injection. PRSS8 inhibited tumors (xenografts) weight and size. B. expression levels of PRSS8 were validated in the tumors by immunohistochemical staining using anti-PRSS8 antibody. C. PRSS8 overexpression resulted in the downregulation of Sphk1/S1P/Stat3/ Akt in nude mouse tumors. Figure 4: PRSS8 inhibited colon cancer cell growth in nude mice. A. 1.5x106 stable cell lines with transfection of empty vector (Vector) or PRSS8 expression plasmid (PRSS8) were injected subcutaneously in nude mice, and the mice were sacrificed 3 weeks post injection. PRSS8 inhibited tumors (xenografts) weight and size. B. expression levels of PRSS8 were validated in the tumors by immunohistochemical staining using anti-PRSS8 antibody. C. PRSS8 overexpression resulted in the downregulation of Sphk1/S1P/Stat3/ Akt in nude mouse tumors. Immunohistochemical staining, staining intensity evaluation and survival analysis Paraffin sections were treated with 3% H2O2, and then incubated with primary antibodies and biotinylated secondary antibodies. The immune complexes were visualized using the Strept Avidin-Biotin Complex kit (Boster Biological Tech. LTD., Wuhan, China). All stained slides were imaged using Aperio ImageScope software (version 11) for immunostaining intensity evaluation. The immunohistochemical staining was scored by three independent researchers, who were blinded to the clinical or pathological information of these patients. A semi- quantitative scale from 0 to 100% was used to grade the percentage of PRSS8-stained epithelial cells relative to all epithelial cells in each tissue core on the TMA slides. For convenient statistic analysis, the percentage scores were transferred as following scored: 0 (<5%), 1 (5–25%), 2 (25–50%) and 3 (>50 staining). Cancer patient survival analysis was performed using Kaplan-Meier method and GraphPad Prism 5.0 software (La Jolla, CA). DISCUSSION www.impactjournals.com/oncotarget Oncotarget 26788 have further demonstrated a tumor suppressing role of PRSS8, including inhibition of tumor growth in nude mice and cancer cell proliferation, and cell cycle arrest. It is strongly suggested that PRSS8 is a tumor suppressor and has important clinical significance in colorectal cancer. purchased from Biomax Inc. (Rockville, MD). The human prostate cancer TMA was purchased from Cooperative Human Tissue Network (CHTN) (Columbus, OH), and the liver cancer TMA was purchased from the Cybrdi Inc. (Potomac, MD). All procedures were approved by the Institutional Review Board of Xinxiang Medical University and Jining Medical University, China. Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive sphingolipid metabolite produced by sphingosine kinase 1 (Sphk1) and regulates numerous cellular processes important for cell growth, survival, invasion, lymphocyte trafficking, vascular integrity, and the production of cytokines and chemokines [8, 28, 29]. Growing evidence has demonstrated that S1P is linked to chronic intestinal inflammation and colitis-associated cancer, in which upregulation of Sphk1 is needed. Recent studies have demonstrated that the Sphk1/S1P axis causes the activation of NF-kB and transcription factor STAT3 and connects chronic inflammation and colitis-associated colorectal cancer, providing a novel mechanism of colitis malignant transformation [8, 30]. We found that PRSS8 inhibited Sphk1/S1P/Stat3 signaling, in terms of negative correlation of PRSS8 and Sphk1 in vitro, in Sphk1 mouse model and in human colorectal cancers. Interestingly, in normal colonic mucosa, PRSS8 expression was higher and Sphk1 was lower, but, in colorectal cancer tissues, PRSS8 expression was decreased and Sphk1 expression was increased, showing an inverse correlation (Figure 3E). The decrease of PRSS8 during carcinogenesis could be resulted from promoter hypermethylation, and the increase of Sphk1 could be activated by upstream signaling (e.g. inflammatory signaling) during colorectal cancer initiation and progression. Moreover, PRSS8 physically bond with Sphk1, evidenced by co-immunoprecipitation assay and co-localized expression in cytoplasm and nucleus. These findings again show tumor inhibition of PRSS8 via its crosstalk with Sphk1/S1p/Stat3 signaling.i Quantitative reverse-transcriptional polymerase chain reaction (qRT-PCR) Total RNA was extracted from the frozen colorectal cancer and normal tissues using Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. qRT-PCR (Applied Biosystem Inc.) was used for mRNA quantification analysis. The primers for mRNA analysis for qRT-PCR are listed in the Supplementary Table 1. In conclusion, we have identified PRSS8 as a tumor suppressor in colorectal cancer formation and progression, and PRSS8 might be one of the key elements that suppress Sphk1/S1p/Stat3/Akt inflammatory signaling during colorectal carcinogenesis. PRSS8 could be a useful biomarker for monitoring colorectal carcinogenesis and progression and for predicting outcomes. Cell culture Human colorectal cancer cell lines HCT116, SW480, HCT8 and Caco2, and human embryonic kidney cells HEK293 from American Type Culture Collection (ATCC) (Manassas, VA), were reserved by Tumor Center of Cancer Hospital, Chinese Academy of Medical Sciences (Beijing, China) for pure research in Jan, 2015. The PCR- based STR analysis was used to characterize these cells by using GenePrint® 10 System Kit (Cat.# B9510) in Jun, 2015. The system contains all materials necessary to amplify STR regions of human genomic DNA, including a hot-start thermostable DNA polymerase. An internal lane standard (ILS) and allelic ladder are genotyping of amplified fragments, and the 2800M Control DNA is supplied as a positive control. The cells were maintained Human cancer samples and tissue microarray (TMA) Human colorectal and esophageal tissues were obtained from the Tissue Bank of the Laboratory for Cancer Signaling Transduction at Xinxiang Medical University, and from the Institute of Precision Medicine of Jining Medical University, China. The human esophageal cancer tissue microarray (TMA) and colorectal cancer TMA with survival information was made in our laboratory, and the human breast cancer TMA were www.impactjournals.com/oncotarget Oncotarget 26789 For co-localization analysis, the cells grown on a chamber slide (BD Biosciences, San Jose, CA) were co-transfected with pEGFP-PRSS8 and pEX-6-Sphk1 plasmids. Twenty-four hours after transfection, the cells were fixed with 4% paraformaldehyde in PBS for 30 min and permeabilized by further treatment with 0.2% Triton X-100 for 10 min, followed by DAPI (4’,6-Diamidine-2’- phenylindole dihydrochloride) staining for 15min. The images were taken using confocal microscopy (Olympus, China). in a complete MEM medium. All cells were free of mycoplasma contamination. All media were supplemented with 10% FBS and antibiotics (10,000 U/ml penicillin, 10 μg/ml streptomycin). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. Expression plasmids construction, small interfering RNA (siRNA) synthesis and transfection The full length of PRSS8 and Sphk1 were cloned from human cDNA and inserted into pEGFP vector (Promega, Madison, WI), pFlag and pEX-6 vector (Shanghai GenePharma, Shanghai, China), to generate pEGFP-PRSS8, pFlag-PRSS8 and pEX-6-Sphk1 expression constructs, respectively. Three 19-nt siRNA oligonucleotides with 3′-dt extensions against PRSS8 and Sphk1 transcripts, and one scrambled siRNA (negative control) were designed, as shown in Supplementary Table 1. siRNAs were synthesized by Shanghai GenePharma Inc. (Shanghai, China). Twenty-four hours before transfection, 1.0×105 cells were seeded in a 6-well plate. 4 μg of PRSS8 or Sphk1 expression plasmid or negative control plasmid was transfected into cells, using Lipofectamine 3000 (Invitrogen, Carlsbad, CA) following the manufacture’s protocol. Co-IP and co-localization analysis The correlation between PRSS8 and Sphk1 was determined by co-immunoprecipitation (co-IP) and co-localization analysis. For co-immunoprecipitation analysis, HEK 293 cells were plated in 10-cm dishes. The cells in Dish 1 and 2 were co-transfected with pFlag-PRSS8 and pEx-6-Sphk1 plasmids. The cells in Dish 3 and 4 were co-transfected with negative control and pEx-6-Sphk1 plasmids. After 48 h, the cells were washed twice with ice-cold phosphate-buffered saline (PBS) and then incubated on ice for 15 min in RIPA lysis buffer supplemented with protease inhibitor cocktail. Total cell lysate was centrifuged at 12 000 r.p.m. for 15 min at 4°C. 300 μg of lysate were incubated with 1μg the anti-Flag antibody (Tianjin Sungene Biotech Co., Ltd) for Dish 1, anti-Sphk1 antibody (Abcam) for Dish 2, anti-Flag antibody for Dish 3 and anti-Sphk1 antibody for Dish 4, for 1 h at 4°C, followed by addition of 20μl agarose beads (Santa Cruz Biotechnology) for overnight at 4°C. Agarose beads were washed five times in RIPA lysis buffer supplemented with protease inhibitor cocktail. Complexes were released from the agarose beads by boiling for 5 min in 2× gel electrophoresis loading buffer. The immunocomplex was separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting was used to detect PRSS8 with anti-flag and to detect Sphk1 with anti- Sphk1 antibody, respectively. Sphk1+/+ and -/- mice Sphk1-deficient mice (Sphk1-/-) and wild-type (Sphk1+/+) sibling mice were generated by mating Sphk1+/- mice (in C57BL/6 background), as described recently [31]. The Sphk1+/+ and -/- mice were sacrificed at about 8 weeks, and colon tissues were collected and fixed in formalin for immunohistochemical staining using anti-PRSS8 antibody. All immunohistochemical staining slides were imaged using Aperio ImageScope software (version 11) for immunostaining intensity evaluation. All procedures were conducted according to the Animal Care and Use guideline approved by University of Illinois at Chicago Animal Care Committee. Cancer cell transplantation (xenograft) in nude mice 1.5x106 HCT116 stably transfected with pFlag- PRSS8 or negative control plasmid were injected subcutaneously into the flank of the normal nude mice at age about 8 weeks (5 mice per group). The animals were maintained in a pathogen-free barrier facility and closely monitored by animal facility staff. 30 days after inoculation, the animals were sacrificed and the xenografts were isolated and observed, the weight (g) and size (cm3) of the xenografts were determined. In addition, half of the xenograft tumors were fixed in 10% buffered formalin, embedded in paraffin and sectioned for hematoxylin and eosin staining and for immunohistochemical staining for PRSS8 expression validation. The another half of the xenograft tumors were frozen in liquid nitrogen for mRNA and protein analysis by qRT-PCR and immunoblotting. All procedures were conducted according to the Animal Care and Use guideline approved by Animal Care Committee of Xinxiang Medical University and Jining Medical University, China. REFERENCES 17. Takahashi S, Suzuki S, Inaguma S, Ikeda Y, Cho YM, Hayashi N, Inoue T, Sugimura Y, Nishiyama N, Fujita T, Chao J, Ushijima T, Shirai T. 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Sphingosine- 1-phosphate links persistent STAT3 activation, chronic intestinal inflammation, and development of colitis- associated cancer. Cancer Cell. 2013; 23:107-120. 22. Gaedcke J, Grade M, Jung K, Camps J, Jo P, Emons G, Gehoff A, Sax U, Schirmer M, Becker H, Beissbarth T, Ried T, Ghadimi BM. Mutated KRAS results in overexpression of DUSP4, a MAP-kinase phosphatase, and SMYD3, a histone methyltransferase, in rectal carcinomas. Genes, chromosomes & cancer. 2010; 49:1024-1034. 9. Yu JX, Chao L, Chao J. Prostasin is a novel human serine proteinase from seminal fluid. ACKNOWLEDGMENTS This work was supported in part by the grants from the National Natural Science Foundation of China (grants # 91229115 and 81272251 to Yang W, #81502105 to Bao Y), the grants for the Outstanding Scientist of Henan Province and for Scientific Research Team for www.impactjournals.com/oncotarget Oncotarget 26790 12. Lopez-Otin C, Matrisian LM. Emerging roles of proteases in tumour suppression. 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https://openalex.org/W2987170546	https://europepmc.org/articles/pmc6828802?pdf=render	English	null	Long-Term Phytoplankton Dynamics in a Complex Temporal Realm	Scientific reports	2,019	cc-by	12,238	OPEN M. Alvarez-Cobelas1, C. Rojo2 & J. Benavent-Corai2 Faced with an environment of accelerated change, the long-term dynamics of biotic communities can be approached to build a consistent and causal picture of the communities’ life. We have undertaken a 25-year monthly-sampling study on the phytoplankton of a meso-oligotrophic lake, paying attention to controlling factors of overall biomass (TB) and taxonomical group biomass (TGBs). Long-term series included decreased trends of TB and TGBs, and multi-scale periodicity. A decadal TB periodicity emerged related to nitrogen concentration and Cryptophytes. Annual periodicities were mainly related to air and water temperature controlling the abundance of Chlorophytes or Dinoflagellates. Intra-annual cycles could arise from autogenic processes. The analysis by periods revealed relevant dynamics (for example, Diatom periodicities), hidden in the analysis of the complete series. These results allow us to establish that: i) two organizational levels of phytoplankton change differently in time scales from months to decades; ii) controlling factors (climate, water physics and chemistry) act at different time scales and on different TGBs, and iii) different combinations of the “taxonomical group-control factor-trend and periodicity” set throughout the studied time explain total biomass dynamics. A holistic approach (multiple complementary analyses) is necessary to disentangle the different actors and relationships that explain non-stationary long-term phytoplankton dynamics. Faced with an environment of accelerated change1, the long-term dynamics of biotic communities can be approached to build a long-term, consistent and causal picture of the communities’ life. They often respond to instability, and this fact makes it necessary to disentangle the time scales involved to predict their future behaviour. When trying to understand long-term ecosystem dynamics, we are confronted with several questions and decisions. How can we identify trends and periodicities in the dependent signals? Do they have any ecological meaning? What are the controlling factors of variable dynamics? Do we have to study ecosystem features (such as biomass), or do we have to deal with other levels of biological complexity (such as groups of species)? Would the controlling factors be the same across all levels of organization? Would they be the same at all temporal scales? Are the responses the same throughout the whole period of study? g y Furthermore, the temporal length of observations along with their high frequency is important to cover all modes of variability. Some phytoplankton studies have reported decadal periodicities2,3. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports OPEN Hence time spans much longer than 10 years are needed to suggest an insightful picture of organismic responses and their controlling factors, providing that the sampling frequency is high enough to disclose some parts of community effects. The idea of multiple scales (from seconds to decades) of responses by different phytoplankton features (from physi- ological rates to community composition and biomass) arose for the first time at the beginning of the eighties4. We therefore focus our approach and discussion on studies longer than one decade that encompass a sampling frequency to cover most community effects. q y yf Recently, there have been many studies dealing with long-term freshwater phytoplankton dynamics, address- ing parts of those aforementioned questions. Some have usually considered biomass, or its chlorophyll-a sur- rogate, as the only signal. Table 1 compiles studies longer than 10 years of observations with frequencies of observation (monthly or shorter) which are useful to detect modes of community variability. A variety of sug- gested causes for the trajectory of long-term (>10 years) phytoplankton biomass exists (Table 1). For example, they include the often expected covariation with phosphorus concentration5,6 which is considered the most likely 1National Museum of Natural History (CSIC), c/ Serrano 115 dpdo, Madrid, E-28006, Spain. 2Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia, c/ Catedrático José Beltrán 2, Paterna, Valencia, E-46980, Spain. OPEN email: malvarez@mncn.csic.es Scientific Reports \| (2019) 9:15967 \| https://doi.org/10.1038/s41598-019-52333-z www.nature.com/scientificreports/ Lake Latitude Period and frequency of study Causes (linked or not) Phytoplanktonic biomass response Reference Balaton 47 °N 1980–2002 (M) P decrease, Cyprinid elimination Decrease 8 Biwa (Northern basin) 35 °N 1962–2003 (BW) Eutrophication until 1985, Global warming Earlier increase, Later decrease 12 Biwa (Northern basin) 35 °N 1979–2009 (BW) Global warming, Wind increase, PAR limitation Earlier increase, Later decrease 44 Crater 43 °N 1984–2000 Global warming, Atmospheric deposition increase, P increase Increase 45 Garda 46 °N 1993–2009 (every 4th week) P increase, EA decrease Increase 15 Geneva 47 °N 1975–2010 (M or BW) P decrease, Daphnia impact Fluctuating (higher variability at the extremes of the series) 46 Grasmere 54 °N 1970–2010 (W or BW) Water renewal increase, P decrease Decrease 47 Greifensee 47 °N 1971–2000 (BW or M) P decrease Fluctuating (high variability) 18 Heiligensee 53 °N 1975–1992 (BW or M) P increase Increase 5 Kinneret 33 °N 1969–2003 (W or BW) N limitation, fungal epidemics Increase 9 Krankesjön 56 °N 1980–2011 (BW or M) Global warming, Nutrient availability, Food web Fluctuating summer biomass 48 Loch Leven 56 °N 1968–1985 (W, BW or M) P decrease Decrease 49 Luzern 47 °N 1955–2000 (W, BW or M) P decrease Decrease 6 Maggiore 46 °N 1984–2005 (BW or M) P decrease Decrease 50 Müggelsee 52 °N 1979–2003 (W or BW) P decrease Decrease 51 Neusiedlersee 48 °N 1968–2007 NAO increase Winter increase 52 North Pine Dam 27 °S 1978–1994 (W) Fluctuating water renewal Fluctuating (high variability) 53 Ontario (Bay of Quinte) 44 °N 1972–2008 (BW or M) P decrease Decrease 54 Ontario (main lake) 44 °N 1975–2000 P decrease Decrease 7 Ontario (main lake) 44 °N 2001–2012 P increase, Global warming, Dreissena increase Fluctuating 7 Orta 46 °N 1984–2008 P decrease Decrease 15 Pyhäjärvi 62 °N 1963–2002 (BW) P decrease Decrease 55 Saidenbach 50 °N 1975–2011 (W or BW) P decrease, global warming Increase 10 Vänern 59 °N 1980–2012 P decrease, Atmospheric deposition decrease, Global warming Increase 13 Washington 47 °N 1975–1999 (W or M) ENSO increase Winter and spring increase 14 Windermere (North basin) 54 °N 1850–2000 NAO increase Increase 56 Table 1. Long-term responses of freshwater phytoplankton biomass and their causes and periodicities in studies longer than 10 years with frequent sampling (BW: biweekly, M: monthly, W: weekly). OPEN EA: Eastern Atlantic Oscillation, ENSO: El Niño-Southern Oscillation, N: nitrogen, NAO: Northern Atlantic Oscillation, P: phosphorus. Table 1. Long-term responses of freshwater phytoplankton biomass and their causes and periodicities in studies longer than 10 years with frequent sampling (BW: biweekly, M: monthly, W: weekly). EA: Eastern Atlantic Oscillation, ENSO: El Niño-Southern Oscillation, N: nitrogen, NAO: Northern Atlantic Oscillation P: phosphorus. Table 1. Long-term responses of freshwater phytoplankton biomass and their causes and periodicities in studies longer than 10 years with frequent sampling (BW: biweekly, M: monthly, W: weekly). EA: Eastern Atlantic Oscillation, ENSO: El Niño-Southern Oscillation, N: nitrogen, NAO: Northern Atlantic Oscillation, P: phosphorus. cause to explain phytoplankton changes in addition to others, such as the intended-or-not species introduction (e.g. mussels7), the exclusion of food-web top species (e.g. cyprinid fish8) and the impact of fungal epidemics9 and/ or atmospheric depositions10. p p One of the main factors believed to act on long-term phytoplankton dynamics is global warming, but its effects are not always as expected11. For example, phytoplankton biomass has been shown to decrease with global warming12, or in other instances its effects are counterbalanced by other proccesses such as reoligotrophication, resulting in resilient phytoplankton structures10,13. There are some studies (Table 1) that deal with long-term biomass responses to local environments, including local warming as one of the drivers5,8. Others also try to link global warming and phytoplankton, using linear relationships of the latter with water temperature to prove this10,13. Many studies have looked for teleconnections11 (i.e. regional climate) as factors controlling long-term phytoplankton dynamics, reporting data gathered from frequent sampling (monthly or shorter14,15).h p y p y p g g q p g y Furthermore, at the organizational level there is a missing link. The dynamics of total biomass is currently explained by species replacement over time driven by changes in local conditions, which often imply a replace- ment of taxonomic groups16. Previous studies have suggested that these groups can sometimes prove to be bet- ter indicators of environmental conditions and effects than species-specific data17. In addition, changes at this class-level of taxonomic composition are usually a good reflection of seasonal changes in the system16, as well as its interannual pattern. Despite this, taxonomical groups have not been considered very often when addressing longer-term (>10 years) phytoplankon changes (but see, for example, studies on lakes Greifensee18, Tahoe19 and Zürich20). Results Long-term trends and periodicities. Total phytoplankton biomass (TB hereafter) experienced a decreas- ing trend in Las Madres lake for the whole period under study (Fig. 1a). The analysis of time series using AEM revealed a negative trend for TB from 1992–2016 (Table 2), as already mentioned, and two further periodicities: a decadal (17 years) and an annual one (Table 2). Wavelet analysis also enabled us to visualize an intra-annual periodicity at least in the first quarter of the studied series (Fig. 1b). Moreover, this analysis was able to identify the temporal variability in the periodicity of TB, as shown by a remarkable annual periodicity up to 1998, later weakening until 2006 when it disappeared (Fig. 1b). g pp ( g ) Throughout the whole series (1992–2016), all biomasses of taxonomical groups (TGBs hereafter), except that of Diatoms, showed a trend and periodicity in their dynamics (Table 2). Chlorophytes and Dinoflagellates had annual cycles and the latter also exhibited an intra-annual periodicity; Cryptophytes had interannual cycle (Table 2). Wavelet analysis complemented this information by highlighting that: i) annual periodicities took place in the early period, ii) Diatom dynamics had weaker annual periodicity over the whole 25-year period, and iii) Cryptophyte periodicity vanished after 1998 (see Supplementary Fig. S1). yp p y p yt pp y g TGBs encompassed the greatest overall biomass over the study period. Cryptophytes dominated during the early years of the series, and two decades later they were co-dominant with Chlorophytes. Diatoms were present throughout the series, whereas Dinoflagellate biomass seemed, sometimes, to be negatively correlated with that of Cryptophytes (Fig. 2a). These changes in TGB composition enabled us to establish three periods of phytoplankton dynamics (Fig. 2a), which were well defined by a cluster analysis (Fig. 2b). y gi y y g The first group of dates occurred from January 1992 to April 1998 (76 months), with TB amount- ing to 0.50 ± 0.43 mg C/L, and this was followed by the period of May 1998-June 2006 (96 months), with 0.17 ± 0.18 mg C/L, and that of July 2006-December 2016 (126 months), with 0.08 ± 0.09 mg C/L. Thus, the aver- age TB in each interval was less than half that of the previous period. TB peaks were also different in these three periods (Fig. 2c), and this resulted from increasing their CVs (first period: 86%; second period: 106%; third period: 113%). www.nature.com/scientificreports/ has been refuted recently for phytoplankton23. In addition, both approaches do not provide information on how much variability is explained by trend and time scales of the signal, and the controlling factors involved. y p y g g Over the last two decades, some methods have been implemented to improve and complement the classical approach (i.e. spectral decomposition), thus enabling to quantify the relative importance of patterns and pro- cesses across time scales of ecosystems, namely, to partition variability at different temporal scales24. The first pro- cedure, called Asymmetric Eigenvectors Maps (AEM hereafter), summarizes temporal scales on a set of vectors that can be used as covariates when modelling ecological responses to environmental variability over time25. The second, known as variance partitioning, has been developed to quantify the individual contribution of temporal scales and environmental factors overcoming the collinearity problem26. A third method (codependence anal- ysis) allows the main factor associated with each temporal scale27 to be identified. These methods consider the studied time series as stationary. Notwithstanding this, ecological communities can be variable through time, and a fourth method, wavelet analysis, enables the emergence and/or disappearance of periodicities over the whole series to be identified28. These methodologies have already been applied successfully to processes in freshwater ecosystems29,30, and usually result in a better description of temporal scales. However, very few studies (e.g.31) are available using all these novel methods to characterize long-term phytoplankton processes, periodicities and controlling factors, as we advocate here. g Our hypotheses to be tested here are the following: 1st) the total biomass of phytoplankton and those of its taxonomical groups show different trends and periodicities that change along the whole time series; 2nd) taxo- nomical composition varies over time exhibiting periodicities longer than seasonal; 3rd) trends and periodicities of both phytoplankton biomass and taxonomical groups biomass depend on the time period, and 4th) there could be different controlling variables of total- and taxonomical groups’ biomass in different time periods and at dif- ferent temporal scales. Therefore, we expect that the response at specific time scales of each taxonomical group to environmental factors at each time period could provide insights into phytoplankton biomass dynamics. p p g p y p y Using 25 years of monthly data on phytoplankton, and its likely controlling factors in an oligo-mesotrophic lake, we aim to address all these issues. www.nature.com/scientificreports/ We will deal with two levels of organization (overall biomass and taxo- nomical composition), testing the occurrence of multiple scales of change (from months to decades) and quanti- fying the explicative value of controlling factors (either regional or local) of phytoplankton dynamics. Our study will demonstrate that there are different trends, periodicities and controlling factors for different levels of organ- ization, and that there can be discontinuities and multiscale changes in long-term phytoplankton dynamics, too. Scientific Reports \| (2019) 9:15967 \| https://doi.org/10.1038/s41598-019-52333-z OPEN ) A further topic worth mentioning in long-term ecological studies is the development of statistical meth- odologies to deal with time series data. Until recently, spectral decomposition of long-term series (detrended series and detection of periodicities)21, and general linear models to disclose the long-term relationship between a dependent biotic signal and an independent environmental factor22, have been the preferred methodologies. These approaches assume that the series is stationary, i.e. constant variance over time, but such an assumption Scientific Reports \| (2019) 9:15967 \| https://doi.org/10.1038/s41598-019-52333-z www.nature.com/scientificreports/ Results In the earlier one, there were two peaks along with a higher monthly variability. The autumnal peak showed a gradual decline, as shown in the second and the third intervals; in the latter monthly variability was also lower than in the second period.l In order to uncover likely periodicities masked by uneven fluctuations over the whole series (1992–2016), trend and periodicity were analyzed in each of the three aforementioned periods. TB dynamics followed a neg- ative trend until 1998, later lacking any trend until 2006 and weakly increasing henceforth up to 2016 (Table 2, Fig. 1a). TB only experienced an annual periodicity and, as the wavelet analysis suggested, more variance was explained in the first period than in the remaining ones (Table 2, Fig. 3).ii i In the first period, only Cryptophyte biomass showed a significant decreasing trend, whereas the lowest biomass variability was experienced by Dinoflagellates. Chlorophytes and Diatoms showed different trends, declining and increasing respectively (Table 2, Fig. 3). The only group with an annual periodicity in the three intervals was Dinoflagellates, which also exhibited intra-annual periodicity during the two earlier intervals. Such an annual periodicity became less important over time (Table 2, Fig. 3), a fact matching an ongoing smoothing of the intra-annual bimodal pattern in the second period, and its waning in the third one (Fig. 2c). Chlorophyte Scientific Reports \| (2019) 9:15967 \| https://doi.org/10.1038/s41598-019-52333-z www.nature.com/scientificreports/ www.nature.com/scientificreports tificreports/ Figure 1. (a) Water column-averaged, monthly phytoplankton biomass in Las Madres lake from January 1992 to December 2016. The series is fitted to a non-linear function, which is also shown along with its statistically significant explained variability (R2, P < 0.001). (b) Continuous wavelet power spectrum showing the periodicity of monthly phytoplankton biomass in Las Madres lake. The thick black contour delimits the significant periodicities (P < 0.05) and the red line denotes the cone of influence, where edge effects may distort the interpretation. Colors reflect the strength of intensity or power (dark red indicates high power; dark blue indicates low power). Figure 1. (a) Water column-averaged, monthly phytoplankton biomass in Las Madres lake from January 1992 to December 2016. The series is fitted to a non-linear function, which is also shown along with its statistically significant explained variability (R2, P < 0.001). (b) Continuous wavelet power spectrum showing the periodicity of monthly phytoplankton biomass in Las Madres lake. Results The thick black contour delimits the significant periodicities (P < 0.05) and the red line denotes the cone of influence, where edge effects may distort the interpretation. Colors reflect the strength of intensity or power (dark red indicates high power; dark blue indicates low power). TB CHLOR CRYP DIAT DINO 1992–2016 Trend (−)25 (−)9 (−)20 (−)16 Cycle in months (years) 201 (17) 13 17 12 (1) 18 15 39 6 (0.5) 6 1992–1998 Trend (−)9 (−)11 Cycle in months (years) 12 (1) 37 27 10 44 6 (0.5) 11 13 1998–2006 Trend (−)6 Cycle in months (years) 12 (1) 26 11 14 29 6 (0.5) 8 6 2006–2016 Trend (+)8 (−)9 (+)12 Cycle in months (years) 12 (1) 21 16 20 Table 2. Adjusted variance (%R2 adj) of biomass models in Las Madres lake in different periods shown when statistically significant (P < 0.05) (see also text and Figs 1, 2). Asymmetric Eigenvector analysis plus forward selection with two stopping criteria were used (see Supplementary Methods). Trends are shown with the sign of the relationship in brackets. TB is overall phytoplankton biomass. CHLOR is Chlorophyte biomass, CRYP – Cryptophyte biomass, DIAT – Diatom biomass, DINO – Dinoflagellate biomass. TB CHLOR CRYP DIAT DINO 1992–2016 Trend (−)25 (−)9 (−)20 (−)16 Cycle in months (years) 201 (17) 13 17 12 (1) 18 15 39 6 (0.5) 6 1992–1998 Trend (−)9 (−)11 Cycle in months (years) 12 (1) 37 27 10 44 6 (0.5) 11 13 1998–2006 Trend (−)6 Cycle in months (years) 12 (1) 26 11 14 29 6 (0.5) 8 6 2006–2016 Trend (+)8 (−)9 (+)12 Cycle in months (years) 12 (1) 21 16 20 TB CHLOR CRYP DIAT DINO 1992–2016 Trend (−)25 (−)9 (−)20 (−)16 Cycle in months (years) 201 (17) 13 17 12 (1) 18 15 39 6 (0.5) 6 1992–1998 Trend (−)9 (−)11 Cycle in months (years) 12 (1) 37 27 10 44 6 (0.5) 11 13 1998–2006 Trend (−)6 Cycle in months (years) 12 (1) 26 11 14 29 6 (0.5) 8 6 2006–2016 Trend (+)8 (−)9 (+)12 Cycle in months (years) 12 (1) 21 16 20 Table 2. Adjusted variance (%R2 adj) of biomass models in Las Madres lake in different periods shown when statistically significant (P < 0.05) (see also text and Figs 1, 2). Asymmetric Eigenvector analysis plus forward selection with two stopping criteria were used (see Supplementary Methods). Results Trends are shown with the sign of the relationship in brackets. TB is overall phytoplankton biomass. CHLOR is Chlorophyte biomass, CRYP – Cryptophyte biomass, DIAT – Diatom biomass, DINO – Dinoflagellate biomass. Table 2. Adjusted variance (%R2 adj) of biomass models in Las Madres lake in different periods shown when statistically significant (P < 0.05) (see also text and Figs 1, 2). Asymmetric Eigenvector analysis plus forward selection with two stopping criteria were used (see Supplementary Methods). Trends are shown with the sign of the relationship in brackets. TB is overall phytoplankton biomass. CHLOR is Chlorophyte biomass, CRYP – Cryptophyte biomass, DIAT – Diatom biomass, DINO – Dinoflagellate biomass. Table 2. Adjusted variance (%R2 adj) of biomass models in Las Madres lake in different periods shown when statistically significant (P < 0.05) (see also text and Figs 1, 2). Asymmetric Eigenvector analysis plus forward selection with two stopping criteria were used (see Supplementary Methods). Trends are shown with the sign of the relationship in brackets. TB is overall phytoplankton biomass. CHLOR is Chlorophyte biomass, CRYP – Cryptophyte biomass, DIAT – Diatom biomass, DINO – Dinoflagellate biomass. Table 2. Adjusted variance (%R2 adj) of biomass models in Las Madres lake in different periods shown when statistically significant (P < 0.05) (see also text and Figs 1, 2). Asymmetric Eigenvector analysis plus forward selection with two stopping criteria were used (see Supplementary Methods). Trends are shown with the sign of the relationship in brackets. TB is overall phytoplankton biomass. CHLOR is Chlorophyte biomass, CRYP – Cryptophyte biomass, DIAT – Diatom biomass, DINO – Dinoflagellate biomass. contribution to overall biomass also varied among periods, with intra or annual periodicities occurring only dur- ing the first two intervals. The annual periodicity of Cryptophytes was lost during the second interval, which was the only period when Diatoms showed any periodicity (Table 2, Fig. 3). contribution to overall biomass also varied among periods, with intra or annual periodicities occurring only dur- ing the first two intervals. The annual periodicity of Cryptophytes was lost during the second interval, which was the only period when Diatoms showed any periodicity (Table 2, Fig. 3). Controlling factors. Regional climate teleconnections did not explain any variability for the overall period. Local climate variables, such as air temperature and solar radiation (Fig. 4a), had a positive relationship with TB Scientific Reports \| (2019) 9:15967 \| https://doi.org/10.1038/s41598-019-52333-z www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Results (a) Changes in the structure of taxonomical group biomass (TGBs) in Las Madres lake from January 1992 to December 2016. (b) Dendrogram of yearly-averaged biomass (%) of taxonomical groups; the main bootstrap values have been included. (c) Average seasonality of total phytoplankton biomass in the intervals reported. Notice the changes of scale between the early interval and the other two, latter ones. Vertical dashed lines separate different temporal intervals. Figure 2. (a) Changes in the structure of taxonomical group biomass (TGBs) in Las Madres lake from January 1992 to December 2016. (b) Dendrogram of yearly-averaged biomass (%) of taxonomical groups; the main bootstrap values have been included. (c) Average seasonality of total phytoplankton biomass in the intervals reported. Notice the changes of scale between the early interval and the other two, latter ones. Vertical dashed lines separate different temporal intervals. dynamics (Table 3), the local factor explaining a 17% of TB variability. Water temperature and the upper mixed layer depth (physical factors, Fig. 4b) explained 15% of overall variability at most (Table 3). Nitrate and ammonia (chemical factors, Fig. 4c) only explained 7% (Table 3). g y p With regard to warming in Las Madres lake, we did not observe this using over 25 years of monthly-averaged air temperature data. However, an increase in the annual average temperature (see Supplementary Fig. S2a) shows an increase of almost 1 °C/decade in air and 0.75 °C/decade in water. The annual average of the upper mixed layer experienced a decreasing trend until 2010, after which it increased again (see Supplementary Fig. S2b). t Chlorophyte biomass was mainly related to radiation and air- and water temperature, hence being related to local factors such as climate and water physics. Cryptophyte dynamics was weakly explained by water chemistry, with an inverse pattern to that of nitrate concentration. Dinoflagellate dynamics was explained by local climate, but also by some physical properties of the water column, such as temperature and mixing depth (Table 3).h y p y p p p g p The partition of variance in the model TB = [LOC] + [PHY] + [CHE] + [AEM], where environmental factors only included abiotic selected variables (those mentioned in Table 3), showed that TB variability was explained by interactions between abiotic factors and AEM periodicities (annual and decadal), amounting to 20% of overall variability and pure AEM (Fig. 5a). Results The relevant interaction with periodicity was also demonstrated by multiple co-dependence analysis (MCA; see Supplementary Table S2), which revealed that variables of local climate were the most influential predictors of TB along with annual periodicity, whereas water chemistry was more influential at the decadal scale. With the aim of highlighting the importance of taxonomical groups for TB dynamics, we analysed the parti- tioning variance of a model having the taxonomical groups as explanatory variables, i.e. TB = [CHLO] + [CRY] + [DIAT] + [DINO] + [AEM]. While the pure variance explained by these groups made up 53% of overall variability, AEM without interac- tion with TGBs did not explain any variance (Fig. 5b). The interaction of TGBs and AEM periodicity amounted to around 15%. MCA revealed that all TGBs, except Cryptophytes, were important for TB at the annual scale. However, the variability of Cryptophyte biomass was remarkable at the decadal scale (see Supplementary Table S2). Scientific Reports \| (2019) 9:15967 \| https://doi.org/10.1038/s41598-019-52333-z www.nature.com/scientificreports/ Figure 3. Wavelet power spectra showing periodicities from the three reported intervals in Las M between 1992 and 2016 (see Fig. 1). (a) Total phytoplankton biomass, (b) Chlorophyte fractional (c) Cryptophyte fractional biomass, (d) Diatom fractional biomass, and (e) Dinoflagellate fractio Explanations as in Fig. 1b. Figure 3. Wavelet power spectra showing periodicities from the three reported intervals in Las Madres lake between 1992 and 2016 (see Fig. 1). (a) Total phytoplankton biomass, (b) Chlorophyte fractional biomass, (c) Cryptophyte fractional biomass, (d) Diatom fractional biomass, and (e) Dinoflagellate fractional biomass. Explanations as in Fig. 1b. When the whole series was split into the three already-envisaged periods (see above), air and water temper- ature explained TB in the first period (1992–1998), also explaining the biomass time course of the taxonomical groups (Chlorophytes, Cryptophytes and Dinoflagellates, Table 4, Fig. 6). In addition, other physical variables, such as underwater light climate and the upper mixed layer, controlled Chlorophyte and Dinoflagellate biomass, respectively. Water chemistry did not explain any variability of either TB or TGBs. Diatom biomass did not appear to be controlled by any factor in this first period. i Local climate (solar radiation; R2 = 25%) and lake physics (water temperature; R2 = 20%) controlled TB during the second period (1998–2006, Table 4, Fig. 6). Results Solar radiation was the variable that best explained Chlorophyte, Diatom and Dinoflagellate biomass, with the upper mixed layer and water temperature also explaining some variability, albeit with an opposite sign. Cryptophyte biomass was not explained by any variable tested in this period (Table 4). p Finally, from 2006 to 2016 a decreasing variance, not greater than 19% of overall variability, was explained by environmental factors. Again, local climate and water physics jointly explained TB and TGBs (Table 4, Fig. 6). Solar radiation and water temperature were also important for Cryptophyte- and Diatom biomass, albeit less Scientific Reports \| (2019) 9:15967 \| https://doi.org/10.1038/s41598-019-52333-z www.nature.com/scientificreports/ Figure 4. Monthly records of some relevant abiotic variables of (a) local climate, (b) water physics and (c) water chemistry in Las Madres lake from 1992 to 2016. Lake variables are water-column averages. The first data is January 1992, the last one is December 2016. Figure 4. Monthly records of some relevant abiotic variables of (a) local climate, (b) water physics and (c) water chemistry in Las Madres lake from 1992 to 2016. Lake variables are water-column averages. The first data is January 1992, the last one is December 2016. Figure 4. Monthly records of some relevant abiotic variables of (a) local climate, (b) water physics and (c) water chemistry in Las Madres lake from 1992 to 2016. Lake variables are water-column averages. The first data is January 1992, the last one is December 2016. than for TB, and the upper mixed layer was also a key variable for Dinoflagellate biomass (Table 4). No regional teleconnections explained any statistically significant variability in any of the periods tested (P > 0.05). Scientific Reports \| (2019) 9:15967 \| https://doi.org/10.1038/s41598-019-52333-z www.nature.com/scientificreports/ (a) Variance partitioning of a model with overall phytoplankton biomass as the dependent variable and local climate, physical and chemical proprieties of water column and AEM (periodicities) as controlling factors in Las Madres lake for 1992–2016. (b) The same for the biomass of taxonomical groups. Only variance related to pure effect of controlling factors can be tested, being its value in bold and underlined lettering when statistically significant (P < 0.05). Only non null variances for both pure and interaction effects are shown to make the figure clearer. For the same reason, Chlorophytes are neglected because their pure adjusted variance was lower than 2%, in spite of the fact that their interaction with Cryptophytes amounted to 15% of explained variability. Figure 5. (a) Variance partitioning of a model with overall phytoplankton biomass as the dependent variable and local climate, physical and chemical proprieties of water column and AEM (periodicities) as controlling factors in Las Madres lake for 1992–2016. (b) The same for the biomass of taxonomical groups. Only variance related to pure effect of controlling factors can be tested, being its value in bold and underlined lettering when statistically significant (P < 0.05). Only non null variances for both pure and interaction effects are shown to make the figure clearer. For the same reason, Chlorophytes are neglected because their pure adjusted variance was lower than 2%, in spite of the fact that their interaction with Cryptophytes amounted to 15% of explained variability. This explanation, for the case in question, would be supported by the negative relationship of TB with the upper mixed layer, since spring algal growth occurs during early stratification after the winter mixing in Las Madres lake. Another process that may be occurring at the same time, and which would explain the negative relation- ship of warming with phytoplankton biomass, is the gradual disappearance of the effects of the internal nutrient dynamics in a newly-formed lake. This process, which that usually occurs over years in recently-built freshwater ecosystems such as reservoirs23,33, reduces the amount and variability of TB as we have observed (Fig. 1). y y g TB dynamics over the whole period of study showed three main types of periodicity: decadal, annual and intra-annual. The decadal change in TB is the result of the decadal change in the taxonomic group of Cryptophytes, whose likely controlling factor is related with nitrate (Fig. 7). Scientific Reports \| (2019) 9:15967 \| https://doi.org/10.1038/s41598-019-52333-z www.nature.com/scientificreports/ Also included, their adjusted explained variance (%R2 adj) if selected by forward selection procedure and the adjusted explained variance of the individual effects of factors (last column), without interactions. This last value results from the RDA and variance partition analysis. Analysis performed for the whole study period (1992–2016) in Las Madres lake. Probability of all variances was lower than 0.001. For more information on statistical tools see Supplementary methods. Diatom biomass was not related to any analysed factor. The “a” letter implies annual co- dependence whereas “d” is decadal co-dependence (see Table S1). While TB is overall phytoplankton biomass, Zm is the thickness of the upper mixed layer. CHE: water chemistry, LOC: local climate, PHY: water physics. Table 3. Control variables that explained more than 5% of total phytoplankton biomass or taxonomical group biomass with the sign of their relation and their percentage of explained variance (R2). Also included, their adjusted explained variance (%R2 adj) if selected by forward selection procedure and the adjusted explained variance of the individual effects of factors (last column), without interactions. This last value results from the RDA and variance partition analysis. Analysis performed for the whole study period (1992–2016) in Las Madres lake. Probability of all variances was lower than 0.001. For more information on statistical tools see Supplementary methods. Diatom biomass was not related to any analysed factor. The “a” letter implies annual co- dependence whereas “d” is decadal co-dependence (see Table S1). While TB is overall phytoplankton biomass, Zm is the thickness of the upper mixed layer. CHE: water chemistry, LOC: local climate, PHY: water physics. Figure 5. (a) Variance partitioning of a model with overall phytoplankton biomass as the dependent variable and local climate, physical and chemical proprieties of water column and AEM (periodicities) as controlling factors in Las Madres lake for 1992–2016. (b) The same for the biomass of taxonomical groups. Only variance related to pure effect of controlling factors can be tested, being its value in bold and underlined lettering when statistically significant (P < 0.05). Only non null variances for both pure and interaction effects are shown to make the figure clearer. For the same reason, Chlorophytes are neglected because their pure adjusted variance was lower than 2%, in spite of the fact that their interaction with Cryptophytes amounted to 15% of explained variability. Figure 5. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 1992–2016 VARIABLES R2 >5% MODELS Response variable Sign Control variable R2 R2adj of selected variable Environmental factor R2adj Total Biomass + Air temperature 15 LOC 17 + Radiation 17 17 (a) − Zm 7 PHY 15 + Water temperature 15 15 (a) − Nitrate 5 5 (d) CHE 7 − Ammonia 5 4 (d) Chlorophyte biomass + Air temperature 12 12 (a) LOC 12 + Radiation 9 + Water temperature 11 11 (a) PHY 11 Cryptophyte biomass − Nitrate 5 5(d) CHE 5 Dinoflagellate biomass + Air temperature 17 LOC 18 + Radiation 18 18 (a) − Zm 11 1 (a) PHY 16 + Water temperature 15 15 (a) Table 3. Control variables that explained more than 5% of total phytoplankton biomass or taxonomical group biomass with the sign of their relation and their percentage of explained variance (R2). Also included, their adjusted explained variance (%R2 adj) if selected by forward selection procedure and the adjusted explained variance of the individual effects of factors (last column), without interactions. This last value results from the RDA and variance partition analysis. Analysis performed for the whole study period (1992–2016) in Las Madres lake. Probability of all variances was lower than 0.001. For more information on statistical tools see Supplementary methods. Diatom biomass was not related to any analysed factor. The “a” letter implies annual co- dependence whereas “d” is decadal co-dependence (see Table S1). While TB is overall phytoplankton biomass, Zm is the thickness of the upper mixed layer. CHE: water chemistry, LOC: local climate, PHY: water physics. 1992–2016 VARIABLES R2 >5% MODELS Response variable Sign Control variable R2 R2adj of selected variable Environmental factor R2adj Total Biomass + Air temperature 15 LOC 17 + Radiation 17 17 (a) − Zm 7 PHY 15 + Water temperature 15 15 (a) − Nitrate 5 5 (d) CHE 7 − Ammonia 5 4 (d) Chlorophyte biomass + Air temperature 12 12 (a) LOC 12 + Radiation 9 + Water temperature 11 11 (a) PHY 11 Cryptophyte biomass − Nitrate 5 5(d) CHE 5 Dinoflagellate biomass + Air temperature 17 LOC 18 + Radiation 18 18 (a) − Zm 11 1 (a) PHY 16 + Water temperature 15 15 (a) Table 3. Control variables that explained more than 5% of total phytoplankton biomass or taxonomical group biomass with the sign of their relation and their percentage of explained variance (R2). Discussion In Las Madres lake, where no anthropogenic stress other than global warming has seemingly occurred31, there have been strong changes in phytoplankton biomass over the last 25 years. In accordance with our first three hypotheses, the total biomass of phytoplankton and those of their taxonomical groups exhibit trends and sev- eral periodicities which do not match entirely across those levels of organization and time periods (Table 2). TB experienced a reduction in the steepness of long-term trend over time, and its variability was strongly dependent upon scale; both facts occurred because TB drivers changed along with time scales, from months to decades. This fact was previously demonstrated but on key abiotic elements of aquatic environments (e.g. dissolved oxy- gen, nutrients)2,32. Taxonomical groups presented periodicities from intra-annual (e.g. Chlorophytes) to decadal (Cryptophytes). These different dynamics crystallize in changes in taxonomic composition that result in three very different periods (6, 8 and 10 years) throughout the time series studied. Thus, TB trends and periodicities are more related to one or another taxonomic group depending on the analysed period (Table 2).h g p p g y p These changes have arisen as a response to the local abiotic environment (Tables 3–4, Figs 5–6). Our study shows that phytoplankton is a complex assemblage whose elements relate to different controlling factors at dif- ferent temporal scales, as we suggested in our fourth hypothesis. Therefore, TB dynamics is the emergent result of the relationship “phytoplankton groups-abiotic controlling factors-periodicities” and of the considerable var- iability of these relationships throughout the series (Fig. 6). We have evidenced that phytoplankton is sensitive to temperature throughout the whole monthly series (Tables 3–4). Moreover, we can suggest that warming, as annual averaged of air- and water temperature is taking place (see Supplementary Fig. S2a). However, such a tem- perature increase does not result in phytoplankton growth, but rather in a decrease in TB, because a higher tem- perature does not imply more TB since the effects of a temperature increase are complex in lakes11. An example of the latter is that a positive effect of a temperature increase on phytoplankton growth could be counterbalanced by another effect of temperature, such as a reduction in the mixing layer (see Supplementary Fig. S2b), which can potentially reduce the necessary nutrient availability for primary production during water column stratification12. www.nature.com/scientificreports/ Also included, their adjusted explained variance (%R2 adj) if selected by forward selection procedure and the adjusted explained variance of the individual effects of factors (last column), without interactions. This last value results from the RDA and variance partition analysis. Analysis performed for three consecutive periods occurring in Las Madres lake for 1992–2016. Probability of all variances was lower than 0.001. Abbreviations as in Table 3. For more information on statistical tools see Supplementary methods. Table 4. Control variables that explained more than 5% of total phytoplankton biomass or taxonomical group biomass with their percentage of explained variance (R2). Also included, their adjusted explained variance (%R2 adj) if selected by forward selection procedure and the adjusted explained variance of the individual effects of factors (last column), without interactions. This last value results from the RDA and variance partition analysis. Analysis performed for three consecutive periods occurring in Las Madres lake for 1992–2016. Probability of all variances was lower than 0.001. Abbreviations as in Table 3. For more information on statistical tools see Supplementary methods. Table 4. Control variables that explained more than 5% of total phytoplankton biomass or taxonomical group biomass with their percentage of explained variance (R2). Also included, their adjusted explained variance (%R2 adj) if selected by forward selection procedure and the adjusted explained variance of the individual effects of factors (last column), without interactions. This last value results from the RDA and variance partition analysis. Analysis performed for three consecutive periods occurring in Las Madres lake for 1992–2016. Probability of all variances was lower than 0.001. Abbreviations as in Table 3. For more information on statistical tools see Supplementary methods. variability, a fact reflected by both the wavelet analysis and the increasingly lower explained variance of annual periodicity (Table 2, Fig. 7). AEM and normalized wavelet analyses of the overall series failed to detect annual periodicities for taxonomical groups that could be otherwise identified when these analyses were applied to each interval (Table 2). The reason for this is that the amplitude of periods also decreased due to the decreasing TB trend over 25 years, another remarkable fact at these time intervals. The higher average value of the signal was associated with a stronger seasonality, compared to what occurred when biomass averages diminished. This lack variability, a fact reflected by both the wavelet analysis and the increasingly lower explained variance of annual periodicity (Table 2, Fig. 7). www.nature.com/scientificreports/ This long-term response to nitrate is very interesting, albeit hard to explain. Cryptophytes are known to uptake ammonia preferably as a nitrogen source34. Since they are known to perform migrations in the water column of lakes35, they might obtain this com- pound from the bacterial reduction of nitrate in the anoxic conditions that prevail in Las Madres hypolimnion in summer. Such conditions might be enhanced by the, already mentioned, weakening of water-column mixing.f g y y g g Inter-annual variability of phytoplankton composition showing three different states, and the existence of different periodicities in the signals, encouraged us to analyse, separately for each time period, the relation- ships of TB and TGBs with the controlling factors. Annual cycles during the earlier studied years showed higher Scientific Reports \| (2019) 9:15967 \| https://doi.org/10.1038/s41598-019-52333-z www.nature.com/scientificreports/ Response variable VARIABLES R2 >5% MODELS Control variable R2 R2adj of selected variables Environmental factor R2adj 1992–1998 Total Biomass Air temperature 41 41 (a) LOC 41 Water temperature 40 40 (a) PHY 40 Chlorophyte biomass Air temperature 33 33 (a) LOC 37 Radiation 16 4 (a) Transparency 18 11 (a) PHY 43 Water temperature 32 32 (a) Cryptophyte biomass Air temperature 18 18 LOC 18 Water temperature 20 20 PHY 20 Dinoflagellate biomass Air temperature 43 43 LOC 43 Radiation 40 Zm 26 PHY 38 Water temperature 38 38 1998–2006 Total Biomass Air temperature 18 LOC 25 Radiation 25 25 Zm 18 PHY 20 Water temperature 20 20 Chlorophyte biomass Air temperature 18 LOC 19 Radiation 19 19 Water temperature 15 15 PHY 15 Diatom biomass Radiation 6 6 LOC 6 Zm 8 8 PHY 8 Dinoflagellate biomass Air temperature 23 LOC 24 Radiation 24 24 Zm 12 PHY 17 Water temperature 17 17 2006–2016 Total Biomass Air temperature 14 LOC 19 Radiation 19 19 Zm 11 PHY 15 Water temperature 15 15 Cryptophyte biomass Air temperature 5 LOC 9 Radiation 9 9 Water temperature 6 6 PHY 6 Diatom biomass Air temperature 5 LOC 7 Radiation 7 7 Water temperature 7 7 PHY 7 Dinoflagellate biomass Air temperature 5 2 LOC 14 Radiation 12 12 Zm 7 7 PHY 7 Water temperature 6 Table 4. Control variables that explained more than 5% of total phytoplankton biomass or taxonomical group biomass with their percentage of explained variance (R2). www.nature.com/scientificreports/ Diagram depicting the most relevant periodicities experienced by each taxonomical group and the sets of environmental variables likely controlling them over 25 years in Las Madres lake. Figure 7. Diagram depicting the most relevant periodicities experienced by each taxonomical group and the sets of environmental variables likely controlling them over 25 years in Las Madres lake. Figure 7. Diagram depicting the most relevant periodicities experienced by each taxonomical group and the sets of environmental variables likely controlling them over 25 years in Las Madres lake. of stationarity in the amplitude of the short time scale would explain why both analyses (i.e. AEM and normal- ized wavelet) enabled us to detect these patterns only during the first time interval. Therefore, when partitioning the time series, the amplitude of annual periods is less variable in the resulting time interval, allowing its detec- tion with AEM and wavelet analysis. Thus, the division of the whole series into periods is a good recommenda- tion for future works that aim to disentangle the drivers of biotic dynamics. As a result new periodicities would emerge, allowing to observe, for example, the annual scale previously undetected for some taxonomical groups as Cryptophytes and Diatoms (Table 2).h yp p y ( ) The dominant mode of TB variability in Las Madres lake is the annual mode (Table 2). Our study gives more weight to local climate (solar radiation, air temperature) and water physics (water temperature) as controlling factors of biomass (Tables 3–4, Fig. 4). Virtually all groups of algae respond to solar radiation and temperature, thus demonstrating an expected seasonality (i.e. annual periodicity). Fundamentally Dinoflagellates and, to a lesser extent, Chlorophytes are the taxonomic groups most related with these physical factors, both groups being considered as characteristic of the period of summer stratification in temperate lakes16. In addition, they are the two dominant groups in the third period of study (2006–2016), when the increase in temperature and its already mentioned consequences (for example, the weakening of water-column mixing) occur.h q p g g The intra-annual bimodal pattern (six months of periodicity) of TB, commonly expressed as the spring and autumn phytoplankton blooms, represents an alternation of periods of stratification and mixing. This alterna- tion is key for the dynamics of motile (i.e. Dinoflagellate) and non-motile (i.e. Diatoms) phytoplankton species, respectively16. www.nature.com/scientificreports/ Both are precisely the taxonomic groups that have been found to be related with mixing depth and intra-annual periodicity of species substitutions in Las Madres lake during the first two periods, when the whole series was partitioned. The intra-annual bimodal pattern disappeared in the last interval of the series, giving way to the unimodal model of a TB peak in spring/summer. As mentioned earlier, thermal changes imply changes in water-column stratification, and it is well known that warming results in stronger stability of the upper mixed layer, thus enabling Dinoflagellates (the only group inversely related to the upper mixed layer in the latter period) to increase over an ongoing longer stratification36. i However, some variability of the studied series remains unexplained. Biotic variables important for phyto- plankton, such as bacteria and zooplankton16,37, are poorly related to TB and TGBs at the monthly scale in Las Madres lake (Alvarez-Cobelas et al., unpublished results), maybe because they are relevant at shorter scales than those used in this study. Another process that could partially explain TB dynamics, and which is not dependent on the abiotic environ- ment, is phytoplankton autogenic succession which commonly runs during the period of thermal stratification when physical stability of the water column remains relatively constant16,23,38. Throughout the 25 years, whenever lake warming reduces the intensity of the autumn-winter mixing period and hence extends the water-column stratification period, a longer auto-organizational period within the year would thus be permitted16. It is in this period that phytoplankton competition, and the ability of its species to distribute themselves in different lay- ers of the water column, would explain the substitution of characteristic groups of phytoplankton species over time16,20,38. This lack of external drivers would agree with the gradual loss of TB variability attributable to the set of factors and periodicities analysed. Therefore, auto-organization remains as another likely, partial explanation for long-term phytoplankton dynamics. g p y p y Having monthly time series of the control variables of phytoplankton and its taxonomic groups over a 25-year period has allowed us to verify that the explanation for its long-term dynamics can be disentangled when we examine the response of taxonomical groups and their relationship with the environment. It also allows us to understand that responses occur at different scales. They are not stationary, but can be due to different agents and scales over time. www.nature.com/scientificreports/ AEM and normalized wavelet analyses of the overall series failed to detect annual periodicities for taxonomical groups that could be otherwise identified when these analyses were applied to each interval (Table 2). The reason for this is that the amplitude of periods also decreased due to the decreasing TB trend over 25 years, another remarkable fact at these time intervals. The higher average value of the signal was associated with a stronger seasonality, compared to what occurred when biomass averages diminished. This lack Scientific Reports \| (2019) 9:15967 \| https://doi.org/10.1038/s41598-019-52333-z 9 www.nature.com/scientificreports/ Figure 6. Diagram relating environmental variables that significantly control the biomass of phytoplankton groups in Las Madres lake in each period. The maximum adjusted variance explaining the biomass of each interval is shown in brackets (see also Table 4). Positions and thickness of each TG band are related with variabilities explained by environmental factors in each period. Zm, upper mixed layer. Figure 6. Diagram relating environmental variables that significantly control the biomass of phytoplankton groups in Las Madres lake in each period. The maximum adjusted variance explaining the biomass of each interval is shown in brackets (see also Table 4). Positions and thickness of each TG band are related with variabilities explained by environmental factors in each period. Zm, upper mixed layer. Figure 7. Diagram depicting the most relevant periodicities experienced by each taxonomical group and the sets of environmental variables likely controlling them over 25 years in Las Madres lake. Figure 6. Diagram relating environmental variables that significantly control the biomass of phytoplankton groups in Las Madres lake in each period. The maximum adjusted variance explaining the biomass of each interval is shown in brackets (see also Table 4). Positions and thickness of each TG band are related with Figure 6. Diagram relating environmental variables that significantly control the biomass of phytoplankton groups in Las Madres lake in each period. The maximum adjusted variance explaining the biomass of each interval is shown in brackets (see also Table 4). Positions and thickness of each TG band are related with variabilities explained by environmental factors in each period. Zm, upper mixed layer. Figure 7. Diagram depicting the most relevant periodicities experienced by each taxonomical group and the sets of environmental variables likely controlling them over 25 years in Las Madres lake. Figure 7. Material and Methods Study site. Las Madres Lake is a seepage, oligo-mesotrophic, gravel-pit lake in an alluvial plain close to Madrid (semi-arid Mediterranean region, Central Spain, 40°18′N3°31′W), with a surface area of 3.4 Ha, and an average and maximum depth of 8 m and 19 m, respectively40. As a new environment created in the seventies, sand and gravel mining was abandoned in 1984. Since then, the lake has been a closed, largely wind-sheltered environment whose water inputs come from the underlying aquifer and rainfall. During winter its water-column temperature has rarely been found below 8 °C. More details on the hydrological, physico-chemical features of this lake and its biota are offered in Supplementary Information. Climate variables, lake sampling and laboratory procedures. Teleconnection variables (NAO, AO, EA and ENSO), compiled by the Climate Prediction Centre of the National Oceanic and Atmospheric Administration, were gathered from http://www.cpc.ncep.noaa.gov/. Local climate variables were recorded at 10 min intervals by a meteorological station located 3 km from Las Madres lake. These variables were air temper- ature (°C) and incoming solar radiation (W/m2); their monthly averages were calculated for this study.h g y g y The lake was sampled monthly from January 1992 to December 2016, between 10:00 and 12:00 (GMT) at a 12 m deep station located in the central area of the lake (see Supplementary Table S1). Physical water-column variables, such as temperature and radiation, were measured with different YSI and LI-COR probes, which were mounted on an SBE-19 Seabird rack from 2006 onwards. Local lake physical variables were: water transparency (Secchi disk depth, m), temperature (°C) and upper mixed layer (Zm, m). Water for chemical analyses and phy- toplankton for this study was collected with a 5 L Niskin PVC bottle at every meter throughout the whole water column. Chemical variables were nitrate, ammonia (mg N/L) and total phosphorus concentrations (mg P/L). They were measured within half an hour after sampling following standard procedures41 up to December 2008 using classical spectrophotometry, and later employing a Seal-3 QuAAtro AQ2 auto-analyzer. Careful checks and intercalibrations were performed between techniques undertaken before and after that date to ensure that chem- ical data are comparable and all series were internally consistent. Silica and DOC data were discarded as variables for further analyses because of their usually high concentration in the lake (see Supplementary Table S1). Most long-term data for Las Madres lake are stored at http://www.sanchezandalvarezlab.es. www.nature.com/scientificreports/ www.nature.com/scientificreports/ phytoplankton, or of any other ecological group. In addition, our results show that the dynamics depends on sev- eral time scales (intra-annual to decadal); thus, it would be acceptable that part of the variance not yet explained could be understood if we had series with higher sampling frequencies (hours, days, weeks). Higher frequencies are related, for example, to metabolic processes or interactions between aquatic ecosystem groups (predation, competition with bacteria). Finally, it is necessary to consider whether the development of eco-evolutionary stud- ies, which link evolutionary change and higher-level ecological variables, such as community composition, could explain the response of species and communities to long-term changes (such as global warming39). p p p g g g g To conclude, the dynamics of long-term phytoplankton biomass appears to be an emergent feature of all the aforementioned processes, also depending upon the time scale involved. Two features of long-term ecological dynamics are demonstrated by our study: 1st) some drivers of long-term lake dynamics can remain hidden, and can only be disentangled by a holistic approach; and 2nd) different levels of community organization, such as total biomass and biomass of taxonomical groups, are helpful to gain insights into the long-term patterns and processes involved. Our methodological approach has provided scientific knowledge on trends, periodicities and controlling factors for different levels of phytoplankton organization and multiscale changes in a warm, temper- ate oligo-mesotrophic lake. Such an approach could be profitably applied to the study of all these features and processes occurring in the long-term phytoplankton dynamics of lakes worldwide. Up to now, these responses had been depicted as (smoothing) trends, either along the oligo-eutrophication axis (or its reversal) or the global warming axis (i.e. stronger thermal effects on lake features over time), but some recent reports have shown that all these effects partly cancelled each other out (see Table 1). It is time to open the gate for more complex views that encompass phenomena, periodicities, interactions and controls previously overlooked in long-term ecology. www.nature.com/scientificreports/ This is the method that must be followed in order to explain the maximum long-term variance of Scientific Reports \| (2019) 9:15967 \| https://doi.org/10.1038/s41598-019-52333-z 10 www.nature.com/scientificreports/ uncorrelated to each other, and could be used as covariates to model phytoplankton responses to environmental variables29. AEM spectral decomposition is well suited for a time series in which periodicities are constant over time, but is unable to characterize non-stationary series, whose main periodicities change over time. Therefore, we also used wavelet analysis which reveals how different scales (periodic components) of the time series emerge and disappear over time28. Controlling factors of phytoplankton dynamics. The target variables (response variables) were: the time series of total phytoplankton biomass (TB hereafter) and the biomass of the main taxonomical groups (TGBs). The five factors that could predict the dynamics of the response variables are five matrices constructed with variables from the set of variables listed in the previous section, selected by applying forward selection with two stopping criteria following43. The variables of regional climate (i.e. teleconnection indices such as NAO, ENSO, etc…) are included in factor [REG], local climate in [LOC], lake physics in [PHY] and lake chemistry in [CHE], as well as the matrix of periodicities [AEM].fi p Multivariate Redundancy Analysis (RDA) was used to establish the effect of the five predictive factors on phy- toplankton dynamics (i.e. TB and TGBs). Variance partitioning reveals how much variance (R2adj) of target biotic variables is explained by pure individual effects of each factor and how much is explained by their interactions24,26. Clearly, this procedure produces much lower values of explained variability than other more commonly employed approaches, such as plain correlation and its derivatives, but this is because temporal components (such as trend and periodicities) and covariation of variables are studied separately. In addition, co-dependency analysis was applied to evaluate which environmental variable was relevant for each phytoplankton periodicity27. Time series partitioning. Since our hypothesis is that there could be different dynamic periods over the whole long-term study, and changes in taxonomical compositions are expected at longer scales, samples were clustered with the UPGMA (unweighted pair group algorithm) applied to the Bray-Curtis similarity matrix calculated on yearly-averaged biomass fractions (%) of the main taxonomic groups. Random bootstrap permutations (999) tested the relevance of each node and a constrained function ordered dates.l A flowchart of statistical methodologies used in this study related to the topics they intend to solve, plus com- plementary information on statistical analyses presented in this section and the software used, are reported in Supplementary Information. Data availability The datasets generated during and/or analysed during the current study are available in the http://www. sanchezandalvarezlab.es repository. Received: 14 December 2018; Accepted: 11 October 2019; Published: xx xx xxxx Received: 14 December 2018; Accepted: 11 October 2019; References 1. IPCC. Climate Change 2014: Impacts, Adaptation, and Vulnerability. Part A: Global and Sectoral Aspects. Contribution of Working Group II to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change (eds Field, C.B. et al.) (Cambridge University Press, Cambridge (UK) and New York, 2014). y g ( ) ) 2. Hanson, P. C., Carpenter, S. R., Armstrong, D. E., Stanley, E. H. & Kratz, T. K. Lake dissolved inorganic carbon and dissolved oxygen changing drivers from days to decades. Ecol. Monogr. 76, 343–363, https://doi.org/10.1890/0012-9615 (2006). 3. Harris, V., Edwards, M. & Olhede, S. C. Multidecadal Atlantic climate variability and its impact on marine pelagic communities. 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Horn, H., Paul, L., Horn, W., Uhlmann, D. & Röske, I., Climate change. Material and Methods For statistical analyses, abiotic factors were water-column averaged. Phytoplankton samples were retrieved at each meter and immediately pooled for the whole water column, thus providing a single sample for each monthly datum. Biomass estimations and their conversion to carbon were calculated following standard methods. Supplementary Information shows more detailed information concern- ing the methods used to study phytoplankton. Phytoplankton taxonomical groups attaining up to 95% of overall biomass in each sample were selected for further analysis; these were Chlorophytes (CHL), Cryptophytes (CRY), Diatoms (DIA) and Dinoflagellates (DINO). Statistical analyses. We use methods implemented to quantify the relative importance of patterns and pro- cesses across time scales of ecosystems24: Asymmetric Eigenvector Maps25, variance partitioning26 and codepend- ence analysis27. Moreover, because ecological communities can change over time and their time series can be non-stationary a fourth method must be taken into account: wavelet analysis28. This tool enables the emergence and/or disappearance of periodicities throughout the whole series to be identified28. Spectral analysis of long term series. Time series were standardized and detrended24. We applied Asymmetric Eigenvectors Maps analyses (AEM) to obtain a set of variables that represents periods with decreasing time scales. For example, these variables represent intra-annual periodicity, annual periodicity, and seasonal and monthly periodicity42. Both the monthly sampling and the 25 years of data enabled us to search for: i) intra-annual perio- dicities longer than a month; ii) annual periodicities; iii) inter-annual periodicities of more than a few years, and iv) decadal periodicities when the cycle is longer than ten years. 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Health Managmn. 14, 33–43, https://doi.org/10.1080/14 634988.2011.550847 (2011). Scientific Reports \| (2019) 9:15967 \| https://doi.org/10.1038/s41598-019-52333-z 13 www.nature.com/scientificreports/ 55. Holopainen, A. L., Lepistö, L., Niinioja, R. & Rämö, A. Spatiotemporal and long-term variation in phytoplankton communities in oligotrophic Lake Pyhäjärvi on the Finnish-Russian border. Hydrobiologia 599, 135–141, https://doi.org/10.1007/s10750-007-9203- 1 (2008). ( ) 56. McGowan, S. et al. Humans and climate as drivers of algal community change in Windermere since 1850. Freshwat. Biol. 57, 260–277, https://doi.org/10.1111/j.1365-2427.2011.02689.x (2012). Acknowledgements A great many people have aided us in undertaking this 25-year period of study in Las Madres lake. The most helpful and lasting ones have been Angel Rubio and José Luis Ayala (CSIC, Madrid, Spain), and Matilde Segura (University of Valencia, Spain). Daniel Sheerin, a native English teacher and editor, improved the language in the manuscript. This study is dedicated to Colin S. Reynolds, the most influential phytoplankton scholar of our time. Competing interestsh g The authors declare no competing interests. Author contributions M.A.C. conceived and designed this research and gathered field and lab data. J.B. carried out data analysis. M.A.C. and C.R. wrote the paper. Additional information dd t o a o at o Supplementary information is available for this paper at https://doi.org/10.1038/s41598-019-52333-z. Supplementary information is available for this paper at https://doi.org/10.1038/s41598-019-52333-z. Supplementary information is available for this paper at https://doi.org/10.1038 Correspondence and requests for materials should be addressed to M.A.C. Correspondence and requests for materials should be addressed to M.A.C. Correspondence and requests for materials should be addressed to M.A.C. 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https://openalex.org/W3175687458	https://www.e3s-conferences.org/10.1051/e3sconf/202127503085/pdf	English	null	Study on Factors Influencing Consumption Potential of Female College Students’	E3S web of conferences	2,021	cc-by	3,268	1 Introduction Nowadays, there are a large number of female college students in school, who have a strong demand for clothing consumption with a unique aesthetic and consumption concept. However, they have not achieved economic independence, and in recent years, the sharing market is booming, and the emerging business model of clothing rental has a huge market potential for female college students. However, in many places such as enterprise development and design, it is still in the initial stage. There are few offline physical stores for female college students' clothing rental, lack of publicity, few varieties of clothing rental, disorderly style and brand, rough workmanship, and it is difficult to meet the needs of the market. How to excavate the potential market is still an important step to develop new market, and starting from the factors influencing the consumption potential of female college students, they can be indirectly open consumer market, to promote domestic consumption patterns change now, launch the consumption concept of Shared economy, make environmental protection save new concept in the field of clothing, let consumer habits costume rental new mode of life ae-mail: luosaisai_2019@qq.com b Correspondence author: Chenggang Li, ccid205@126.com, Professor, Post-doctoral © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Study on Factors Influencing Consumption Potential of Female College Students' 1Beijing Institute of Fashion Technology Business School of BIFT Beijing, China 2Beijing Institute of Fashion Technology Business School of BIFT Beijing, China Abstract. Under the background of supply-side structural reform and the concept of sustainable development, the clothing rental market cannot be ignored, and its market consumption potential is still promising. Enterprises should improve the hygienic quality of rental clothing, broaden the types of clothing supply and increase the economic source of female college students to fully tap the consumption potential of rental clothing market among female college students. In order to open the market, it is essential to conform to the trend of digital economy and sharing economy in the new era, and promote the green and sustainable development of the clothing industry. achievements. On the one hand, scholars pointed out in their reports that the six factors affecting consumption level include income, price, family structure, social security system, consumer psychology and commodity factors[1].in Long Teng's How to Sorrow the Consumption Potential of Residents, he proposed that in recent years, China's consumption has gradually shifted from basic subsistence consumption of food, clothing, housing and transportation to high-quality consumption with cultural entertainment, green environmental protection and individual pursuit [2].In the supply-side structural reform, Wang Yun, a scholar, emphasized that to provide high quality and effective supply, it is necessary to improve the quality of products and services to develop the garment rental market [3].Secondly, the scholar Lixin Guan proposed that income is the premise and basis of consumption. The development of the clothing rental market should increase the income of target customers, improve their consumption ability, and make consumers have money to spend [4]. Research on the Impact of the Improvement of Consumption Environment on the Consumption Potential of Residents points out that under the consumption environment of COVID-19, green and sustainable consumption is becoming a new consumption growth point.[5] https://doi.org/10.1051/e3sconf/202127503085 https://doi.org/10.1051/e3sconf/202127503085 E3S Web of Conferences 275, 03085 (2021) EILCD 2021 3.1 income The income factor will affect the consumer's consumption choice, consumption concept and consumption mode. And the impact of economic income on consumer behavior is different, but also constantly changing, it will inevitably affect consumers' consumption quantity, quality, structure and consumption mode. Through a survey of 125 female college students across the country, I obtained their average monthly income and average monthly clothing consumption. As shown in figure 1 3 Study on the influencing factors of consumption potential This paper selected female college students nationwide as research objects, conducted a questionnaire survey on Fig. 1 Average monthly income and average monthly clothing consumption of female college students Fig. 1 Average monthly income and average monthly clothing consumption of female college students (1) female students' average monthly income is concentrated in 1301-1700 interval, average monthly clothing consumption is mostly between 301-500, and in this interval is the highest proportion of female college students' clothing consumption accounts for income 43%, through these data can be analyzed out, female college students' clothing consumption is proportional to income, the higher the income the clothing consumption also increases. Female college students do not have independent income. In the economic downturn, they belong to the low-income group, so on the supply side, they will pay more attention to the price and quality of clothes, and cheap and fine clothes are their first choice. Lower prices will increase their clothing rental behavior, thus expanding domestic demand and opening the market. In addition to reducing the price of clothing rental, increasing the economic income of female college students is also a feasible way to open the clothing rental market for female college students in terms of income factors. 100 to 300, and the number of people depending on the situation also accounts for 48%, while those within 100 and 300-500 are respectively 26.4% and 14.4%, and those above 500 only account for 7.2%. Therefore, most female college students choose the price as "believe that you get what you pay for" and purchase clothes without planning. Effective interval pricing of rental clothes can promote their consumption demand. 2.1 Research status of consumption potential factors at home and abroad Wang Bei, a scholar, believes that the emergence of clothing rental comes with the development of social economy [6]. However, with the arrival of the "Internet +" era, China began to see the emergence of new online rental business models such as doraemon and beauty Rent, whose users are targeted at women who have just entered the society and have just realized independent sources of Many domestic experts have made a lot of research results on the factors affecting consumption potential and clothing rental. There are also many achievements in these two aspects. I have sorted out and summarized these E3S Web of Conferences 275, 03085 (2021) EILCD 2021 https://doi.org/10.1051/e3sconf/202127503085 them in March 2021, and conducted in-depth interviews with some of the students. In this study, a random sampling survey was adopted, 130 questionnaires were issued and 130 questionnaires were received, among which 125 were valid, with an effective recovery rate of 96%. I will focus on the analysis of the research results from four factors that affect the consumption potential: income, consumption psychology, goods and family structure. income [7] As an indispensable group of college students in clothing consumption, there is also a platform for college students to rent and customize clothing in China - campus clothing C2C platform. [8] Above all, the author thinks that costume rental market has not yet appeared in view of the female college student groups to pursue personality and unique, and is dedicated to daily wearing costumes rental market consumption potential of study. Based on the factors affecting the potential consumption of female college students' clothing rental, I will summarize the existing problems of female college students' consumption in the clothing rental market, so as to provide theoretical basis and direction for the market development of rental clothing among female college students 3.2 Psychological factor of consumption Psychological factors reflect consumers' shopping preference motivation, which directly affects consumers' shopping decisions. Psychological factors include but are not limited to purchase motivation, product awareness, purchase attitude. I find out the demand of female college students for rental services mainly through investigating the demand of motivation in the psychological factors of female college students' consumption. The seven demand indicators affecting female college students' rental clothing are shown in Figure 2 (2) The survey data on pricing shows that 48% of people can accept the price range of a single product from 2 2 E3S Web of Conferences 275, 03085 (2021) EILCD 2021 https://doi.org/10.1051/e3sconf/202127503085 Figure 2 Indicators of demand affecting female college students' rental clothing Figure 2 Indicators of demand affecting female college students' rental clothing According to the data, hygiene is the primary consideration of almost all clothing rental consumers. The proportion of people who care about sharing clothes with others is as high as 70.4%, which shows that they are not willing to accept the clothes of others out of psychological mind, which may also include the fear of infectious diseases.58.4 percent said it was less convenient to wear their own clothes, and 66.67% were worried about the source of their clothes. The consideration of price is between income and whether renting clothes is a better option than buying clothes. attach great importance to the service of selling. The packaging and label of clothing can attract the attention of consumers and thus attract the customer flow. In the survey, 70.9% people said they had been attracted by interesting labels, and 43% said they had chosen to buy again because of the excellent packaging of clothing. Female college students pursue the trend, love new things, and have their own understanding of the product logo. Therefore, enterprises to develop a product in line with their own LOGO, to provide good packaging, is another commodity factor to seize consumers. ommodity factor to seize consumers. Fig. 3 Indicators of factors affecting commodities Secondly there are 71.2% of the female college students said don't wear clothes to buy after, 11.2% said they often have to buy the clothes of regret, the reason for more impulsive consumption, through the depth interview, female college students think through clothing leasing can provide a "consider yourself exactly like the dress? Do the clothes fit me?" To reduce their impulse consumption. 3.4 Family structure factor The family is the most important product consumer in the society, and the family members constitute the most influential primary reference group. But the family economic income restricts the individual purchase ability, purchase way, consumption structure and living habit and so on. This survey mainly examines the influence of the consumption concept of family structure on the clothing rental of female college students. 3.2 Psychological factor of consumption Hygiene and safety problems are inevitable and must be solved in the process of clothing rental. Female college students can not directly know the degree of disinfection and cleanliness of clothes when renting clothes, but will indirectly judge the cleanliness of clothes through flagship stores and so on. For those who are psychologically concerned, businesses can reassure them by spreading the idea that recycling is an environmentally friendly, green economy practice. For the problem of clothing source, merchants can use the digital recycling - cleaning - leasing - recycling cycle to accelerate the creation of new consumer behavior and consumer product demand. Fig. 3 Indicators of factors affecting commodities 4 the current problems needs of female college students, choose to develop the market for the daily wear of female college students, provide personalized and stylized clothing for daily use, and formulate the price range of rental clothing that meets the economic conditions of female college students. (1) The price range of rental clothing is small, and female college students have less disposable income. The choice of clothing rental platform mostly stays on the light luxury and some designer brands, and there are not many clothes for female college students to choose in terms of price. On the other hand, the living expenses of female college students in this survey are basically from their parents, and they have not achieved economic independence. Therefore, their consumption does not match their economic ability, and the acceptable clothing rental price is limited, which can provide a platform for them to satisfy their purchase desire with the least amount of money. Provide high quality rental clothing, establish and perfect cleaning and disinfection procedures, to ensure the hygiene and safety of clothing, dispel consumer concerns. Enterprises should win consumers' trust and good reputation with high quality products. On the other hand, it is necessary to establish a sound collection, sorting, storage, cleaning procedures of second-hand clothes, through alliance with traditional dry cleaners, to achieve the international standards of health of leased clothes. Provision of short-term clothing to simplify the rental process. In this survey, more than half of the respondents hope that the clothing rental cycle is measured in days. Female college students pursue the freshness and novelty of clothing, so their clothing rental period is relatively short. Enterprises should consider the time range of clothing rental accepted by female college students, increase the links of collection, cleaning, disinfection and recycling of rental clothing, and strive to improve its convenience, so as to ensure hygiene and simplify the process, so that rental clothing can be available at any time. (2) Family consumption concept has a profound impact, and consumer habits are difficult to change. Consumers have a deep-rooted traditional concept of family, so their acceptance of renting clothes is not high. Although most people in the questionnaire are willing to try the service of renting clothes, not many people actually use it, which can be reflected from the number of users and the activity of the APPs of the major clothing renting platforms. 3.3 commodities g Among the female college students surveyed, 99.2% of their personal income is given by their parents, and their consumption values and habits are greatly influenced by family economic conditions.3.6% said their families were relatively well-off, they said they had no need to rent clothes and would not consider renting clothes;52.44% of them said that their family conditions were ordinary and their parents were economical, so renting clothes could be considered.29.16% of people from middle class families said that their parents think that wearing rental clothes will make others misunderstand their family's economic situation, so renting clothes is not considered; Only 14.85% of the respondents said their family was poor and their parents were frugal in spending money. Renting clothes could reduce expenses and reduce family burden. [9]. In terms of the frequency of female college students buying clothes, 44.8% of them buy clothes every month, and 42.4% buy clothes in different seasons. Businesses can launch high-quality clothes rental in certain seasons, which can not only improve the utilization rate of clothes, but also achieve the purpose of using the value of goods. On the other hand, the pre-sale, in-sale and after-sale service of clothing is an important indicator to attract consumers to buy the goods again. The first reflection of customers on the company is pre-sale service. In-sale service is an important part of product marketing, and after-sale service is an important push for products to enter the market. Seventy-three percent of female college students said they attach great importance to the follow- up service of clothing rental, and 21.4 percent said they 3 3 https://doi.org/10.1051/e3sconf/202127503085 E3S Web of Conferences 275, 03085 (2021) EILCD 2021 E3S Web of Conferences 275, 03085 (2021) 6 Conclusion: The COVID-19 epidemic has balanced consumers' demand for clothing in many ways. At the same time, consumers have realized that the clothing industry is also an important force supporting the environmental cause, and expanding the clothing rental industry is conducive to environmental protection. However, the development of female college students' clothing rental in China is still backward, the construction of clothing rental platform is not perfect, the service level needs to be improved, and the hygiene and safety of clothing is not guaranteed. But on the whole, female college students' clothing rental consumption conforms to the trend of the development of The Times, which is a very environmentally friendly and sustainable way for individuals and the whole society. 4 the current problems On the other hand, trust issues, cleanliness and hygiene issues in the clothing flow also prevent people from trying something new. The general public clothing, others wear always have psychological barriers, do not conform to people's consumption habits and cultural habits, especially clothes such as more personal things. Seize the emerging consumption point of environmentalism and break the traditional concept of renting consumption. The epidemic has enhanced people's awareness of environmental protection. Sustainable clothing is not only a business, but also a parallel clothing consumption between consumerism and environmentalism, showing the development characteristics from temporary rental to return to nature. Environmental clothing has become an inevitable trend. According to young consumers' sensitivity to sustainable development, businesses can promote the concept of environmental protection and convey the trend of clothing rental. Sustainable consumption will get closer and closer to us, and choosing to rent clothing is also an important step to the cause of environmental protection. (3) Inadequate hygiene and quality of leased clothing. It often happens that there is a big gap between the hygiene of leased clothes and the introduction of the platform. The washing standard of the leasing platform is not standardized and there is a lack of strong supervision measures, which makes people worry about whether the cleaning and disinfection are thorough. Rental clothing promotes green and sustainable development, so second- hand clothes are the main source of clothing for rental service. However, there is no fixed industry standard for purchasing second-hand clothes, and the quality of clothes is uneven. (4) The utilization rate of clothing is low and the convenience is insufficient. In the survey, 58.4% of people said that renting is not as convenient as wearing their own clothes. The cycle between the platform sending out clothes and the renter returning the clothes is too long, so that most people have a resistance to renting clothes. How to shorten the middle link of clothing is a major problem that cannot be ignored in the development of female college students' clothing rental market. 5 Suggestions: Clarifying the needs of target customers, we will provide a range of low-price clothes rental that meets the needs of female college students. For college students, most of the market provides short-term theatrical performance costumes, competitions and formal clothes for professional interviews. The market of female college students' daily wear clothing rental has not been deeply developed. Businesses can clarify the clothing rental 4 E3S Web of Conferences 275, 03085 (2021) EILCD 2021 https://doi.org/10.1051/e3sconf/202127503085 E3S Web of Conferences 275, 03085 (2021) ACKNOWLEDGMENT Fund Project: In 2021, Beijing Institute of Fashion Technology Undergraduate Innovation and Entrepreneurship Training Program Project - Feasibility Study of Clothing Exchange and Leasing Mode under the Shared Economy Environment. Beijing Social Science Foundation - Capital Garment Culture and Garment Industry Research Base Project - Research on Intelligent Business Form Innovation of Beijing Fashion Culture Industry (19JDYJA007) Reference 1. Wu J Y. Influencing factors and promoting mechanism of cultural consumption [J]. Shandong Social Sciences,2019(06):94-99. 2. Long Teng. How to stimulate residents' consumption potential [J]. People's Forum,2018(34):88-89. 3. Wang Yun. How to further promote the release of consumption potential under the new situation [J]. People's Forum Academic Frontiers,2019(02):28-39 4. Guan Lixin. Consumption becomes the main driving force of national economic cycle [N]. International Business Daily,2019-03-22(002). 5. Long Shaobo, Zhang Rui.The Impact of Consumption Environment Improvement on Residents' Consumption Potential -- Based on the Perspective of Current Residual Consumption Potential [J]. Forum on Statistics and Information, 201,36(01):79-89 6. Wang Bei, Zhou Yang, Si Xingyu, Zhang Shangyan, He Lingyue. Research on the development status and development strategy of apparel rental platform in China [J]. Light Textile Industry & Technology,2020,49(05):72-74. 7. Li Ling, Ye Jing, Zhang Beibei, Zhou Jiani.Research on the development status and development strategy of Internet clothing rental market in China [J]. Light Textile Industry & Technology,2017,46(05):65-67. 8. Guo Yanjie, Hou Dongyu.Research on the clothing consumption behavior of female college students today [J]. Peony,2019(03):124-126. 9. Chen H. Habit formation, consumption heterogeneity and consumption potential release of urban residents in China [D].Shandong University,2019. 5 5
https://openalex.org/W4391750126	https://naradidik.ppj.unp.ac.id/index.php/nara/article/download/79/77	Indonesian	null	Kendala Dalam Mencapai Standar Penilaian KKM Mata Pelajaran Sosiologi Pada Masa Pandemi di SMA Negeri 4 Kota Tanjungpinang	Naradidik	2,023	cc-by	4,634	ERROR: type should be string, got "https://naradidik.ppj.unp.ac.id/index.php/nar\na \nNaradidik: Journal of Education & Pedagogy \nVolume 2 Nomor 3 2023, pp 218-224 \nISSN: 2827-864X (Online) – 2827-9670 (Print) \nDOI: https://doi.org/10.24036/nara.v2i3.80 \n \nReceived: September 1, 2022; Revised: September 29, 2023; Accepted: September 30, 2023 https://naradidik.ppj.unp.ac.id/index.php/nar\na \nNaradidik: Journal of Education & Pedagogy \nVolume 2 Nomor 3 2023, pp 218-224 \nISSN: 2827-864X (Online) – 2827-9670 (Print) \nDOI: https://doi.org/10.24036/nara.v2i3.80 \n \nReceived: September 1, 2022; Revised: September 29, 2023; Accepted: September 30, 2023 http\na \nNaradidik: Journal of Education & Pedagogy \nVolume 2 Nomor 3 2023, pp 218-224 \nISSN: 2827-864X (Online) – 2827-9670 (Print) \nDOI: https://doi.org/10.24036/nara.v2i3.80 \n \nReceived: September 1, 2022; Revised: September 29, 2023; Accepted: September 30, 2023 https://naradidik.ppj.unp.ac.id/index.php/nar https://naradidik.ppj.unp.ac.id/index.php/nar Keywords: KKM; Sociology learning; The covid 19 pandemic. Keywords: KKM; Sociology learning; The covid 19 pandemic. How to Cite: Nusa, E.P. & Sylvia, I. (2023). Kendala Dalam Mencapai Standar Penilaian KKM \nMata Pelajaran Sosiologi Pada Masa Pandemi di SMA Negeri 4 Kota Tanjungpinang. Naradidik: \nJournal of Education & Pedagogy, 2(3), 218-224. This is an open access article distributed under the Creative Commons 4.0 Attribution License, which permits unrestricted use, distribution, \nand reproduction in any medium, provided the original work is properly cited. ©2023 by author. Abstrak Penelitian ini bertujuan untuk mendeskripsikan kendala-kendala yang dihadapi dalam pembelajaran \nsosiologi agar siswa dapat mencapai KKM pada masa pandemi di kelas XI SMA N 4 \nTanjungpinang. Penelitian ini menggunakan pendekatan kualitatif dengan jenis penelitian studi \nkasus. Teknik pengumpulan data dilakukan melalui observasi, wawancara, dan studi dokumen. Informan dalam penelitian ini berjumlah 25 orang yang ditentukan berdasarkan kriteria tertentu. Peneliti melakukan triangulasi sumber dan triangulasi teknik. Analisis data dilakukan dengan \nmenggunakan model analisis interaktif Miles dan Huberman. Hasil penelitian mengungkapkan \nbahwa kendala pencapaian KKM dalam pembelajaran sosiologi yang diperoleh pada hasil \npenelitian ini yaitu kurangnya akses terhadap sumber belajar dan tugas karena jaringan internet yang \ntidak stabil, rendahnya motivasi siswa di masa pandemi, siswa tidak memahami materinya. Kata kunci : KKM; Pembelajaran sosiologi; Pandemi Covid-19. Kendala Dalam Mencapai Standar Penilaian KKM \nMata Pelajaran Sosiologi Pada Masa Pandemi \ndi SMA Negeri 4 Kota Tanjungpinang Eka Patria Nusa1, Ike Sylvia2* \n1,2Universitas Negeri Padang \n*Corresponding author, e-mail: ikesylvia@fis.unp.ac.id. Abstract This study aims to describe the obstacles encountered in studying sociology so that students can \nachieve the KKM during the pandemic in class XI SMA N 4 Tanjungpinang. This study uses a \nqualitative approach with a case study type of research. Data collection techniques were carried out \nthrough observation, interviews, and document studies. There were 25 informants in this study who \nwere determined based on certain criteria. Researchers conducted source triangulation and \ntechnique triangulation. Data analysis was performed using the Miles and Huberman interactive \nanalysis model. The results of the study revealed that the obstacles in achieving KKM in sociology \nlearning were those obtained in the results of this study, namely the lack of access to learning \nresources and assignments due to unstable internet networks, students had low motivation during a \npandemic, students did not understand the material. Pendahuluan Dalam pelaksanaan pembelajaran pada kurikulum 2013, siswa dikatakan berhasil jika telah \nmenguasai kompetensi tertentu yang telah ditetapkan dalam kurikulum. Indikator bahwa siswa telah \nmenguasai kurikulum yakni kemampuan hasil belajar yang diukur telah mencapai kriteria ketuntasan \nminimal (KKM) yang telah ditetapkan, atau melampaui KKM. Dengan KKM ini, siswa yang telah berhasil \ndapat melanjutkan belajar untuk menguasai kompetensi selanjutnya, dan yang belum menguasai dapat \nmemperdalam materi yang belum dikuasai melalui melalui program remedial. Hal ini menunjukkan \npentingnya KKM dalam menentukan keberlanjutan belajar siswa. Selama ini, kriteria ketuntasan minimal \natau dalam istilah pengukuran sering disebut dengan batas lulus (cut of score) yang ditentukan menggunakan \nkebijakan. 218 Nusa, E.P & Sylvia, I. 219 Setiap sekolah menentukan KKM yang berbeda dengan sekolah lain. Hal ini disebabkan karena, \nsekolah yang menentukan KKM yang berdasarkan musyawarah guru berdasarkan intake, kompleksitas, dan \ndaya dukung yang dimiliki sekolah. Sebagai akibatnya KKM setiap mata pelajaran di suatu daerah \nkabupaten/kota memiliki KKM akan berbeda-beda, sehingga sulit dibandingkan kualitas pembelajarannya. Pembelajaran pada saat pendemi covid 19 membawa dampak proses pembelajaran tidak dapat \nberjalan dengan maksimal, sehingga proses penyampaian materi maupun pengalaman belajar siswa tidak \ndapat dioptimalkan. Kondisi ini mengakibatkan banyak siswa yang tidak mampu mencapai KKM yang telah \nditetapkan. KKM yang telah ditetapkan untuk pembelajaran sosiologi pada kelas XI di SMA N 4 Tanjung \nPinang adalah 77. Berdasarkan hasil pengamatan yang telah dilakukan pada pembelajaran sosiologi kelas XI SMA N 4 \nTanjung Pinang, terdapat kekurangan dalam proses pembelajaran dan siswa kesulitan beradaptasi dengan \ncepat mengikuti proses pembelajaran pada masa pandemi. Dalam prakteknya, pembelajaran terkadang \nberjalan tidak efektif, hal ini disebabkan karena proses pembelajaran pada pada masa pandemi, lebih \nmenekankan kemandirian siswa untuk mengakses sumber belajar sendiri. Banyak siswa yang tidak disiplin \nmembaca sumber belajar dan hanya asal dalam mengerjakan tugas, sehingga materi yang seharusnya \ndipelajari tidak dapat dikuasai dengan baik, hasil belajar yang diperoleh tidak sesuai dengan tujuan yang \ndiharapkan. Hal ini terbukti dari nilai ulangan formatif dan sumatif pada pembelajaran sosiologi yang hanya \nsebagian orang siswa dapat telah mencapai standar ketuntasan yang ditetapkan di SMA N 4 Tanjung Pinang \nyaitu sebesar 77. Rendahnya hasil belajar siswa ini, terbukti dari hasil nilai ulangan semester yaitu rata hasil \nbelajar siswa berkisar antara 60 sampai 68 dengan presentase ketuntasan masih dibawah 50%. Pendahuluan j\np\ng\np\nDalam belajar seseorang dipengaruhi oleh banyak faktor, terdapat beberapa faktor kemungkinan yang \ndapat dijadikan bahan kajian diantaranya adalah faktor internal dan faktor eksternal yang mempengaruhi \nsiswa dalam mencapai hasil belajar yang diharapkan. Penelitian Akila Afra Santika, yang berjudul Strategi Guru Dalam Pencapaian Kriteria Ketuntasan \nMinimal (KKM) di Masa Pandemi Covid-19 Pada Mata Pelajaran Geografi Kelas XI IPS di MAN 1 Kerinci \nDan SMAN 6 Kerinci menemukan beberapa permasalahan yang dapat mempengaruhi pencapaian KKM, \npermasalahan tersebut disebabkan oleh beberapa faktor, baik faktor internal maupun faktor eksternal. Faktor \ninternal adalah strategi dan media pembelajaran yang digunakan guru belum menarik dan belum dapat \nmemvisualkan materi pembelajaran, terdapat sikap sebagian siswa yang dapat mempengaruhi teman \nsekelasnya sehingga berdampak pada tidak efektifnya pembelajaran. Sementara faktor eksternal adalah letak \nsekolah menentukan perilaku belajar siswa (Santika, 2022). Sementara itu menurut Uno menjelaskan bahwa \nkualitas pembelajaran dikelaslah yang menentukan kualitas pendidikan. Tingkat kualitas pembelajaran \ndapat diperlihatkan oleh tingginya keterlibatan siswa dalam pembelajaran (Uno, 2008). Salah satu cara yang \ndapat membantu guru dalam upaya meningkatkan kualitas pembelajaran adalah implementasi standar \nproses dalam pembuatan rencana pelaksanan pembelajaran. Pembuatan rencana pelaksanaan pembelajaran \nperlu menitikberatkan pada aktivitas siswa, guru sebagai fasilitator membantu siswa jika mendapatkan \nkesulitan, membimbing diskusi agar mampu membuat kesimpulan yang benar (Yamin, 2009). Selain \npelaksanaan proses pembelajaran menentukan hasil belajar siswa, proses penilaian juga ikut serta \nmenentukan luaran hasil belajar siswa, Parwati dalam tulisannya Strategi Guru Dalam Melaksanakan \nPenilaian Pembelajaran Pada Masa Pandemi Covid-19 di Kelas Tinggi Sekolah Dasar melaksanakan \ninstrument penilaian berperan penting terhadap hasil belajar yang dicapai siswa, hasil belajar akan baik jika \ndilaksanakan guru melaksanakan strategi : (1) melakukan pembelajaran tatap muka secara langsung. (2) \nPenilaian berdasarkan tugas harian siswa. (3) penilaian berdasarkan pengalaman siswa. (4) Mengacu pada \nnilai rapor semester sebelumnya (Parwati, 2021). p\ny (\n,\n)\nBerdasarkan uraian di atas, peneliti memandang penting untuk meneliti masalah kendala yang \ndihadapi dalam mencapai standar penilaian KKM mata pelajaran Sosiologi pada masa pandemi di SMA \nNegeri 4 Kota Tanjung Pinang. Naradidik: Journal of Education & Pedagogy Hasil dan Pembahasan Standar penilaian pendidikan adalah suatu kriteria mengenai lingkup, tujuan, manfaat, prinsip, \nmekanisme, prosedur, dan instrumen dalam melakukan penilaian hasil belajar peserta didik dalam satuan \npendidikan dasar dan pendidikan menengah (Hurrahma & Sylvia, 2022). Guru dalam pelaksanaan \npembelajaran berperan sebagai pengajar dan melakukan penilaian melalui proses pengumpulan dan \npengolahan informasi untuk mengukur pencapaian pemahaman materi serta hasil belajar peserta didik \nselama mengikuti pembelajaran yang diberikan guru. Dalam menentukan tingkat kelulusan harus menggunakan istilah kriteria dalam penilaian, kriteria \nmerupakan suatu tingkatan atau batasan minimal untuk mengukur penilaian proses atau hasil belajar dengan \nmelalui hal tersebut dapat diketahui ketuntasan belajarnya. Kriteria atau tingkatan terendah dalam \nmenentukan ketuntasan dalam belajar disebut Kriteria Ketuntasan Minimal (KKM) (Mardapi, 2012). Guru dalam hal ini hanya berperan dalam memfasilitasi siswa untuk mengembangkan minat belajar \ndan dapat memahami materi yang telah diberikan dengan baik, selanjutnya peran guru sebagai mediator \nyaitu sebagai perantara kepada siswa untuk mendapatkan informasi atau materi ajar yang diberikan dalam \nbentuk power point maupun word dalam pelaksanaan pembelajaran secara daring berlangsung sehinga dapat \nmembantu siswa memahami materi belajar sehingga tugas dan ujian yang diberikan dapat dikerjakan dengan \nbaik sehingga nilai KKM yang ditentukan sebelumnya dapat tercapai, dan peran guru sebagai motivator \nadalah untuk membangkitkan minat dan semangat siswa dalam mengikuti pembelajaran yang diberikan oleh \nguru walaupun pelaksanaan pembelajaran tidak dilakukan secara tatap muka. Dalam penerapan standar penilaian dimulai dengan penetapan oleh guru mata pelajaran terlebih \ndahulu untuk menentukan KKM yang harus dicapai siswa atau peserta didik, dalam mata pelajaran sosiologi \nyang ditetapkan oleh guru dan sekolah SMA Negeri 4 Tanjungpinang pada mata pelajaran sosiologi dengan \nnilai KKM yang harus dicapai oleh siswa yaitu 77, standar penilaian yang dilakukan oleh guru disesuaikan \ndengan kegiatan pembelajaran di masa pandemi. Pelaksaanaan pembelajaran yang dalam hari biasanya dilaksanakan secara tatap muka harus dirubah \nmenjadi pembelajaran secara daring atau online, hal ini berdasarkan anjuran pemerintah melalui kementrian \npendidikan untuk mengurangi penyebaran kasus positif di masa pandemi, karena hal itu pemerintah \nmemberlakukan social distancing terhadap segala sektor termasuk dalam pelaksanaan pembelajaran di \nsekolah. Siswa dihimbau agar tetap mengikuti pembelajaran dari guru di rumah rumah masing – masing \ndengan menggunakan perangkat seperti laptop, HP dan perangkat lainnya yang dapat terkoneksi internet \nagar kegiatan pembelajaran secara online yang diberikan guru kepada siswa dapat terlaksana walaupun tidak \nsecara tatap muka. Metode Penelitian Penelitian ini dalam pelaksanaannya menggunakan metode penelitian kualitatif dengan \nmenggunakan pendekatan penelitian ini ialah studi kasus. Studi kasus yang peneliti maksud ialah studi kasus \nsebagai strategi penelitian yang fokus terhadap peristiwa yang terjadi saat ini (Sukardi, 2008). Informan \ndalam penelitian ini ditentukan dengan menggunakan teknik purposive sampling, yaitu merupakan teknik \ndalam memilih informan. Dalam penelitian ini informan berjumlah 25 orang yang terdiri dari wakil \nkurikulum, guru mata pelajaran sosiologi, siswa dan orang tuanya yang dipilih sesuai kriteria. Teknik \npengumpulan data menggunakan metode observasi, wawancara dan dokumen. Dalam mendapatkan \nkeabsahan data yang terpercaya peneliti menggunakan teknik triangulasi sumber terkait kendala dalam \nmencapai standar penilaian KKM siswa pada pembelajaran sosiologi di kelas XI SMA N 4 Tanjung Pinang. Naradidik: Journal of Education & Pedagogy 220 ISSN: 2827-864X (Online) Dalam melakukan analisis, data yang bersumber dari informan, dianalisis dengan cara mendeskripsikan dan \nmengkategorikan serta mencari persamaan dan perbedaan pendapat antar informan. Data yang telah \ndianalisis, selanjutnya menghasilkan suatu kesimpulan dari hasil wawancara. Selain itu peneliti juga \nmelakukan triangulasi teknik dilakukan dengan cara mengecek data kepada sumber yang sama dengan \nteknik yang berbeda (Yusuf, 2016). Triangulasi teknik dalam penelitian ini dilakukan dengan mengecek data \nhasil penelitian yang diperoleh dari observasi, wawancara, dan dokumentasi. Data yang diperoleh dari \nobservasi informan, dibandingkan dengan data yang diperoleh dari hasil wawancara terhadap kepala \nsekolah, guru dan siswa, orang tua siswa terkait kendala kendala yang dihadapi dalam mencapai standar \npenilaian KKM mata pelajaran Sosiologi pada masa pandemi di SMA Negeri 4 Kota Tanjung Pinang. Data yang diperoleh dalam penelitian ini kemudian dianalisis, kemudian dikelompokkan-\nkelompokkan, diinterpretasikan kemudian untuk memperoleh kesimpulan yang dilakukan dengan prinsip \ninduksi yang mengedepankan penggambaran yang berawal dari dan hal yang spesifik. Kesimpulan yang \ndiperoleh dalam penelitian ini berupa deskripsi atau gambaran mengenai keadaan kasus yang diteliti melalui \ninforman yang berjumalh 25 orang. Deskripsi atau gambaran akhir yang diperoleh dari penelitian ini yaitu \nmengenai kendala kendala yang dihadapi dalam mencapai standar penilaian KKM mata pelajaran Sosiologi \npada masa pandemi di SMA Negeri 4 Kota Tanjung Pinang. Hasil dan Pembahasan Faktor kendala yang dihadapi oleh guru serta siswa dalam penerapan pembelajaran online \nyang mempengaruhi terhadap pemahaman terhadap pencapain KKM sehingga tidak dapat tercapai tujuan \ndari standar penilaian yang telah ditetapkan sebelum pembelajaran dimulai. Terdapat beberapa kendala \ndalam mencapai KKM sesuai standar penilaian yang ditetapkan. Kendala ini menyebabkan guru dan siswa \nterganggu dalam melanjutkan kegiatan proses pembelajaran. Adapun kendala tersebut dijelaskan pada \nbagian di bawah ini: Minim Akses Sumber Belajar dan Penugasan karena Jaringan Internet yang Tidak Stabil \nKetika pandemi terjadi sekolah mengeluarkan kebijakan agar siswa dapat belajar dari rumah secara \nonline. Dalam pelaksanaan pembelajaran secara online siswa menggunakan media komunikasi yang harus Akses Sumber Belajar dan Penugasan karena Jaringan Internet yang Tidak Stabil (Kendala Dalam Mencapai …) Nusa, E.P & Sylvia, I. 221 terkoneksi dengan internet seperti handphone android atau perangakat elektronik lainnya yang memiliki \nkamera sebagai media penghubung antara guru dan siswa. Oleh karena itu dalam proses pembelajaran yang \ntelah dilaksanakan selama masa pandemi, siswa diharapkan memili perangkat tersebut dan akses yang lancar \nuntuk dapat mengakses sumber belajar dan penugasan yang diberikan. p\ng\nj\np\ng\ny\ng\nSalah satu kendala yang ditemui adalah sulitnya mendapatkan sinyal internet yang lancar (Nafrin & \nHudaidah, 2021). Hal ini diungkapkan oleh siswa AD siswa kelas XI IPS 5 dalam wawancara berikut ini. p\ng\nj\np\ng\ny\ng\nSalah satu kendala yang ditemui adalah sulitnya mendapatkan sinyal internet yang lancar (Nafrin &\nHudaidah, 2021). Hal ini diungkapkan oleh siswa AD siswa kelas XI IPS 5 dalam wawancara berikut ini. “…saya sebagai siswa ingin sekali selalu mengikuti kegiatan belajar yang dilaksanakan oleh \nguru secara online tetapi terkadang saya tidak dapat diikuti karena koneksi jaringan yang sering \nterganggu terjadi di daerah tempat tinggal, saya sehingga saat mencoba memasuki aplikasi \npembelajaran melalui link yang diberikan oleh guru tidak dapat dilakukan karena sinyal \ninternet berulangkali hilang” (Wawancara dengan AD, 27 Juli 2022). Hal ini diperkuat dari wawancara dengan siswa lainnya yang bernama PI kelas XI IPS 4 yang\nberpendapat: “…sebagai siswa diwajibkan untuk dapat terus mengikuti pembelajaran materi yang diberikan \noleh guru ketika mengajar, tetapi karena adanya pandemi ini sangat berdampak terhadap saya \nkarena ketika mengikuti pembelajaran kondisi sinyal internet sering menghilang hal ini terjadi \nkarena di wilayah rumah saya tidak didukung oleh kualitas jaringan internet yang baik. Oleh \nsebab itu saya sering terputus atau tertinggal dalam memahami materi yang diajarkan guru \ndalam media pembelajaran berbasis jaringan internet” (Wawancara dengan PI, 26 Juli 2022). Hasil dan Pembahasan Kendala jaringan internet menyebabkan siswa untuk tidak dapat mengikuti kegiatan secara maksimal \nsehingga banyak siswa yang tidak memahami materi yang diajarkan atau disampaikan oleh guru sehingga \ntujuan dalam pelaksanaan pembelajaran tidak dapat dicapai. Kesulitan siswa dalam mengakses internet ini \nmembuat mereka tidak membaca materi dan membuat penugasan. Hal ini diungkapkan oleh guru sosiologi \nkelas XI, yaitu ibu GS: “Saya memang memberikan materi sebagai sumber belajar kepada siswa yang harus mereka \nakses dari rumahnya. Kadangkala siswa beralasan tidak dapat membaca materi dan tugas \nkarena jaringan yang tidak lancar atau kuota internet yang tidak ada” (Wawancara dengan Ibu \nGS, 30 Juli 2022). Peneliti juga menanyakan kepada orang tua siswa terhadap pernyataan siswa tersebut. Berikut pernyataan orangtua PI. “Anak saya sudah memeiliki handphone android yang dapat dipergunakannya ketika belajar \npada masa covid ini, tetapi karena keadaan rumah kami yang berada di dekat pantai, sering \nkali jaringan hilang ketika anak saya harus menyelesaikan tugas sekolahnya” (Wawancara \ndengan orangtua PI, 28 Juli 2022). “Anak saya sudah memeiliki handphone android yang dapat dipergunakannya ketika belajar \npada masa covid ini, tetapi karena keadaan rumah kami yang berada di dekat pantai, sering \nkali jaringan hilang ketika anak saya harus menyelesaikan tugas sekolahnya” (Wawancara \ndengan orangtua PI, 28 Juli 2022). Prosedur penilaian hasil belajar siswa yang telah ditetapkan yaitu KKM 77, diharapkan dapat dicapai \noleh siswa, namun karena ada siswa yang tidak membaca materi dan mengumpulkan tugasnya di waktu \nyang ditentukan, sehingga proses penilaian yang dilakukan hanya dapat diberikan kepada siswa yang \nmampu melngkapi penugasan. Kendala jaringa internet yang tidak stabil menjadi kendala pertama yang \nmembuat siswa tidak dapat belajar dengan baik dan nilai mereka tidak mencapai KKM. Artinya kendala jaringan internet yang tidak stabil dijadikan alasan oleh siswa, sementara diharapkan \nsiswa mampu beradaptasi untuk mengantisipasi kendala jaringan ini (Putria et al., 2020). Siswa seharusnya \nmampu beradaptasi dengan perubahan sistem belajar yang mengalami transisi dari pembelajaran tatap muka \nmenjadi pembelajaran daring karena pandemi. Naradidik: Journal of Education & Pedagogy Tidak Mengerjakan Tugas Dalam proses memberikan penilaian, tugas merupakan salah satu faktor penting untuk melihat\npenguasaan materi siswa. Ketika masa pandemi guru memberikan tugas secara online dan siswa\nmengantarkan tugas tersebut langsung ke sekolah sesuai waktu yang telah ditentukan, namun masih ada\nsiswa yang tidak mengerjakan. Hal ini diketahui melalui wawancara dengan Ibu GS: “…Ketika pelaksanaan pembelajaran melalui aplikasi tugas atau LKPD yang saya berikan \ndalam bentuk word yamg harus dikerjakan siswa dengan pengetikan dan diprint setelahnya \nbaru diantar disekolah, dalam proses pengumpulan tugas masih banyak siswa yang tidak \nmengerjakan atau mengantarkan tugas yang telah disuruh’’ (Wawancara dengan Ibu GS, \ntanggal 26 Juli 2022). “…Ketika pelaksanaan pembelajaran melalui aplikasi tugas atau LKPD yang saya berikan \ndalam bentuk word yamg harus dikerjakan siswa dengan pengetikan dan diprint setelahnya \nbaru diantar disekolah, dalam proses pengumpulan tugas masih banyak siswa yang tidak \nmengerjakan atau mengantarkan tugas yang telah disuruh’’ (Wawancara dengan Ibu GS, \ntanggal 26 Juli 2022). Hal ini didukung dari wawancara dengan YD, siswa kelas XI IPS 5 yang mengungkapkan: “…saya sebagai siswa sebenarnya ingin mengerjakan tugas yang telah dikirimkan guru melalui \nmedia pembelajaran daring seperti zoom dan google classroom akan tetapi tugas tersebut tidak \ndapat saya akses karena terkendala oleh sinyal jaringan internet di tempat saya tinggal sehingga \nkarena hal tersebut saya tidak dapat mendownload file tugas tersebut disebabkan oleh itu saya \njadi tidak dapat mengumpulkan tugas yang telah diberikan oleh guru” (Wawancara dengan \nYD, tanggal 20 Juli 2022). “…saya sebagai siswa sebenarnya ingin mengerjakan tugas yang telah dikirimkan guru melalui \nmedia pembelajaran daring seperti zoom dan google classroom akan tetapi tugas tersebut tidak \ndapat saya akses karena terkendala oleh sinyal jaringan internet di tempat saya tinggal sehingga \nkarena hal tersebut saya tidak dapat mendownload file tugas tersebut disebabkan oleh itu saya \njadi tidak dapat mengumpulkan tugas yang telah diberikan oleh guru” (Wawancara dengan \nYD, tanggal 20 Juli 2022). Hasil wawancara dengan Ibu GS sebagai guru sosiologi dan siswa, dalam hal ini guru selalu \nmemberikan tugas dan memberikan waktu dengan batas waktu yang telah ditentukan, tetapi masih banyak \nsiswa yang tidak mengantarkan tugas yang telah diberikan, padahal tugas merupakan salah satu aspek \npenting dalam standar penilaian untuk mencapai nilai KKM yang telah ditetapkan sebelumnya. Berdasarkan \npenilaian dari tugas siswa, guru dapat mengetahui pemahaman siswa terhadap materi yang diajarkan, dan \nmemberikan penguatan untuk materi selanjutnya (Mardianto & Prayitno, 2020). Namun banyak siswa yang \ntidak mengerjakan tugas karena kemampuan adaptasinya dengan perubahan rendah. Hal ini didukung dari dari wawancara dengan Ibu GS sebagai guru sosiologi: Hal ini didukung dari dari wawancara dengan Ibu GS sebagai guru sosiologi: “…ketika saya memulai pengajaran pada awalnya masih banyak siswa yang memperhatikan \nmateri pelajaran yang saya terangkan atau ajarkan dengan menghidupkan kamera videonya, \nakan tetapi menjelang pertengahan hingga akhir proses pembelajaran banyak siswa yang \nmematikan kameranya sehingga saya tidak dapat mengetahui siswa yang masih memiliki \nmotivasi mengikuti pembelajaran” (Wawancara dengan Ibu GS, 19 Juli 2022). “…ketika saya memulai pengajaran pada awalnya masih banyak siswa yang memperhatikan \nmateri pelajaran yang saya terangkan atau ajarkan dengan menghidupkan kamera videonya, \nakan tetapi menjelang pertengahan hingga akhir proses pembelajaran banyak siswa yang \nmematikan kameranya sehingga saya tidak dapat mengetahui siswa yang masih memiliki \nmotivasi mengikuti pembelajaran” (Wawancara dengan Ibu GS, 19 Juli 2022). Berdasarkan hasil wawancara dan observasi dapat diketahui bahwa faktor yang menyebabkan \nkurangnya motivasi siswa mengikuti pembelajaran secara daring siswa kurang memiliki kesadaran belajar \nmemalui zoom itu adalah sama fungsinya dengan pemberian materi di kelas, namun berbeda medianya, \nyaitu tatap muka dan melalui media digital. Guru sudah berusaha mengantisipasi agar siswa memahami \nmateri dengan menjelaskannya secara online melalui zoom meeting, namun siswa belum memiliki \nkesadaran diri sendiri dalam mengikuti pembelajaran (Marbun, 2021). Tidak Mengerjakan Tugas Proses adaptasi siswa \ndengan lingkungan pembelajaran yang mengalami transisi dari pembelajaran tatap muka menjadi \npembelajaran daring menyebabkan pemberian materi, tugas, atau PR dalam bentuk file, dan sering dianggap \nberbeda dengan tugas yang diberikan secara langsung di sekolah. Guru sebagai pemberi tugas sudah \nmengingatkan bahwa penting bagi siswa untuk mengerjakan tugasnya karena hal itu berkaitan dengan \npenilaian dan ketuntasan siswa. Memiliki Motivasi yang Rendah Dalam pelaksanaan pembelajaran daring, kendala lain yang dirasakan oleh guru yaitu siswa masih \nkurang dalam hal motivasi untuk mengikuti pembelajaran yang dilaksanakan oleh guru (Sari et al., 2021). Motivasi mengikuti pembelajaran selama pandemi ADS, siswa kelas XI IPS 5 dapat dilihat pada wawancara \nberikut. “…saya sendiri sebagai siswa merasakan perbedaan antara proses pembelajaran secara tatap \nmuka dengan secara online seperti dalam memperhatikan materi lebih fokus secara langsung \ntetapi jika menggunakan media seperti handphone saya tidak dapat fokus karena sambil \nmemperhatikan pembelajaran saya dapat memainkan media sosial sehingga menggangu fokus \natau motivasi saya dalam mengikuti materi yang diberikan” (Wawancara dengan ADS, tanggal \n18 Juli 2022). “…saya sendiri sebagai siswa merasakan perbedaan antara proses pembelajaran secara tatap \nmuka dengan secara online seperti dalam memperhatikan materi lebih fokus secara langsung \ntetapi jika menggunakan media seperti handphone saya tidak dapat fokus karena sambil \nmemperhatikan pembelajaran saya dapat memainkan media sosial sehingga menggangu fokus \natau motivasi saya dalam mengikuti materi yang diberikan” (Wawancara dengan ADS, tanggal \n18 Juli 2022). Naradidik: Journal of Education & Pedagogy ISSN: 2827-864X (Online) 222 Remedial Sulit dilakukan di Masa Pandemi Remedial (perbaikan) merupakan suatu bentuk pembelajaran yang memperbaiki berdasarkan hasil \npengajaran yang telah dilakukan, dengan tujuan menjadi lebih baik. Secara umum tujuan pembelajaran \nremedial sama dengan tujuan pembelajaran biasa, yaitu membantu siswa untuk mencapai tujuan \npembelajaran atau kompetensi yang telah ditetapkan berdasarkan kurikulum yang berlaku (Prasetiyo, 2021). Secara khusus tujuan pembelajaran remedial adalah untuk membantu siswa yang belum menguasai materi \npelajaran tertentu melalui kegiatan pembelajaran tambahan untuk mendapatkan hasil yang maksimal dan \nguru memperbaiki cara mengajarnya.” Remedial diartikan sebagai pengobatan, penawaran serta \npenyembuhan yang berkaitan dengan perbaikan. Pada pengertian yang lebih luas pengajaran remedial yaitu \npengajaran yang bersifat kuratif (penyembuhan) atau korektif (perbaikan). Pengajaran remedial merupakan \nbentuk khusus pengajaran yang bertujuan untuk menyembuhkan masalah atau kesulitan belajar bagi siswa \n(Prayitno, 2008). Dalam program remedial siswa dituntut untuk dapat memperbaiki nilainya dengan \nmengikuti ujian ulang. Sehingga dengan begitu nilai siswa dapat sesuai KKM yang sudah ditetapkan \n(Muksin, 2020). Guru juga diharapkan dapat mengetahui kesulitan yg dialami siswa. Dengan begitu peserta \ndidik dapat memahami atau mengetahui materi apa yang belum dipahaminya, sehingga siswa dapat \nmeningkatkan kemampuannya. Remedial yang awalnya dilakukan di sekolah secara bertatap muka dengan \nguru, karena situasi pandemi yang tidak memungkinkan untuk dilakukan di sekolah sehingga berganti \ndengan dilakukan di rumah masing-masing atau bisa disebut remidial online. Pelaksanaan program remedial online pada mata pelajaran sosiologi di SMA N 4 Tanjungpinang \ndilakukannya menngunakan metode memberikan soal kembali kepada siswa yang belum tuntas KKM. Soal \nyang diberikan kepada siswa yaitu berupa vidio pembelajaran, siswa dapat mengaksesnya dengan internet \natau youtube kemudian siswa dapat menyimpulkannya menggunakan bahasa sendiri. Metode lain yang \ndigunakan Ibu GS adalah dengan memberikan tugas kelompok antar siswa atau teman sebayanya sehingga \ndapat saling membantu kesulitan siswa yang lainnya, tak jarang juga Ibu GS memberikan soal atau tugas \nkemudian siswa menggumpulkannya melalui google classroom atau google form. Namun program remedial \nyang dilakukan Ibu GS juga tidak memberikan hasil yang signifikan. Hal ini disebabkan kendala yang \ndialami oleh siswa seperti jaringan internet dan motivasi siswa yang rendah selama masa pandemi. Kemudian BPT menjelaskan pendapat yang serupa Kemudian BPT menjelaskan pendapat yang serupa Kemudian BPT menjelaskan pendapat yang serupa “Soal itu sebenarnya mudah, namun jawabannya yang sulit karena saya tidak membaca, tidak \nbelajar sungguh-sungguh, jadi saya tidak tahu mau menjawab apa, membuat tugas ssepeerti \napa, nanti tinggal saya cari tugas teman dan menyalinnya” (Wawancara dengan BPY, 2 \nAgustus 2022). Pernyataan siswa ini peneliti konfirmasi kepada guru sosiologi Pernyataan siswa ini peneliti konfirmasi kepada guru sosiologi “Banyak sekali tugas siswa yang sama selama pandemi ini, mereka menjadi terbiasa copy paste \ntugas teman dan menyalin dari internet” (Wawancara dengan Ibu GS, tanggal 6 Agustus 2022). Kenyataan ini juga peneliti tanyakan kepada Wakil Kurikulum, yang menyebutkan sebagai \nberikut: “Ada beberapa siswa yang selama pandemi ini dapat mengerjakan tugas dengan baik, tepat \nwaktu dan benar, namun juga sebagian siswa selalu telat mengantarkan tugas atau tugas ini \nmereka ambil dari internet, sehingga ketika ujian mereka sulit untuk menguasai materi yang \ntelah dipelajari”. (Wawancara dengan Waka Kurikulum, tanggal 6 Agustus 2022). Beberapa pernyataan di atas memperlihatkan bahwa siswa tidak memahami soal dan \npenugasan yang diberikan karena tidak membaca sumber belajar yang tepat dan sering melakukan \ncopy paste untuk melengkapi tagihan penugasan. Naradidik: Journal of Education & Pedagogy Siswa Tidak Memahami Materi Kesesuaian materi dalam soal ulangan harian yang diujikan kepada siswa penting untuk diperhatikan \n(Nurshella & Sylvia, 2022). Berdasarkan wawancara dengan RDG, AHM, dan BPT, mereka mengatakan \nmateri dalam soal ulangan sudah sesuai dengan yang telah diajarkan. “Saya mengerti maksud soal yang diberikan oleh Ibu, tapi karena saya tidak membaca \nmaterinya sehingga saya tidak mengerti harus menjawab apa, jadinya saya mengarang dalam \nmenjawab soal” (Wawancara dengan RDG, 28 Juli 2022). AHM juga mengatakan sebagai berikut: AHM juga mengatakan sebagai berikut: “Bingung mau menjawab apa soalnya, karena ketika ibu menerangkan di zoom saya matikan \nkamera dan mengerkajan kegiatan lain, lalu saya tidak mendownload materi apalagi \nmembacanya, saya tidak suka belajar online” (Wawancara dengan AHM, 1 Agustus 2022). (Kendala Dalam Mencapai …) Nusa, E.P & Sylvia, I. 223 Daftar Pustaka Hurrahma, M., & Sylvia, I. (2022). Efektivitas E-LKPD Berbasis Liveworksheet dalam Meningkatkan Hasil \nBelajar Sosiologi Peserta Didik di Kelas XI IPS SMA N 5 Padang. Jurnal Sikola: Jurnal Kajian \nPendidikan Dan Pembelajaran, 4(1), 14–22. https://doi.org/10.24036/sikola.v4i1.193 Marbun, T. (2021). Peningkatan Partisipasi dan Hasil Belajar Siswa dalam Pembelajaran Sejarah di Masa \nPandemi. Khazanah Pendidikan, 15(1), 22. https://doi.org/10.30595/jkp.v15i1.9595 ( )\np\ng\nj p\nMardapi, D. (2012). Pengukuran Penilaian & Evaluasi Pendidikan. Bandung: Nuha Medika. Mardianto, M. F. F., & Prayitno, P. (2020). Peningkatan Hasil Evaluasi Pembelajaran Daring saat Pandemi \nCovid-19 Berdasarkan Media Powerpoint Interaktif. MUST: Journal of Mathematics Education, Science \nand Technology, 5(2), 171. https://doi.org/10.30651/must.v5i2.6119 Muksin, M. (2020). Meningkatkan kemampuan Guru Membuat Program Remedial Melalui Pembinaan \nGuru dengan Diskusi Kelompok di SDN Telok Jolo 3 Semester II Tahun Ajaran 2018 / 2019. E- \nJurnal Mitra Pendidikan, 4(8), 554–568. https://doi.org/10.52160/e-jmp.v4i8.770 Muri, A. Y. (2016). Metode Kuantitatif, Kualitatif & Penelitian Gabungan. Kencana. Nafrin, I. A., & Hudaidah, H. (2021). Perkembangan Pendidikan Indonesia di Masa Pandemi Covid-19. Edukatif : Jurnal Ilmu Pendidikan, 3(2), 456–462. https://doi.org/10.31004/edukatif.v3i2.324 Muri, A. Y. (2016). Metode Kuantitatif, Kualitatif & Penelitian Gabungan. Kencana. Nafrin, I. A., & Hudaidah, H. (2021). Perkembangan Pendidikan Indonesia di Masa Pandemi Covid-19. Edukatif : Jurnal Ilmu Pendidikan 3(2) 456 462 https://doi org/10 31004/edukatif v3i2 324 Nushella, N., & Sylvia, I. (2022). Evaluasi Pembelajaran Sosiologi secara Daring pada Masa Pandemi \nCovid-19 di SMA Negeri 1 Pangkalan Kuras. Naradidik: Journal of Education and Pedagogy, 1(2), 127–\n136. https://doi.org/10.24036/nara.v1i2.35 p\ng\nParwati, S. (2021). Strategi Guru dalam Melaksanakan Penilaian Pembelajaran pada Masa Pandemi Covid-\n19 \ndi \nKelas \nTinggi \nSekolah \nDasar. Proseding \nSeminar \nNasional \nPascasarjana. https://proceeding.unnes.ac.id/index.php/snpasca/article/view/920 p\np\ng\np p\np\nPrasetiyo, J. (2021). Evaluasi dan Remediasi Belajar. Jakarta: Trans Info Media Prayitno, P. (2008). Dasar-dasar Bimbingan Konseling. Jakarta: Rineka Cipta. Putria, H., Maula, L. H., & Uswatun, D. A. (2020). Analisis Proses Pembelajaran dalam Jaringan \n(DARING) Masa Pandemi Covid- 19 Pada Guru Sekolah Dasar. Jurnal Basicedu, 4(4), 861–870. https://doi.org/10.31004/basicedu.v4i4.460 Santika, A. A. (2022). Strategi Guru Dalam Pencapaian Kriteria Ketuntasan Minimal (KKM) di Masa Pandemi \nCovid-19 Pada Mata Pelajaran Geografi Kelas XI IPS di MAN 1 Kerinci Dan SMAN 6 Kerinci. Universitas \nNegeri Padang. Sari, R. P., Tusyantari, N. B., & Suswandari, M. (2021). Dampak Pembelajaran Daring Bagi Siswa Sekolah \nDasar \nSelama \nCovid \n19. Prima \nMagistra: \nJurnal \nIlmiah \nKependidikan, \n2(1), \n9–15. https://doi.org/10.37478/jpm.v2i1.732 Sukardi, S. (2008). Metodelogi Penelitian Pendidikan. Jakarta: Bumi Aksara. Uno, H. B. (2008). Orientasi Baru dalam Psikologi Pembelajaran. Kesimpulan Pembelajaran dimasa pandemi memiliki banyak kendala, salah satunya adalah tingkat pencapaian \nsiswa terhadap KKM rendah. Terdapat beberapa kendala yang diperoleh dalam hasil penelitian ini yaitu \nMinim Akses Sumber Belajar dan Penugasan karena Jaringan Internet yang Tidak Stabil, Memiliki Motivasi \nyang Rendah, Siswa Tidak Memahami Materi, Remedial Sulit dilakukan di Masa Pandemi. Naradidik: Journal of Education & Pedagogy Naradidik: Journal of Education & Pedagogy ISSN: 2827-864X (Online) 224 Daftar Pustaka (Kendala Dalam Mencapai …) Daftar Pustaka Jakarta: Bumi Aksara. Yamin, M. dan M. (2009). Manajemen Pembelajaran Kelas Strategi Meningkatkan Mutu Pembelajaran. Jakarta: \nGaung Persada Press. (Kendala Dalam Mencapai …)"
https://openalex.org/W4296405186	https://zenodo.org/records/6411633/files/922.pdf	English	null	Acid Catalysed lsomerisation of 3-Epi-Moretenyl Acetate	Zenodo (CERN European Organization for Nuclear Research)	1,977	cc-by	796	J, INDIAN CHBM. SOC., VOL. UV, SliPTIIMIII!R 1977 J, INDIAN CHBM. SOC., VOL. UV, SliPTIIMIII!R 1977 2-Methyl-benzimlnazole (Ill): Hot PPA ( 12ml; 20g) was added to a reaction mass of the diamine (5. 4g.) and the Jl·ketoester (7.0ml) containing a drop of concentrated hydrochloric acid. Turbid layer, separated during reaction, disappeared on stirring. The reaction mass was stirred at 120-130" for 21 hours. It was treated with ice and just neutralised ; the product (3.2g.) was crystallised from hot water; m. p. 175"; its mixed m.p. with the authentic specimen of 2-methyl·benzimi- nazole was undepressed. hydrolysis (Motbanolic-KOH) gave. the corresponding alcohol-hop-17{21)-en•3ot-olM (Ill), m. p. 185·86", [<JD+47• which on oxidation with Cr03-Py complex yielded hopenone-1 (identified by m. m. p and JR com,?arison with an authentic sample preplTed from moretenone1 ). The alcohol (III) was also prepared from hopenono·l by Meerwein·Pondortr reduction' and was found to be identical (m. m. p. and IR). hydrolysis (Motbanolic-KOH) gave. the corresponding alcohol-hop-17{21)-en•3ot-olM (Ill), m. p. 185·86", [<JD+47• which on oxidation with Cr03-Py complex yielded hopenone-1 (identified by m. m. p and JR com,?arison with an authentic sample preplTed from moretenone1 ). The alcohol (III) was also prepared from hopenono·l by Meerwein·Pondortr reduction' and was found to be identical (m. m. p. and IR). ;~, ·OAc It, R• 'H .. ·lllf Ill, A• 'H Hopenone-I,R • 0 l 4th IV 1 R• <':" Y 1 R• <!" ttop•none,.ll , R • 0 Mo;~, ·OAc It, R• 'H .. ·lllf Ill, A• 'H Hopenone-I,R • 0 l 4th IV 1 R• <':" Y 1 R• <!" ttop•none,.ll , R • 0 #~N-CCH, c/N I I "cH D )c.cH,COCHi V'-N-e/ • "N H " H 0 (I) A/N.., ' I I )C·CH• ~/'N H (DI) c/N D )c.cH,COCHi "N H #~N-CCH, I I "cH V'-N-e/ • H " 0 (I) A/N.., ' I I )C·CH• ~/'N H (DI) A.cetoacet·o-aminoani/ide( IJI) : o-Phenylenedia- mine(5.4g) was added to a solution of ortbophosphoric acid (12 ml) containing Jl·ketoester (6.8 ml), externally cooled with ice ; phosphorous pentoxide(20g) was added to this solution in small lots ; the mixture was then heated on steam-bath for i hour. The reaction mass was treated with ice and neutralised with ammonium and IOdium hydroxide giving yellow product. Yellow needles from aqueous ethanol (2.68g; m.p. 105" dec). Refereneet I. W. A. SIXtON, J. Chem. Soc., 1942, 303. 2. 0. HI!I:SBI!RO and P. KOLLIR, Ber, 1896, 29, tsoo. 3. G. W. WHELAI'{D, Advanced Organic Chemistry, John Wtley and Sons, p 408. 3. R. S. MoRRELL and A. E. BELLARS. /. Chem. &e. 1904, 85, 34S and 1520. 4. F. UHLIG and H. R. SNYDBR ; Advance, in Organi~: CbcmistiY·Methods and Results; 1960, I, 35. Jntorscience publishers, IDC. N.:w York. J, INDIAN CHBM. SOC., VOL. UV, SliPTIIMIII!R 1977 Analysis: C10Hu01N 51 requires N, 14.58% found N, 14.50% Analysis: C10Hu01N 51 requires N, 14.58% found N, 14.50% It coupled with diazotlsed amines giving deee red' colour1 its benzene solutic.n on boiling gave benziminazolo-2- acetone (m.p. 1 18°). Hop-17(21 )·en-3ot-yl acetate on further isomerllatloa under strong acid condition of Fazakerloy and co-workers• (AcOH-C6H 8·H2SO.) yielded an acetate, hop-13(18)-en·lo(.-yl acetate, (IV), m. p. 179·81", [ado ·32 1°, which on hydrolysis (Methanolic-KOH) furnished tho alcohol, bop•l3(18)·en·3ot·ol, (V), m.p. 219·21",[-t]o· 14"8°, Oxidation {Cr08 ·P~) of tho alcoboi(V) furnished a ketone, m. p. 148·50°, [ot}D 49", identical with hope: none•II (m m. p). Meerwein-Pondortr reduction' of Hoponone·ll yielded an alcohol which was found to' be identical with the alcohol (V) and its acetate with (IV) (m.m.p) H N. KHA'JTGIR" and B. P. PRADH~~ 1. H N. ICRASTOIR, B. P. P&ADHAN, A. M. DUPIIILD 1111d L. J. DuRHAM, Chem. Comm, 1967, 1217. lkpartmont of Cbem;stry, University of North Beopl, Dt. Darjeeting lkpartmont of Cbem;stry, University of North Beopl, Dt. Darjeeting 2. H. FAZAXERLBY, T. G. HAuALL, and E. R. H.JoNIS, J, Chem. Soc., 1959, 1877. 3, H. R. ARTHUR, S. W. TAM and V. AuosuslNG, Afllt,J. Chem.,l960, 506. Mollllscrlpt r-cteved 1 September 19'16, acupted 2/une 1971 3-BPI·MORBTENYL acetate1 (I) on acid isomerisatiun under mild condition of FazJkerley and co-workerss (AcOH-H,SO.,) furaisb~ bt>p·l7(2l)·on-3>t-yl acetate {ll), m. P· 11:1·21", (<lD+ll.l0 • Toe aaetate (rJ) on Mollllscrlpt r-cteved 1 September 19'16, acupted 2/une 1971 3-BPI·MORBTENYL acetate1 (I) on acid isomerisatiun under mild condition of FazJkerley and co-workerss (AcOH-H,SO.,) furaisb~ bt>p·l7(2l)·on-3>t-yl acetate {ll), m. P· 11:1·21", (<lD+ll.l0 • Toe aaetate (rJ) on 3-BPI·MORBTENYL acetate1 (I) on acid isomerisatiun under mild condition of FazJkerley and co-workerss (AcOH-H,SO.,) furaisb~ bt>p·l7(2l)·on-3>t-yl acetate {ll), m. P· 11:1·21", (<lD+ll.l0 • Toe aaetate (rJ) on 4. WAI·HAAN HUI and MAN•MOON Ll, Phytochlmlllt1• 1976, 15,427. 4. WAI·HAAN HUI and MAN•MOON Ll, Phytochlmlllt1• 1976, 15,427. s. A. c. PATON, F. s. SI'RINO and a. StsVBNsoM, J. Chfm., Soo.,1958,;2640. s. A. c. PATON, F. s. SI'RINO and a. StsVBNsoM, J. Chfm., Soo.,1958,;2640. 922
https://openalex.org/W4388899928	https://aestheticinvestigations.eu/article/download/15315/18571	English	null	Introduction to the Special Issue on Philosophy and Architecture	Aesthetic investigations	2,023	cc-by	1,345	Aesthetic Investigations Vol 6, No 1 (2023), 1-4 Special Issue – Architecture beyond the building Special Issue – Architecture beyond the building Architecture beyond the building buildings would be a very narrow conception of architecture. To get anything like its full potential out of the philosophy of architecture we need a broader conception of philosophy and a broader conception of architecture and its aims. Without prejudice for or against any of its recent methodologies, whether analytic, phenomenological, or otherwise, philosophy can be understood as reflection at the deepest or most abstract level – whichever metaphor might be preferred – on the fundamental concepts and principles of any and all of the areas of human thought, practice, and experience, from the nature of speech and communication in general, to formal reasoning as in mathematics and logic, empirical investigation in natural scientific and historical inquiry, systems of moral obligations and virtues, political organisation including ju- dicial and penal systems, the creation, experience, and comprehension of art, and more, such as the nature of religious belief and practice for those to whom that is important. Architecture, too, can and should be broadly understood, to include the design and construction of a wide range of the built environment, including not just particular building-types such as single- and multifamily dwellings, office buildings, shopping malls, and distribution facilities, public facilities from courthouses to stadiums, but also many works of civil engineering such as highways and bridges, landscape architecture, city planning, and more – in real life, as we know, most works of architecture are the products of col- laboration among many disciplines, not only exterior and interior designers but also structural engineers, lighting and HVAC engineers, landscape ar- chitects, zoning and environmental experts, and more; although the licensed architect often gets all the glory, in the case of almost all large-scale building projects today, he or she ought to be thought of as more like the lead author on a scientific paper that involved the efforts of many specialists. The word ‘architect’, although grammatically singular, should now typically be taken as the name for a group, like ‘Bourbaki’ was really the name for a group of mathematicians. Architecture beyond the building Affiliation Brown University Author Paul Guyer Brown University Abstract: Introduction to the Special Issue: Architecture beyond the building This issue focuses on the philosophy of architecture. What does that mean? Obviously, it depends on what is meant by ‘philosophy’ and by ‘architecture’. Both terms have to be understood broadly to make the topic interesting and important, and both are taken broadly by the essays included here. If phi- losophy is understood as above all the theory of meaning and reference, then philosophy of architecture might end up being confined to Nelson Goodman’s question, ‘how do buildings mean?’ or, ‘what kind of symbol-system is archi- tecture?’ If architecture is understood, as John Ruskin suggested, not as the whole art of building but as the art of decorating buildings, then philosophy of architecture might end up as a narrow theory of the beauty or beautifica- tion of buildings, ‘aesthetics’ in the popular sense rather than in the sense of a philosophical discipline. But to limit philosophy to a theory of meaning, therefore to limit aesthetics to a theory of meaning in art, but also to limit aesthetics to a theory of beauty, would be an excessively narrow approach to philosophy. And to limit the role of architecture to the beautification of Architecture beyond the building Architecture beyond the building Further, unlike other disciplines that often leave their his- tory only in memory, perhaps aided by written records, architecture produces physical objects that typically endure for a long time, intact or in many dif- ferent stages of decline, and even the remains of earlier works in all of these categories, thus ancient and recent ruins, can also count as architecture and as subject to the reflections of philosophy of architecture. If both philosophy and architecture are so broadly understood, the philos- ophy of architecture will necessarily become a broad inquiry into the nature of the goals of architecture, both the practical and aesthetic dimensions of the experience of architecture, and the interaction between these or the con- straints that one of these might exercise upon another. The philosophy of architecture can and should be the effort to understand in all the ways avail- able to us architecture in all its complexity – although again at a high level of 2 Paul Guyer Paul Guyer abstraction, so that it is not identical with either the history of architecture or architectural theory, understood as the discourse or rhetoric advocating a particular style or a way of building, even though it might draw upon these. The essays included in this special issue all both depend upon and il- lustrate this broad approach to the philosophy of architecture. My own es- say focuses on the treatment of architecture by the now relatively neglected mid-twentieth century American philosopher Susanne Langer, who cast her philosophy as a whole as a theory of symbols, yet she understood symbol- ism more broadly than Nelson Goodman did, and her interest was by no means restricted to anything like a semantics of architecture. Rather, her concepts of ‘virtual space’ and ‘ethnic domains’ led her to explore the ways in which aesthetic, functional and cultural considerations interact in the case of architecture. Elizabeth Scarbrough’s essay ‘Contemporary Urban Ruins’ discusses a variety of recent theories of the aesthetics of ruins, and brings them to bear in a case study of both aesthetic and political issues concerning a derelict sports facility in Miami. Saul Fisher’s paper explores the interplay between architecture and politics. Fisher argues against excessively ideologi- cal claims that all architecture is political, since much architecture is public, often subject to political considerations, it is hardly all political in intent or meaning. Architecture beyond the building By contrast, Rossen Ventzislavov’s paper is focused more on purely aesthetic issues, yet he gains insight about architecture in a surprising comparison with performance art. Likewise focused on the aesthetic status of architecture, Jörg Gleiter’s contribution proposes the idea of a “surplus of form” as a way of conceptualising the aesthetic aspect of architecture. All of these papers illustrate the merits of an open-minded, pluralistic approach to the values at play in architecture. Finally, two pieces on our theme are included in the ‘Arts and Artists’ section of this issue. Sue Spaid’s piece on the ‘Bilbao effect’ of Frank Gehry’s Guggenheim Museum Bilbao explores the impact of architecture on tourism, but then, through a common comparison of Gehry’s buildings in Bilbao and elsewhere to bouquets or corsages of flowers, turns to a discussion of the envi- ronmental impact of the cut-flower industry and flower shows. She concludes with a discussion of the model for environmentally-conscious development planning afford by Floriade Expo 2022 in Almere, NL. Spaid’s piece shows how useful broad conceptions of both architecture and philosophy can be. The other piece in ‘Arts and Artists’ is an interview that I conducted with my cousin Laure van Heijenoort, who after a childhood started in Greenwich Village, New York, made her career as a labour and health care lawyer in Albuquerque, New Mexico. Including this interview might seem like sheer nepotism, but Laure had the extraordinary experience in between New York and New Mexico of living and going to school in buildings by two great twentieth-century architects, Mies van der Rohe and Eliel Saarinen. Her thoughtful reminiscences of those years in her life provide a sample of the 3 3 Architecture beyond the building kind of experiential data that broad-minded philosophers of architecture can use to their advantage. paul_guyer@brown.edu COPYRIGHT: © 2023 Paul Guyer © 2023 Paul Guyer This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See https://creativecommons.org/licenses/by/4.0/ p // g/ / y/ / Aesthetic Investigations is a peer-reviewed open access journal published by the Dutch Association of Aesthetics. 4
https://openalex.org/W4232576605	https://zenodo.org/records/3393511/files/14933__1_224660_LE_334131.pdf	Dutch	null	INHAALAFSCHRIJV1NGEN	MAB	1,970	cc-by	3,210	INHAALAFSCHRIJV1NGEN door A. F. A. Cheizoo Met grote belangstelling heb ik kennis genomen van het artikel van Prof. van der Schroeff in het nummer van M.A.B. van j.1. februari. Mij b l t lli d i h fd k kt d h t f it d t P f j Mijn belangstelling werd in hoofdzaak gewekt door het feit, dat Prof. van der Schroeff bij de behandeling van het probleem van de z.g. „inhaalafschrij- vingen”, het accent ten dele verlegt naar het terrein van de financiering, hetgeen naar mijn mening de enig juiste benadering van het probleem is. R d l b di d ij d ij h t bl d i h l g j g gj g p Reeds lang bevredigde mij de wijze waarop het probleem van de inhaal- afschrijvingen in de literatuur wordt behandeld in het geheel niet, daar deze behandeling mij irreëel en onlogisch voorkomt en de beredenering daarvan gewrongen terwijl daarentegen verleggen van het probleem naar het terrein van de financiering, waar het m.i. ook thuishoort, een volkomen reële en logische oplossing biedt. D b ik h d P f d S h ff i ij ik l d l i h g p g Daarom betreur ik het, dat Prof. van der Schroeff in zijn artikel de logische consequentie niet tot het uiterste heeft doorgetrokken. Hij zegt in M.A.B. blz. 59: „Was b.v. in de gegeven casus de verhouding ƒ 400.000,— eigen vermogen en ƒ 1.600.000.— vreemd vermogen geweest, dan zou een verlies van de inhaalafschrijvingen zich voor het eerst aan het einde van het 9e jaar voordoen. Indien de installatie met een lening, derhalve geheel met vreemd vermogen zou zijn gefinancierd, dan komt de noodzaak van inhaalafschrij vingen te vervallen. Hieruit blijkt dat deze afschrijvingen samenhangen met de financiering uit eigen vermogen. De vermogensstructuur en de verhou dingen bij de financiering zijn voor het betreffend vraagstuk bepalend.” Dit is volkomen juist maar dan volgt hieruit, dat de oorzaak van het z.g. „verlies” niet bij de niet meer aanwezige werkeenheden te zoeken is maar bij „de vermogensstructuur en de verhoudingen bij de financiering”. Bedrijfseconomisch kan men de duurzame produktiemiddelen beschouwen als voorraden „werkeenheden”. Prof. van der Schroeff zegt hiervan in Kosten en kostprijs: „Economisch kan het duurzame produktiemiddel worden ge zien als een voorraad prestaties, die het produktiemiddel gedurende zijn levensduur aan de voortbrenging bewijst” en Prof. INHAALAFSCHRIJV1NGEN D d k h d b d t 1 200 000 t k D h d i Herwaardering van een aanwezige voorraad is volkomen logisch maar her waardering van een niet meer aanwezige voorraad werkeenheden van 800.000 stuks lijkt mij toch wel in strijd met de logica. Hoe kan men een voorraad die er niet is, herwaarderen? D d k h d b d 1 200 000 k D h d i , De voorraad werkeenheden bedraagt 1.200.000 stuks. De herwaardering kan derhalve nooit meer bedragen dan ƒ 120.000,—. N d h d i t ƒ 200 000 d t l l t d d Na de herwaardering met ƒ 200.000,— redeneert men als volgt verder: de afschrijvingen blijken nu in vorige jaren te klein te zijn geweest en er moet een „inhaalafschrijving” worden toegepast van 10% van ƒ 800.000,— = ƒ 80.000,—. Deze inhaalafschrijving moet dan ten laste van de winst gebracht worden van het boekjaar waarin blijkt dat deze inhaalafschrijving nodig is. O d t l t ik ill k d t d f h ij i i i j j j g g Op de eerste plaats zou ik willen opmerken, dat de afschrijvingen in vorige jaren, indien zij gebaseerd waren op de toen geldende vervangingswaarde, volkomen juist zijn geweest; er is niet te weinig afgeschreven. O d t d l t i f h ij i i t b t d j j g g g Op de tweede plaats is een afschrijving op een niet meer bestaande voor raad een moeilijk verteerbare zaak en bovendien komt het mij voor, dat afschrijven op een niet meer bestaande voorraad eveneens in strijd met de logica is. Inhaalafschrijvingen zijn, als men de naam niet misverstaat, afschrijvingen. Nu is afschrijven het tot uitdrukking brengen van een waardevermindering. Uit het betoog van Prof. van der Schroeff blijkt echter, dat de inhaalafschrij vingen in grootte afhankelijk zijn van de wijze van financiering van de desbe treffende duurzame produktiemiddelen. H d d i d i d d k i idd l fh k p Hoe nu de waardevermindering van duurzame produktiemiddelen afhanke lijk kan zijn van de wijze van financiering is een ondoorgrondelijk probleem en nog vreemder wordt het als waardevermindering van niet meer bestaande werkeenheden afhankelijk is van de wijze van financiering. INHAALAFSCHRIJV1NGEN Limperg zegt hiervan: „Het werktuig is dus in die zin een voorraad werkeenheden, zoals men ook een voorraad grondstoffen economisch als een voorraad werkeenheden kan zien.” G l id lijk d i d l d ijd d d k h d Geleidelijk worden in de loop van de tijd de voorraden werkeenheden verbruikt; de in de duurzame produktiemiddelen opgehoopte voorraad wordt dus steeds kleiner en het in deze produktiemiddelen geïnvesteerde vermogen komt geleidelijk weer in geld ter beschikking. T ill t ti ij hi l d b t ik it t k h g j g g Ter illustratie van mijn hiernavolgend betoog ga ik uit van een sterk gesche matiseerd getallenvoorbeeld. Bij een door Prof. van der Schroeff gegeven casus is sprake van een installatie met een aanschaffingswaarde van ƒ 2.000.000,—, welke 10 jaar gebruikt wordt en daarna geen restwaarde meer heeft. De jaarlijkse afschrijving bedraagt ƒ 200.000,—. m a b blz. 70 Deze casus kan worden uitgebreid met het gegeven, dat de in de installatie opgehoopte voorraad 2.000.000 werkeenheden bedraagt, welke voorraad evenredig met de tijd wordt verbruikt. N i j i d f h ƒ800 000 ij 800 000 k g j Na vier jaar is dan afgeschreven ƒ 800.000,—, zijn er 800.000 werkeen heden verbruikt en bedraagt de aanwezige voorraad nog 1.200.000 werkeen heden. Nu blijkt - en ik volg hier de voorbeelden van Prof. van der Schroeff-, dat aan het eind van het vierde jaar de vervangingswaarde van de installatie met 10% is gestegen. V l d b d li k i d b ld P f d g g Volgens de bestaande literatuur en ook in de voorbeelden van Prof. van der Schroeff heeft nu op de eerste plaats een herwaardering plaats van de instal laties met 10% van ƒ 2.000.000,— of ƒ 200.000,—. Boekhoudkundig kan deze correctie aangegeven worden met de journaalpost: I ll i ƒ200 000 ƒ 200.000,— Aan Herwaardering Installaties Herwaardering van een aanwezige voorraad is volkomen logisch maar her waardering van een niet meer aanwezige voorraad werkeenheden van 800.000 stuks lijkt mij toch wel in strijd met de logica. Hoe kan men een voorraad die er niet is, herwaarderen? INHAALAFSCHRIJV1NGEN V lk d h ijf ik h t P f d S h ff bl 55 t j j g Volkomen onderschrijf ik, hetgeen Prof. van der Schroeff op blz. 55 zegt, n.1. „dat er ten opzichte van de verbruikte werkeenheden een uitgestelde vervanging optreedt, welke een vervangingsverplichting doet ontstaan. Deze vervangingsverplichting moet bij gestegen vervangingswaarde hoger worden gewaardeerd.” m a b blz. 71 In verband met de waardestijging van die vervangingsplicht moeten voor zieningen getroffen worden, waarvan de aard en de grootte afhankelijk zijn van de wijze van financiering. I di bij t i d li k k In verband met de waardestijging van die vervangingsplicht moeten voor zieningen getroffen worden, waarvan de aard en de grootte afhankelijk zijn van de wijze van financiering. I di bij t i d li k k j g Indien er bij gestegen vervangingswaarde van een verlies gesproken kan worden, spruit dit verlies uitsluitend voort uit het feit, dat de uit afschrijving vrijgekomen middelen niet in de waardestijging delen. Niet de niet meer aanwezige werkeenheden maar de daarvoor in de plaats gekomen liquidi teiten veroorzaken het verlies. De consequenties van een algemene stijging van het prijsniveau Deze kwestie wil ik gaarne het eerst behandelen omd De consequenties van een algemene stijging van het prijsniveau Deze kwestie wil ik gaarne het eerst behandelen omd Deze kwestie wil ik gaarne het eerst behandelen, omdat de gestage geldont waarding een belangrijke oorzaak is van de stijging van de vervangingswaarde der produktiemiddelen. Ik it h t hi b ld bij ik d t d p Ik ga uit van het hiervoor gegeven voorbeeld, waarbij ik aanneem, dat de installatie geheel met eigen vermogen is gefinancierd. Aan het eind van het vierde jaar blijkt, dat de vervangingswaarde van de installatie met 10% is gestegen. Voorts zijn de vrijgekomen middelen opnieuw geinvesteerd en heb ben deel aan de prijsstijging. V d t l ik d t ll d i k h d h d d k p j jg g Verder stel ik, dat alleen de aanwezige werkeenheden herwaardeerd kun nen worden en dat op niet aanwezige werkeenheden niet kan worden afge schreven. De balansopstellingen in de achtereenvolgende jaren zijn dan als volgt (in duizenden guldens): Uitgangs Eind 4e Eind 4e Eind 10e Positie positie jaar voor jaar na jaar na ver herw aard. herw aard. vanging Installatie ƒ 2.000 ƒ 2.000 ƒ 2 .1 2 0 ƒ 2 .1 2 0 ƒ 2 .2 0 0 Herinvestering vrijgekom en m iddelen 800 880 2.200 ƒ 2.000 ƒ 2.800 ƒ 3.000 ƒ 4 .3 2 0 ƒ 2.200 Eigen verm ogen Afschrijving Herw aardering Installatie H erw aardering herinvestering ƒ 2.000 ƒ 2.000 800 ƒ 2.000 800 120 80 ƒ 2.000 2.120 120 80 ƒ 2.200 ƒ 2.000 ƒ 2.800 ƒ 3.000 ƒ 4 .3 2 0 ƒ 2.200 Installatie Herinvestering vrijgekom en m iddelen Eigen verm ogen Afschrijving Herw aardering Installatie H erw aardering herinvestering Er is hier geen sprake van een inhaalafschrijving. Een verlies kan optreden, indien men er niet in slaagt met de herinvestering een voldoende groot ver- mogensaccres te verkrijgen maar er bestaat geen causaal verband tussen een dergelijk verlies en de hoeveelheid niet meer aanwezige werkeenheden. Tenslotte het geval, dat de installatie gefinancierd is voor ƒ 1.500.000,— met eigen vermogen en ƒ 500.000,— met vreemd vermogen. De lening wordt in 10 jaar met gelijke bedragen afgelost. m ab blz. 12 De balansopstellingen in de achtereenvolgende jaren zijn dan als volgt: Uitgangs- Eind 4e Eind 4e Eind 10e Positie positie j. voor j. na jaar na ver- herw aard. herw aard. vanging Installatie ƒ 2.000 ƒ 2 .0 0 0 ƒ 2 .1 2 0 ƒ 2 .1 2 0 ƒ 2 .2 0 0 Herinvestering vrijgekom en m iddelen V rijgekom en m iddelen 600 660 1.680 30 ƒ 2.000 ƒ 2.600 ƒ 2.780 ƒ 3 .8 0 0 ƒ 2 .2 3 0 Eigen verm ogen ƒ 1.500 ƒ 1.500 ƒ 1.500 ƒ 1.500 ƒ 1.650 Lening 500 300 300 550 Afschrijving H erw aardering 800 800 2.120 Installatie H erw aardering her- 120 120 investering Financieringsw inst 60 60 30 ƒ 2.000 ƒ 2.600 ƒ 2.780 ƒ 3.800 ƒ 2.230 Ook hier geen inhaalafschrijving. Met het oog op het gestegen prijsniveau is het reëel, dat bij vervanging een lening van ƒ 550.000,— gesloten wordt. N i blijft li id idd l b hikb ƒ1 680 000 Ook hier geen inhaalafschrijving. Met het oog op het gestegen prijsniveau is het reëel, dat bij vervanging een lening van ƒ 550.000,— gesloten wordt. N i blijft li id idd l b hikb ƒ1 680 000 j g g g ƒ g Na vervanging blijft aan liquide middelen beschikbaar ƒ1.680.000,— + ƒ 550.000,- - ƒ 2.200.000,- = ƒ 30.000,-. Dit b d i it d 10% h t b d d l i h t i d j g g g ƒ g Na vervanging blijft aan liquide middelen beschikbaar ƒ1.680.000,— + ƒ 550.000,- - ƒ 2.200.000,- = ƒ 30.000,-. Dit b d i it d 10% h t b d d l i h t i d ƒ ƒ ƒ Dit bedrag is uiteraard 10% van het bedrag van de lening aan het eind van het vierde jaar. Naar gelang de waardestijging was opgetreden aan het eind van het le, 2e, 3e enz. jaar zou deze fjnancieringswinst resp. hebben bedra gen ƒ 45.000,—, ƒ 40.000,—, ƒ 35.000,— enz. Inhaalafschrijvingen en diversiteit I d l t t f T t Inhaalafschrijvingen en diversiteit In de laatste paragraaf, „Tot besluit”, van zijn artikel maakt Prof. van der Schroeff nog een opmerking, die ik hier duidelijkheidshalve geheel citeer en waarop ik gaarne nog even zou willen inhaken. Prof. van der Schroeff zegt: „Zowel de diversiteitsfactor als de wijze van financiering - zo mag de eind conclusie luiden - doen hun invloed gelden met betrekking tot de noodzaak inhaalafschrijvingen ten laste van de resultatenrekening te brengen. Bij ge noegzame diversiteit van levensduur en/of tijdstip van aanschaffing en ver vanging, alsook in het geval waarin duurzame produktiemiddelen geheel of gedeeltelijk met vreemd vermogen zijn gefinancierd is de „klassieke” winst- theorie op basis 'van de vervangingswaarde ten aanzien van bedoelde afschrij vingen - ziehier mijn huidig oordeel - „plus royaliste que le roi”.” Indien een groep duurzame produktiemiddelen een zodanige leeftijdsopbouw heeft, dat de jaarlijkse vervanging gelijk is aan de jaarlijkse afschrijving - wanneer dus de constante leeftijdsopbouw is bereikt en de diversiteit maxi maal is - is er niet alleen geen sprake van inhaalafschrijvingen bij gestegen m a b blz. 73 In de laatste paragraaf, „Tot besluit”, van zijn artikel maakt Prof. van der Schroeff nog een opmerking, die ik hier duidelijkheidshalve geheel citeer en waarop ik gaarne nog even zou willen inhaken. Prof. van der Schroeff zegt: „Zowel de diversiteitsfactor als de wijze van financiering - zo mag de eind conclusie luiden - doen hun invloed gelden met betrekking tot de noodzaak inhaalafschrijvingen ten laste van de resultatenrekening te brengen. Bij ge noegzame diversiteit van levensduur en/of tijdstip van aanschaffing en ver vanging, alsook in het geval waarin duurzame produktiemiddelen geheel of gedeeltelijk met vreemd vermogen zijn gefinancierd is de „klassieke” winst- theorie op basis 'van de vervangingswaarde ten aanzien van bedoelde afschrij vingen - ziehier mijn huidig oordeel - „plus royaliste que le roi”.” Indien een groep duurzame produktiemiddelen een zodanige leeftijdsopbouw heeft, dat de jaarlijkse vervanging gelijk is aan de jaarlijkse afschrijving - wanneer dus de constante leeftijdsopbouw is bereikt en de diversiteit maxi maal is - is er niet alleen geen sprake van inhaalafschrijvingen bij gestegen m a b blz. 73 prijsniveau maar men handelt dan volkomen correct, indien men de jaarlijkse vervanging ten laste van de resultatenrekening brengt. Dit past volkomen bij mijn stelling, dat men niet aanwezige voorraden werkeenheden niet kan her waarderen en dat men op niet aanwezige voorraden werkeenheden niet kan afschrijven. Inhaalafschrijvingen en diversiteit I d l t t f T t T ill i di h l d k h i d b ld prijsniveau maar men handelt dan volkomen correct, indien men de jaarlijkse vervanging ten laste van de resultatenrekening brengt. Dit past volkomen bij mijn stelling, dat men niet aanwezige voorraden werkeenheden niet kan her waarderen en dat men op niet aanwezige voorraden werkeenheden niet kan afschrijven. T ill i di h l d k h i d b ld j Ter illustratie diene het volgende sterk geschematiseerde voorbeeld. E d i h ft bij h i hti 3 lk lijk t g g Een onderneming heeft bij haar oprichting 3 volkomen gelijke sets machi nes aangeschaft voor ƒ 50.000,— per set. De sets hebben een levensduur van 5 jaar, worden ieder jaar met gelijke bedragen afgeschreven en hebben geen restwaarde. I d t t lt d 50 000 k h d d t d Iedere set stelt een voorraad van 50.000 werkeenheden voor, zodat de onderneming over een voorraad werkeenheden beschikt van 150.000. Een investering van ƒ 150.000,— komt derhalve overeen met deze voorraad werk eenheden. D it f h ij i ijk d idd l d b ikt itb idi De uit afschrijving vrijkomende middelen worden gebruikt voor uitbreiding van het aantal sets. E di b k i t t d t 6 j d d i b Een eenvoudige berekening toont aan, dat na 6 jaar de onderneming be schikt over 5 sets en dat dit aantal constant blijft. De samenstelling is dan n.1. 1 t i l t 50 000 k h d j g 1 set waarin opgesloten 50.000 werkeenheden 1 set waarin opgesloten 40.000 werkeenheden 1 set waarin opgesloten 30.000 werkeenheden 1 set waarin opgesloten 20.000 werkeenheden 1 set waarin opgesloten 10.000 werkeenheden Totaal 150.000 werkeenheden Ieder jaar wordt ƒ 50.000,— afgeschreven, wordt een set buiten gebruik ge steld en vervangen door een nieuwe. De investering is nog steeds ƒ 150.000,— hetgeen overeenkomt met de voorraad werkeenheden en deze blijft ook ƒ 150.000,—. De balans van de onderneming vertoont dan de volgende cijfers: Machines ƒ 250.000,-. Eigen vermogen ƒ 150.000, Afschrijvingen 100.000, ƒ 250.000,-. Eigen vermogen ƒ 150.000, Afschrijvingen 100.000, Indien het prijsniveau niet verandert blijven deze cijfers van jaar tot jaar gelijk; de afschrijving neemt toe met ƒ 50.000,— en neemt af met de aan schaffingswaarde van de buiten gebruik gestelde set. Inhaalafschrijvingen en diversiteit I d l t t f T t Men bereikt hetzelfde resultaat door de cijfers onveranderd te laten en de aanschaffing jaarlijks ten laste van de resultatenrekening te brengen. A h t i d k j blijkt d i d d t t Indien het prijsniveau niet verandert blijven deze cijfers van jaar tot jaar gelijk; de afschrijving neemt toe met ƒ 50.000,— en neemt af met de aan schaffingswaarde van de buiten gebruik gestelde set. Men bereikt hetzelfde resultaat door de cijfers onveranderd te laten en de aanschaffing jaarlijks ten laste van de resultatenrekening te brengen. A h t i d k j blijkt d i d d t t g g Aan het eind van zeker jaar blijkt de vervangingswaarde van de sets met 20% gestegen te zijn. Al d th d h d i i h l f h ij i dt t g g j Als nu de methode van herwaardering en inhaalafschrijving wordt toege past, dan ziet, na herwaardering enz. de balans er als volgt uit; Machines ƒ 300.000,- Eigen vermogen ƒ 150.000,- Verlies door in- Reserve herwaar- haalafschrijving 20.000,- dering 50.000,- Afschrijvingen 120.000,- m ab blz. 74 Hier wordt een bedrag van ƒ 250.000,—, dat overeenkomt met een voorraad werkeenheden van 250.000, met 20% verhoogd. De onderneming heeft echter nooit over een voorraad van 250.000 eenheden beschikt; 100.000 eenheden die er nooit geweest zijn worden herwaardeerd en op deze zelfde schimmige eenheden wordt een inhaalafschrijving van ƒ 20.000,— toegepast. De o.i. juiste balansopstelling is: Machines ƒ 180.000,- Eigen vermogen ƒ 150.000,- Reserve herwaard. 30.000,- Machines ƒ 180.000,- Eigen vermogen ƒ 150.000,- Reserve herwaard. 30.000,- De reserve herwaardering bedraagt hier 20% van de werkelijke voorraad werkeenheden ad ƒ 150.000,—. Ik h i l d tik l i d l h id t ij h t bl t De reserve herwaardering bedraagt hier 20% van de werkelijke voorraad werkeenheden ad ƒ 150.000,—. Ik h i l d tik l i d l h id t ij h t bl t ƒ , Ik hoop in een volgend artikel in de gelegenheid te zijn het probleem te behandelen ingeval van partiële prijsstijging. m ab blz. 75
https://openalex.org/W3036277307	https://europepmc.org/articles/pmc7303182?pdf=render	English	null	New international guidance on quality, safety and efficacy of DNA vaccines	npj vaccines	2,020	cc-by	2,013	COMMENT OPEN New international guidance on quality, safety and efﬁcacy of DNA vaccines action to prevent epidemics has identiﬁed alignment of regulatory expectations for the testing and evaluation of novel vaccine candidates as a key enabler for effective outbreak responses4. In 2005, WHO developed “Guidelines for assuring the quality and nonclinical safety evaluation of DNA vaccines”5 and, based on the subsequent progression of several DNA vaccines to clinical evaluation and increasing awareness of the potential utility of DNA vaccines in outbreak responses, initiated a process for revision of those guidelines in February, 2018 from which a draft was publicly disseminated in mid-2019 for comment6. The revised Guidelines focus on biologically manufactured bacterial plasmid DNA for use in humans, and address aspects related to control of manufacture and characterization, approaches to nonclinical and clinical testing, and information that may be required by national regulatory authorities for approval of clinical trials or licensure. The revised Guidelines are considered unlikely to be applicable to RNA vaccines, and development by WHO of separate document(s) covering RNA vaccines is in progress6. In this issue of npj Vaccines, Sheets et al.7 provide an overview of the revision process to date, report the outcomes from an Informal Consultation of subject matter experts held in December, 2019 to review the draft revisions and discuss public comments that had been received, and describe the timeline for further revision and subsequent approval and implementation of the new guidelines by the WHO Expert Committee on Biological Standardization during 2020. In response to other recent global public health emergencies, such as the 2014–2015 Ebola virus epidemic in West Africa and the widespread emergence of Zika virus in 2015–2016, several national and international health policy and funding agencies have prepared lists of priority pathogens considered to have potential to cause pandemic disease, and promoted the develop- ment of platform technologies that can speed the production of new vaccines against those agents, or against a previously unidentiﬁed emerging pathogen, often termed “Disease X”. One such group of platform technologies that is considered by many to be a key component of rapid vaccine development are nucleic acid vaccines, in which the genetic sequences for potentially immunogenic and protective antigens from the pathogen are delivered as DNA or RNA molecules. www.nature.com/npjvaccines 1Department of Microbiology and Immunology, Institutional Ofﬁce of Regulated Nonclinical Studies, Institute for Human Infections and Immunity, Sealy Institute for Vaccine Sciences, and WHO Collaborating Center for Vaccine Research, Evaluation and Training on Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX, USA. ✉email: d.beasley@utmb.edu COMMENT OPEN New international guidance on quality, safety and efﬁcacy of DNA vaccines David W. C. Beasley 1✉ npj Vaccines (2020) 5:53 ; https://doi.org/10.1038/s41541-020-0199-0 action to prevent epidemics has identiﬁed alignment of regulatory expectations for the testing and evaluation of novel vaccine candidates as a key enabler for effective outbreak responses4. In 2005, WHO developed “Guidelines for assuring the quality and nonclinical safety evaluation of DNA vaccines”5 and, based on the subsequent progression of several DNA vaccines to clinical evaluation and increasing awareness of the potential utility of DNA vaccines in outbreak responses, initiated a process for revision of those guidelines in February, 2018 from which a draft was publicly disseminated in mid-2019 for comment6. The revised Guidelines focus on biologically manufactured bacterial plasmid DNA for use in humans, and address aspects related to control of manufacture and characterization, approaches to nonclinical and clinical testing, and information that may be required by national regulatory authorities for approval of clinical trials or licensure. The revised Guidelines are considered unlikely to be applicable to RNA vaccines, and development by WHO of separate document(s) covering RNA vaccines is in progress6. In this issue of npj Vaccines, Sheets et al.7 provide an overview of the revision process to date, report the outcomes from an Informal Consultation of subject matter experts held in December, 2019 to review the draft revisions and discuss public comments that had been received, and describe the timeline for further revision and subsequent approval and implementation of the new guidelines by the WHO Expert Committee on Biological Standardization during 2020. Over the past two decades, outbreaks of novel and re-emerging infectious diseases, particularly caused by zoonotic viruses, have prompted an international push to develop strategies addressing research, nonclinical and clinical testing, manufacturing, and regulatory evaluation that can increase the rapidity of medical countermeasure development to control outbreaks and prevent resurgence of those diseases. Most recently, the emergence of a novel coronavirus, SARS-CoV-2, and its explosive dissemination— resulting in a global disease pandemic (termed “coronavirus disease 2019”, or COVID-19) that, as of late April 2020, is associated with almost 3.1 million conﬁrmed infections and 220,000 deaths1—has prompted an unprecedented response from the scientiﬁc and public health communities to develop therapies and vaccines. The publicly stated timeframes of 12–18 months for availability of licensed vaccines targeted against SARS-CoV-2 are optimistic, but would have been considered fantastic even a decade ago. Published in partnership with the Sealy Center for Vaccine Development COMMENT OPEN New international guidance on quality, safety and efﬁcacy of DNA vaccines Compared with more established approaches for immunization using injection of live attenuated or inactivated pathogens, or protein subunits of those pathogens, nucleic acid-based approaches offer several advantages for rapid response including rapid adaptability, simpler manufactur- ing processes, enhanced physical stability, and robust safety2,3. In addition, they may beneﬁt from simpliﬁed requirements for nonclinical safety evaluation when alternative novel sequences are inserted into a previously well characterized platform construct. Some questions remain regarding the immunogenicity of nucleic acid vaccines compared with other more traditional vaccine approaches, but signiﬁcant efforts have been made to address those through optimization of vaccination routes and timing of doses, and the use of vehicles and devices that facilitate uptake of the nucleic acid by host cells, resulting in improved performance of this class of vaccines in large animal models and in clinical use2,3. This communication is timely, given the current global impacts of COVID-19 and the rapid development of a large number of candidate vaccines, including several based on DNA or other nucleic acid platforms. At the time of this report (late April, 2020), and only 2–3 months following initiation of their development, a candidate DNA vaccine (ClinicalTrials.gov identiﬁer NCT04283461) and a candidate RNA vaccine (NCT04336410) are in Phase 1 clinical trials, and more than a dozen other nucleic acid vaccine candidates are in development8. For the two vaccines now in Phase 1, it seems that the rapid path to clinical testing was facilitated by both the short timelines for manufacturing of initial clinical lots and streamlined requirements for nonclinical safety testing based on prior nonclinical and clinical experience with those platforms8,9. Evaluation of vaccine efﬁcacy in newly developed animal models for COVID-19 can be expected to proceed in parallel with clinical safety testing. Vaccine development against SARS-CoV-2 is beneﬁting from prior experience with other closely related emerging coronaviruses associated with severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS). COMMENT OPEN New international guidance on quality, safety and efﬁcacy of DNA vaccines Currently there are no licensed vaccines for coronaviruses that cause respiratory diseases in humans, and induction of robust and durable protective immunity against infection by SARS-CoV-2 Concurrent with facilitating new approaches and technologies that may speed vaccine development, the WHO’s R&D Blueprint for Department of Microbiology and Immunology, Institutional Ofﬁce of Regulated Nonclinical Studies, Institute for Human Infections and Immunity, Sealy Institute for Vaccine ciences, and WHO Collaborating Center for Vaccine Research, Evaluation and Training on Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX, USA. email: d.beasley@utmb.edu ublished in partnership with the Sealy Center for Vaccine Development Published in partnership with the Sealy Center for Vaccine Development D.W.C. Beasley 2 through vaccination may be challenging. Furthermore, the out- comes of nonclinical efﬁcacy testing of some candidate vaccines for SARS and MERS suggests that another signiﬁcant concern is the potential for immune potentiation of COVID-19 lung disease9–12. The anticipated demand for hundreds of millions of doses for SARS-CoV-2 vaccines and their use in a wide range of target populations, coupled with the likely limitations of manufacturing capacity and scalability for any single vaccine type, suggests that engagement and coordination of multiple international partners for development of a portfolio of licensed vaccines based on multiple platforms will ideally be needed13. 8. Thanh Le, T. et al. The COVID-19 vaccine development landscape. Nat. Rev. Drug Discov. https://doi.org/10.1038/d41573-020-00073-5 (2020). 9. Lurie, N., Saville, M., Hatchett, R. & Halton, J. Developing Covid-19 Vaccines at Pandemic Speed. N. Engl. J. Med. https://doi.org/10.1056/NEJMp2005630 (2020). 10. Lu, S. Timely development of vaccines against SARS-CoV-2. Emerg. Microbes Infect. 9, 542–544 (2020). 11. Chen, W. H., Strych, U., Hotez, P. J. & Bottazzi, M. E. The SARS-CoV-2 Vaccine Pipeline: an Overview. Curr. Trop. Med. Rep. https://doi.org/10.1007/s40475-020- 00201-6 (2020). 12. Hotez, P. J., Corry, D. B. & Bottazzi, M. E. COVID-19 vaccine design: the Janus face of immune enhancement. Nat. Rev. Immunol. https://doi.org/10.1038/s41577- 020-0323-4 (2020). 12. Hotez, P. J., Corry, D. B. & Bottazzi, M. E. COVID-19 vaccine d 13. Berkley, S. COVID-19 needs a big science approach. Science. https://doi.org/ 10.1126/science.abb8654 (2020). The current COVID-19 pandemic will provide nucleic acid vaccine platforms their greatest opportunity to demonstrate in practice their proposed value as key components in the rapid response to controlling global or regional emerging infectious disease threats. COMPETING INTERESTS The author declares no competing interests. Reprints and permission information is available at http://www.nature.com/ reprints Reprints and permission information is available at http://www.nature.com/ reprints Reprints and permission information is available at http://www.nature.com/ reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. COMMENT OPEN New international guidance on quality, safety and efﬁcacy of DNA vaccines The availability of the revised WHO Guidelines related to DNA vaccines, currently as a draft and subsequently in a ﬁnal form, should facilitate communications between vaccine developers and regulatory agencies as nucleic acid vaccine candidates progress towards possible licensure. ADDITIONAL INFORMATION Correspondence and requests for materials should be addressed to D.W.C.B. Correspondence and requests for materials should be addressed to D.W.C.B. Correspondence and requests for materials should be addressed to D.W.C.B. Received: 4 May 2020; Accepted: 14 May 2020; Published in partnership with the Sealy Center for Vaccine Development © The Author(s) 2020 REFERENCES 1. WHO. Coronavirus disease 2019 (COVID-19) Situation Report – 101. https://www. who.int/docs/default-source/coronaviruse/situation-reports/20200430-sitrep- 101-covid-19.pdf (2020). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. 2. Lee, J., Arun Kumar, S., Jhan, Y. Y. & Bishop, C. J. Engineering DNA vaccines against infectious diseases. Acta Biomater. 80, 31–47 (2018). 3. Rauch, S., Jasny, E., Schmidt, K. E. & Petsch, B. New vaccine technologies to combat outbreak situations. Front Immunol. 9, 1963 (2018). 4. WHO. An R&D Blueprint for Action to Prevent Epidemics: Plan of Action, May 2016. https://www.who.int/blueprint/about/r_d_blueprint_plan_of_action.pdf (2016). https://www.who.int/blueprint/about/r_d_blueprint_plan_of_actio 5. WHO. Guidelines for assuring the quality and nonclinical safety evaluation of DNA vaccines. WHO Tech. Rep. Ser. 941, 57–81 (2007). 6. WHO. Guidelines for Assuring the Quality, Safety, and Efﬁcacy of DNA Vaccines. http:// www9.who.int/biologicals/WHO_DNA_vaccine_HK_26_July_2019.pdf (2019). 7. Sheets, R. et al. WHO informal consultation on the guidelines for evaluation of the quality, safety, and efﬁcacy of DNA vaccines, Geneva, Switzerland, December 2019. NPJ Vaccines 5, 52 (2020). © The Author(s) 2020 Published in partnership with the Sealy Center for Vaccine Development npj Vaccines (2020) 53 Published in partnership with the Sealy Center for Vaccine Development

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